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Ph.D Thesis
ESTIMATION OF ALUMINUM, MANGANESE, IRON, COPPER AND ZINC IN BIOLOGICAL SAMPLES OF
HUMAN SUBJECTS HAVING DIFFERENT NEUROLOGICAL AND PSYCHIATRIC DISORDERS
THESIS SUBMITTED TOWARDS THE PARTIAL FULFILMENT OF THE
REQUIREMENT OF THE UNIVERSITY OF SINDH, FOR THE AWARD OF
DOCTOR OF PHILOSOPHY DEGREE IN ANALYTICAL CHEMISTRY
MARIAM SHAHZADI
National Centre of Excellence in Analytical Chemistry, University of Sindh, Jamshoro – PAKISTAN
2017
CERTIFICATE
This is to certify that the work present in this thesis entitled “ESTIMATION OF
ALUMINUM, MANGANESE, IRON, COPPER AND ZINC IN BIOLOGICAL
SAMPLES OF HUMAN SUBJECTS HAVING DIFFERENT NEUROLOGICAL
AND PSYCHIATRIC DISORDERS” has been carried out by Ms. Mariam
Shahzadi D/O Muhammad Aslam under our supervision. The work is genuine,
original and, in our opinion, suitable for submission to the University of Sindh
for the award of degree of doctor of philosophy in analytical chemistry.
SUPERVISOR
___________________________________________
Dr. Hassan Imran AfridiAssociate ProfessorNational Centre of Excellence in Analytical ChemistryUniversity of Sindh, Jamshoro Pakistan
CO-SUPERVISOR
__________________________________________
Dr. Tasneem Gul KaziProfessorNational Centre of Excellence in Analytical ChemistryUniversity of Sindh, Jamshoro Pakistan
DEDICATION
This endeavor is dedicated to my beloved family, companionable
supervisors Dr. Hassan Imran Afridi and Prof. Dr. Tasneem Gul Kazi.
All I have and will accomplish are only possible due to their
continuous prayer and sacrifices. It is their unconditional love that
motivates me to set higher target.
ACKNOWLEDGEMENTS
I praise ALMIGHTY ALLAH, the most merciful and the most gracious, who is the entire and
only source of every knowledge and wisdom endowed to mankind and who guides me in the
darkness, help me in difficulties and blessed me with the ability to do this work and his
PROPHET HAZRAT MUHAMMAD (Salallah-o-Allaehe Wasallim) .
I wish to acknowledge the NCEAC University of Sindh Jamshoro for providing the financial
support, which made this study possible. First and foremost I would like to take this opportunity
to express my sincerest thanks to my worthy, respectful and devoted supervisors Dr. Hassan
Imran Afridi and Prof. Dr. Tasneem Gul Kazi without their constant help, deep interest kind
and attentive guidance, the completion of this thesis was not possible. I appreciate all their
contributions of time, ideas, immense intellectual input, patience and sympathetic behavior.
With due respect, I am deeply and strongly obliged to Director Prof. Dr. Shahabuddin Memon
for his encouraging attitude, providing good research facilities, excellent research environment
to carry out this research work. I am so grateful to Prof. Dr. Sirajuddin, Prof. Dr. Syed Tufail
Hussain Sherazi, Dr. Jamel Ahmad Baig, Dr. Amber Rehena Solangi, Dr. Najma Memon, Dr.
Farah Naz Talpur, Dr. Amna Baloch, Dr. Huma Ishaq, Dr. Sarfaraz Mehasar and Dr. Ayaz
Memon for their research consultancy.
I really acknowledge and offer my heartiest gratitude to my parents and brothers for their moral
support, cooperation, encouragement, patience, tolerance and prayers for my health and success
during this work
I want to extend my sincere and heartfelt thanks and appreciation to my colleagues, Dr.
Naeemullah, Ms. Salma Aslam Arain, Dr. Sadaf Sadia Arain, Jamshed Ali, Asma Akhtar, Mr.
Abdul Haleem Panwhar, Mr. Kapil Dev for their assistance, good company, stunning behavior,
friendly attitude and keeping excellent healthy and competitive environment for learning
purpose in the research Labs. Special thanks to other research fellows specially Shahnawz
Blaoch, Sehrish Talpur, Mr. Mustafa Khan, Mr. Noman Khan and Mr. Muhammad Bilal and rest
of my research fellows for their help and transparency during my research. They always
encouraged and cooperated with me to provide the invaluable input for the improvement of this
study. Among the administrative staff of the center, I am highly grateful to Mr. Pir Ziauddin,
Mr. Pir Sirajuddin, Mr. Akhtar Ali Vighio, Mr. Mudasir Ahmed Arain, Mr. Nasrullah, Mr.
Jawad Ahmad and the rest staff members of center.
.
Mariam Shahzadi
ABSTRACT
Metals play main role in the living beings, maintain cell structure and neurotransmission, regulate gene expression, and antioxidant response, and other physiological functions. Though, higher metal accumulate in the nervous system might be toxic, cause oxidative damage, disrupting mitochondrial function, and impairing the activity of various enzymes. Neurodegenerative diseases are thought to be multifactorial, while metals efficiency/deficiency of some metals. Aluminum, manganese, iron copper and zinc (Al, Mn, Fe, Cu & Zn) can be involved as cofactors in abnormalities or suspected of being risk factors for this disorder. The Al and Mn can be involved as cofactors in abnormalities or suspected of being risk factors for this disorder. Same as in neurological disorders very limited information available on the role of trace elements in psychiatric disorders. Immense pieces of evidence support the idea that high exposure to trace and toxic elements, such as Al, Mn, Fe, Cu while deficiency of Zn may be factors or cofactors in the etiopathogenesis of a variety of psychiatric disorders.
Epidemiological and clinical studies have indicates a strong correlation among high elemental exposure and a number of neuro and psychiatric disorders, including Multiple sclerosis, Alzheimer’s disease, , Parkinson’s disease, stroke, schizophrenia, and bipolar disorders. Excess intake of nonessential and essential trace metals can occur from dietary intake as well as occupational and environmental exposures.
Industrial society yield several ease foods with aluminum additives that increase many food properties and usage of alum (aluminum potassium sulfate or aluminum sulfate) in water purification to enable distribution of greater volumes of drinking water to millions of urban users.
The current determination assesses the extent to which the routine, life-long consumption, and metabolism of Al compounds leads to Alzheimer’s disease. Exposed to Mn from mining, working in Mn metal and alloy smelters, dry cell battery manufacture, work with fungicides & fertilizers containing this element, and welding are examples of industrial work which may cause damaging to the central and peripheral nervous systems and psychological disorders which can be progressive and irreversible. The accumulation of Fe and Cu in brain region causes degeneration of dopaminergic neuron and form complex with neuromelanin inducing oxidative stress which leads to different nervous system.
Analysis of trace elements in human tissues and fluids were used to acquire information on nutritional level for diagnosis of diseases, indicating of systemic intoxication, and environmentally exposed. In the many cases, whole blood, serum, urine and plasma were determine. For present study, biological samples were collecting (Blood, scalp hair and serum) of patients having different neurological disorders (Parkinson’s, Alzheimer’s, Dementia, Multiple sclerosis, Brain tumor, Brain hemorrhage, Stroke) and psychiatric disorders (Schizophrenia, Bipolar disorder, Depression). To comparing the biological samples were collecting from healthy controls/ referents of similar age groups, socioeconomic status of both genders.
Generally, the levels of these elements are very lower in biological samples, so sensitive and sophisticated analytical techniques are required for their analysis. Whereas for common techniques such as flame atomic absorption spectrometry, lower level of elements and complex of matrix of biological samples requires enrichment and separation of analyte before their analysis. In this regard, a variety of techniques have been proposed for the separation and preconcentration of study analytes such as modified cloud point extraction method, temperature controlled ionic liquid-dispersive liquid phase microextraction, dual dispersive ionic liquid based on ultrasound assisted microextraction, modified dispersive liquid-phase microextraction, switchable solvent extraction, deep eutectic solvent extraction.
All of the above mentioned advanced preconcentration procedure were applied for Al, Mn and Fe, Cu, Zn in scalp hair and blood serum samples. The validity and accuracy of developed procedures were carried out by analysis of certified reference material of human hair (NCS ZC81002), human blood (Seronorm Trace Elements Whole Blood (LOT 1103128) and serum from Clincheck control lyophilized ® human serum. Authenticity of the different developed methodology was also checked by the standard addition method in a real sample, which gave satisfactory results. Validity of the proposed procedure was checked by relative standard deviation (%RSD), which was obtained to be <5%. In some developed method the effects of different variables/factors were optimized by multivariate techniques.
The mean concentration values of Al was observed to be higher in scalp hair samples of different types of male psychiatric patients, schizophrenia (13.6±1.02 μg g-1) and bipolar disorder (12.3±1.57 μg g-1) as compared with normal referent (6.73±1.69 μg g-1). Whereas Mn concentration was found to be significantly higher (p=0.01–0.001), in schizophrenia (4.71±0.46 μg g-1) and bipolar disorder (5.83±0.85 μg g-1) normal referent (3.60±0.47) μg g-1. In Alzheimer’s, stroke and dementia disease patients the concentration of Al in scalp hair, blood samples was two folds higher than normal referent (11.3±2.03 µg g-1), (10.3±1.76 µg L-1). The resulted data indicate that the accumulation and metabolism of Al are altered in Alzheimer’s patients.
The level of Mn in scalp hair samples of Parkinson's male and female patients was found to be significantly higher (p<0.01) at confidence intervals 95% CI (9.64–10.5) µg g-1 versus referents CI (3.65–4.09) µg g-1. In Parkinson's, neurons produce dopamine in the substantia nigra die due to high exposure of Mn, decreasing the overall supply of dopamine and influence the brain's capability to effects movement.
Whereas level of Mn in blood samples of Parkinson’s patients, dementia, multiple sclerosis was found to be higher 56% , 69% and 81% than normal referent .
The mean levels of Fe in serum samples of different neurological disorders have Alzheimer’s patients are significantly higher (p<0.001) than the controls (CI 660±50.5 µg L-1) of same age group. The concentration of Cu ion in blood serum of different neurological disorders was found to be greater (P<0.001) at 95% confidence intervals (CI) for Alzheimer’s (CI: 1650±21.4), depression (CI 1430±10.9), dementia (CI 1530±8.38) μg L-1 versus normal referents (CI: 801±54.6) μg L-1. Higher concentration of Cu ion varies the level of neurotransmitter, which leads to dys-functioning of brain and chronic mental disorder.
The resulting data indicate that the Zn levels are significantly lower (p<0.001), such as 11%, 15% and 19% in serum samples of schizophrenia, depression and bipolar disorder respectively than controls of same age group at 95% confidence intervals (CI 0.423±0.08 mg L-1). Zn deficiency may alter its homeostasis in the brain created different dysfunctions. Consequently, for proper brain functioning and vesicular Zn is an essential nutrient for neuronal signaling factor.
TABLE OF CONTENTS
CERTIFICATE............................................................................................................II
DEDICATION............................................................................................................III
ACKNOWLEDGEMENTS.......................................................................................IV
ABSTRACT.................................................................................................................V
TABLE OF CONTENTS........................................................................................VIII
LIST OF TABLE..........................................................................................................1
LIST OF FIGURES......................................................................................................3
ABBREVIATIONS......................................................................................................5
CHAPTER 1..................................................................................................................8
INTRODUCTION........................................................................................................8
1.1 MOTIVATION..................................................................................................................8
1.2 CONTRIBUTIONS OF THE THESIS...................................................................................9
1.3 STRUCTURE OF THE THESIS.........................................................................................10
1.3.1 CHAPTER 1...........................................................................................................10
1.3.2 CHAPTER 2................................................................................................................10
MULTIVARIATE OPTIMIZATION.........................................................................................10
1.3.3 CHAPTER 3...........................................................................................................10
1.3.4 CHAPTER 4...........................................................................................................10
1.3.5 CHAPTER 5...........................................................................................................11
1.4 NEUROLOGICAL DISORDERS........................................................................................11
1.5 PSYCHOLOGICAL DISORDERS......................................................................................12
1.6 ROLE OF METALS IN PATHOGENESIS OF NEUROLOGICAL/PSYCHIATRIC
DISORDERS……………………………………………………………………………..13
1.6.1 ALUMINUM...........................................................................................................14
1.6.2 MANGANESE........................................................................................................16
1.6.3 IRON.....................................................................................................................17
1.6.4 COPPER.................................................................................................................17
1.6.5 ZINC......................................................................................................................18
1.7 BIOLOGICAL SAMPLES.................................................................................................19
1.8 ANALYTICAL TECHNIQUES AND EXTRACTION METHOD...........................................20
1.9 MULTIVARIATE STUDY................................................................................................26
1.10 AIMS AND OBJECTIVES..............................................................................................26
1.11 SUMMARY...................................................................................................................28
LITERATURE REVIEW/BACKGROUND............................................................29
2.1 NEUROLOGICAL DISORDERS........................................................................................29
2.2 PSYCHOLOGICAL DISORDERS......................................................................................31
2.3 ROLE OF METALS IN PATHOGENESIS OF NEUROLOGICAL DISORDERS.....................32
2.3.1 ALUMINUM...........................................................................................................34
2.3.2 MANGANESE........................................................................................................34
2.3.3 IRON.....................................................................................................................35
2.3.4 COPPER.................................................................................................................36
2.3.5 ZINC......................................................................................................................37
2.4 BIOLOGICAL SPECIMENS..............................................................................................38
2.5 ANALYTICAL TECHNIQUES AND EXTRACTION METHODS..........................................38
2.6 MULTIVARIATE STUDY................................................................................................41
2.7 SUMMARY.....................................................................................................................42
CHAPTER 3...............................................................................................................43
RESEARCH METHODOLOGY...............................................................................43
3.1 PLAN OF WORK............................................................................................................43
3.2 STUDY POPULATION.....................................................................................................43
3.2.1 QUESTIONNAIRE EMPLOYED IN SAMPLING CAMPAIGN..................................46
3.2.2 SAMPLING............................................................................................................46
SCALP HAIR...................................................................................................................46
3.3 CHEMICALS AND REAGENTS......................................................................................47
3.4 INSTRUMENTATION......................................................................................................48
3.5 STATISTICAL ANALYSIS...............................................................................................49
3.7 DEVELOPED ADVANCED EXTRACTION METHODOLOGIES..........................................52
PROCEDURE...................................................................................................................52
ANALYTICAL FIGURES OF MERIT.................................................................................52
3.7.2 DUAL CLOUD POINT EXTRACTION (D-CPE) METHODOLOGY TO DETERMINE
ZINC IN SERUM SAMPLES..............................................................................................53
PROCEDURE...................................................................................................................53
ANALYTICAL FIGURES OF MERIT.................................................................................54
3.7.3 TEMPERATURE CONTROLLED IL-BASED DISPERSIVE MICRO-EXTRACTION (TIL-
DLLME) USING TWO COMPLEXING AGENTS, TO ANALYZE AL IN SCALP HAIR
SPECIMENS OF AD PATIENTS: A MULTIVARIATE STUDY............................................56
PROCEDURE...................................................................................................................56
EXPERIMENTAL DESIGN...............................................................................................57
CALIBRATION AND SENSITIVITY..................................................................................57
3.7.4 PRECONCENTRATION OF TRACE LEVEL OF CU IN SERUM SPECIMENS OF
PATIENTS HAVING NEURO DISEASED USING ULTRASOUND ENERGY.........................60
ANALYTICAL FIGURES OF MERIT.................................................................................60
3.7.5 AN INNOVATIVE MODIFIED DISPERSIVE LIQUID-PHASE EXTRACTION OF IRON
IN SERUM SPECIMENS OF NEURO DISEASED PATIENTS...............................................61
3.7.6 DEVELOPMENT OF GREEN, SWITCHABLE SOLVENT EXTRACTION METHOD FOR
ENRICHMENT OF ALUMINUM IN BLOOD SAMPLES OF DIFFERENT NEUROLOGICAL
DISORDERS PATIENT......................................................................................................63
PROCEDURE OF SS-E....................................................................................................63
METHOD VALIDATION..................................................................................................65
3.7.7 PRECONCENTRATION OF TRACE LEVEL MANGANESE IN BLOOD SAMPLES
USING A DEEP EUTECTIC SOLVENT EXTRACTION (DES) METHOD..............................66
PREPARATION OF DES...................................................................................................66
DES-ASSISTED EXTRACTION METHOD.........................................................................66
ANALYTICAL FIGURES OF MERIT.................................................................................68
3.8 SUMMARY...............................................................................................................69
RESULTS AND DISSCUSSION.............................................................................70
4.1 ANALYSIS OF MANGANESE IN SCALP HAIR SPECIMENS OF PD PATIENTS................70
GENERAL REMARKS......................................................................................................70
4.1.1 RESULTS...............................................................................................................70
EFFECT OF PH...............................................................................................................71
EFFECT OF PAN CONCENTRATION................................................................................71
TRITON X-114................................................................................................................72
4.2 ZINC LEVELS IN SERUM SAMPLES OF PSYCHIATRIC PATIENTS.................................77
4.3 ANALYSIS OF AI IN SCALP HAIR SPECIMENS OF AD PATIENTS BY ADVANCE
EXTRACTION METHODOLOGY: A MULTIVARIATE STUDY................................................81
GENERAL REMARKS......................................................................................................81
OPTIMIZING BY CENTRAL 23+ STAR ORTHOGONAL COMPOSITE DESIGN (CCD)........83
4.4 DETERMINATION OF TRACE LEVEL OF COPPER IN SERUM SAMPLES OF PATIENTS
HAVING NEUROLOGICAL DISORDERS................................................................................92
GENERAL REMARKS......................................................................................................92
4.4.1 OPTIMIZATION OF EXPERIMENTAL FACTORS.....................................................93
PAN CONCENTRATION...................................................................................................93
AMOUNT OF IL...............................................................................................................94
SONICATION TIME.........................................................................................................95
EFFECT OF MATRIX ION................................................................................................95
4.4.2 APPLICATION.......................................................................................................97
4.5 A DISPERSIVE LIQUID-PHASE MICRO-EXTRACTION METHODOLOGY FOR TRACE
LEVEL OF IRON IN SERUM SAMPLES OF NEURO DISORDERS PATIENTS...........................98
GENERAL REMARKS......................................................................................................98
4.5.1 OPTIMIZED EXPERIMENTAL FACTORS................................................................98
EFFECT OF PH................................................................................................................98
BACK EXTRACTING SOLVENT.......................................................................................99
4.6.1 CHARACTERIZATION OF SS...............................................................................102
DESCRIPTION OF SS.....................................................................................................102
4.6.2 OPTIMIZATION OF FACTORS.............................................................................103
4.7 PRECONCENTRATION OF TRACE LEVEL MANGANESE IN BLOOD SAMPLES OF
PATIENTS WITH DIFFERENT NEUROLOGICAL DISORDERS USING A DEEP EUTECTIC
SOLVENT EXTRACTION.....................................................................................................108
GENERAL REMARKS....................................................................................................108
4.7.1 OPTIMIZATION OF DES-E METHOD...................................................................108
EFFECT OF PH.............................................................................................................108
EFFECT OF PAN CONCENTRATION..............................................................................109
EFFECT OF MOLAR RATIO OF EUTECTIC MIXTURES FOR DES..................................110
EFFECT OF DEEP EUTECTIC SOLVENT VOLUME.........................................................110
EFFECT OF DECANOL AND HEXANOL VOLUME.........................................................111
INTERFERENCE STUDY................................................................................................111
4.8 SUMMARY...................................................................................................................113
CHAPTER 5.............................................................................................................114
CONCLUSION AND FUTURE DIRECTIONS....................................................114
5.1 CONCLUSION..............................................................................................................114
5.2 SOCIOECONOMIC IMPACT..........................................................................................117
5.3 RECOMMENDATIONS..................................................................................................118
5.4 SUMMARY...................................................................................................................119
REFRENCES............................................................................................................120
LIST OF TABLE
TABLE 3-1 INSTRUMENTAL CONDITIONS FOR FLAME ATOMIC ABSORPTION
SPECTROMETRY……..49
TABLE 3-2 DETERMINATION OF Al3+, Mn2+ CERTIFIED HUMAN HAIR SAMPLES BY CDM AND
MWD………………………………………………………………………………………………….5
1TABLE 3-3 PERFORMANCE CHARACTERISTICS OF THE PRESENTED d-CPE
METHOD……………….53
TABLE 3-4 ANALYSIS OF Mn2+ IN CERTIFIED REFERENCE MATERIAL (µg g-1) BY d-CPE (n
=10).....53
TABLE 3-5 CHARACTERISTICS PERFORMANCE OF THE PRESENTED d-CPE PROCEDURE…………...55
TABLE 3-6 PRECONCENTRATION OF Zn2+, IN CERTIFIED REFERENCE MATERIAL (mg L-1) BY
CONVENTIONAL CPE AND d-CPE METHODS (n=10)
………………………………………………...55
TABLE 3-7 VARIABLES AND LEVELS USED IN THE FACTORIAL DESIGN FOR EXTRACTION OF Al3+…
57
TABLE 3-8 ANALYSİS OF Al3+ İN CERTIFIED REFERENCE MATERIAL AND SPİKED SAMPLE OF SCALP
HAİR USİNG TIL-DLLME METHOD.............................................................................................59
TABLE 3-9 CHARACTERISTICS PERFORMANCE OF THE DEVELOPED UDIL-µE
PROCEDURE………..61
TABLE 3-10 PRECONCENTRATION OF CU ION IN CERTIFIED REFERENCE MATERIAL (µg L-1) BY
UDIL- µE (n=4)
…………………………………………………………………………………………….61
TABLE 3-11 CHARACTERISTICS PERFORMANCE OF THE DEVELOPED MDLP-ΜE
PROCEDURE……..63
TABLE 3-12 PRECONCENTRATION OF Fe IN CERTIFIED REFERENCE MATERIAL (µg L-1) BY MDLP-
µE METHOD (n=10)
………………………………………………………………………………………63
TABLE 3-13 ANALYSIS OF Al3+ IN CERTIFIED REFERENCE MATERIAL AND SPIKED SAMPLE OF
BLOOD USING (SS-E) METHOD……………...
…………………………………………………………….....66
TABLE 3-14 CHARACTERISTIC PERFORMANCE OF THE PROPOSED DES-E
PROCEDURE…………………………………………………………………………………….….....68
TABLE 3-15 ANALYSIS OF Mn2+ IN CERTIFIED REFERENCE MATERIAL AND SPIKED BLOOD SAMPLE
USING DES-E METHOD……………………………………………………………………………....68
TABLE 4-1 INFLUENCE OF SELECTED FOREIGN IONS ON THE %RECOVERY OF THE Mn2+
DETERMINED BY APPLYING THE d-CPE
METHOD………………………………………………………………….74
TABLE 4-2 CONCENTRATION OF Mn2+ IN SCALP HAIR SAMPLES OF PARKINSON'S PATIENTS AND
HEALTHY CONTROL SUBJECTS (µg g-1)……………………………..……………………………….75
TABLE 4-3 COMPARATIVE DATA OF ANALYTICAL PARAMETERS FOR Mn2+ WITH AND WITHOUT
PRECONCENTRATION METHODS COUPLED WITH DIFFERENT INSTRUMENTAL
TECHNIQUES……......76
TABLE 4-4 THE CONCENTRATION OF Zn2+ IN SERUM SAMPLES OF PSD MALE PATIENTS AND
HEALTHY CONTROL SUBJECTS (mg L-1) ……………………………………………………………...
………...81
TABLE 4-5 PLACKETT–BURMAN DESIGN FOR THE SIGNIFICANT VARIABLE DETERMINATION (n=5)
………………………………………………………………………………………………....83
TABLE 4-6 CENTRAL 23+ STAR ORTHOGONAL COMPOSITE DESIGN (N=16) FOR THE SET OF (IL), (L1)
AND (P)…………………………………………………………………………………………….…85
TABLE 4-7 THE ESTIMATED EFFECTS AND INTERACTION OF VARIABLES BY ANOVA FOR
RECOVERY TEST…………………………………………………………………………………….
…………....86
TABLE 4-8 EFFECTS OF THE MATRIX IONS ON THE RECOVERIES OF THE Al3+
……………………….89
TABLE 4-9 THE CONCENTRATION OF Al3+ IN SCALP HAIR SAMPLES OF REFERENCES AND
ALZHEIMER’S PATIENTS USING TIL-DLLME
METHOD……………………………………………………………….91
TABLE 4-10 COMPARATIVE DATA OF ANALYTICAL CHARACTERISTICS OF TIL-DLLME
FOR Al3+ WITH PREVIOUS REPORTED PRECONCENTRATION TECHNIQUE……………………….….92
TABLE 4-11 COMPARATIVE DATA OF ANALYTICAL CHARACTERISTICS OF UDIL-ΜE
FOR CU ION WITH PREVIOUS REPORTED PRECONCENTRATION TECHNIQUES……….
……………....96
TABLE 4-12 THE CONCENTRATION OF Cu ION IN SERUM SAMPLES OF NEUROLOGICAL
DISORDERS MALE PATIENTS AND NORMAL REFERENT (µg L-1)……..…………….……….............97
TABLE 4-13 THE CONCENTRATION OF Fe IN SERUM SAMPLES OF NEUROLOGICAL
DISORDERS MALE PATIENTS AND HEALTHY CONTROL (µg L-1).………………….
………….........101
TABLE 4-14 THE CONCENTRATION OF Al3+ IN BLOOD SAMPLES OF REFERENTS AND
DIFFERENT NEURO DISORDERS MALE PATIENTS USING SS-E
METHOD……………………...........108
TABLE 4-15 CONCENTRATION OF Mn2+ IN BLOOD HEALTHY REFERENCES SAMPLES OF AND
DIFFERENT NEURO DISORDERS IN MALE PATIENTS USING DES-E METHOD.……………………
113
LIST OF FIGURES
FIGURE 3-1 GRAPHICAL REPRESENTATION OF d-CPE METHOD
FIGURE 3-2 GRAPHICAL DIAGRAM OF TIL-DLLME METHOD
FIGURE 3-3 GRAPHICAL DIAGRAM OF UDIL-ΜE METHOD
FIGURE 3-4 GRAPHICAL REPRESENTATION OF MDLP-ΜE METHOD
FIGURE 3-5 VISUAL REPRESENTATION OF SS OF [DBUH][DECANOL] IN AQUEOUS MEDIUM (A) UPPER
IMMISCIBLE SS AND LOWER AQUEOUS PHASE (B) CONVERTED TO A CLEAR HOMOGENOUS MONOPHASIC
SOLUTION OF SS IN AQUEOUS MEDIUM BY EXPOSING TO 4 MPA OF CO2 WHILE STIRRER FOR 5 MIN AT 500
RPM (C) THE POLAR SS/WATER MONOPHASIC SYSTEM WAS SEPARATED INTO ITS BIPHASIC RESPECTIVE SS
AND AQUEOUS LAYERS BY BUBBLING WITH N2 AND HEATING AT 55°C.
FIGURE 3-6 GRAPHICAL REPRESENTATION OF SS-E METHOD………………………………………
FIGURE 3-7 PREPRATİON AND THEİR CHECKED MİSCİBİLTY OF DES İN WATER
FIGURE 3-8 GRAPHICAL REPRESENTATION OF DES-E METHOD
FIGURE 4-1 EFFECT OF pH. ON THE % RECOVERY OF Mn2+ USING d-CPE METHOD
FIGURE 4-2 EFFECT OF PAN CONCENTRATION ON THE % RECOVERY OF Mn2+ USING d-CPE METHOD…
FIGURE 4-3 THE EFFECTS OF THE SURFACTANT TRITON X-114 ON THE % RECOVERY OF
Mn2+ USING d-CPE METHOD
FIGURE 4-4 EFFECT OF pH ON PRECONCENTRATION OF Zn2+ BY d-CPE
FIGURE 4-5 EFFECT OF PAN CONCENTRATION ON % RECOVERY OF Zn2+ BY d-CPE
FIGURE 4-6 EFFECT OF SURFACTANT CONCENTRATION ON % RECOVERY Zn2+ BY d-CPE
FIGURE 4-7A THREE DIMENSION (3D) SURFACE RESPONSE FOR % RECOVERY OF Al3+ BY
TIL-DLLME. INTERACTION BETWEEN IONIC LIQUID [IL (ΜL)] AND OXINE [L1 (mol L-1)]
FIGURE 4-7B INTERACTION BETWEEN IL (ΜL) AND PH FOR L1
FIGURE 4-8A THREE DIMENSION (3D) SURFACE RESPONSE FOR % RECOVERY OF Al3+ BY TIL-DLLME.
INTERACTION BETWEEN IL (ΜL) AND MORIN [L2 (mol L-1)]
FIGURE 4-8B INTERACTION BETWEEN IL (ΜL) AND PH FOR L2
FIGURE 4-9 EFFECT OF pH ON PRECONCENTRATION OF Cu ION BY UDIL-ΜE
FIGURE 4-10 EFFECT OF PAN CONCENTRATION ON % RECOVERY OF Cu ION BY UDIL-ΜE
FIGURE 4-11 EFFECT OF AMOUNT OF IL ON % RECOVERY Cu ION BY UDIL-ΜE
FIGURE 4-12 EFFECT OF SONICATION TIME ON % RECOVERY Cu ION BY UDIL-ΜE
FIGURE 4-13 EFFECT OF pH ON THE %RECOVERY OF Fe BY MDLP-ΜE
FIGURE 4-14 EFFECT OF OXINE CONCENTRATION ON % RECOVERY OF Fe BY MDLP-ΜE
FIGURE 4-15 EFFECT OF ASPIRATING/DISPENSING CYCLES ON THE % RECOVERY OF Fe BY MDLP-ΜE….
4
FIGURE 4-16 IN-SITU IR SPECTRA OF THE SPS SYSTEM OF (A) [DBUH][DECANOL] (B) FORMED [DBUH]
[DECANOLCO2] BY CO2 BUBBLING INTO THE MIXTURE AND (C) RECYCLING [DBUH][DECANOL] BY CO2
REMOVAL FROM THE MIXTURE BY BUBBLING WITH N2 AND HEATING AT 55°C
5
FIGURE 4-17 EFFECT OF PH ON THE RECOVERY (%) OF Al3+ SS-E
FIGURE 4-18 TIME PERCENT CONCENTRATION PROFILES OF CONVERSION OF DBU/DECANOL TO
DBU/DECANOL CO2 BY EXPOSING TO 4 MPA OF CO2 WHILE STIRRER AT 500 RPM
FIGURE 4-19 PERCENTAGE STRIPPING (% S) OF Al3+ FROM SS TO ACIDIC MEDIUM
FIGURE 4-20 EFFECT OF PH ON PRECONCENTRATİON OF Mn2+ BY DES-E
FIGURE 4-21 EFFECT OF PAN CONCENTRATİON ON % RECOVERY OF Mn2+ BY DES-E
FIGURE 4-22 EFFECT OF DES VOLUME ON % RECOVERY Mn2+ BY USİNG DES-E
FIGURE 4-23 EFFECT OF DECANOL VOLUME ON % RECOVERY Mn2+ USİNG DES-E.......................
6
ABBREVIATIONS
AD Alzheimer’s disorder
PD Parkinson’s disorder
MS Multiple sclerosis
PSD Psychiatric disorder
Al Aluminum
Mn Manganese
Fe Iron
Cu Copper
Zn Zinc
FAAS Flame atomic absorption spectrometry
ETAAS Electrothermal atomic absorption spectrometry
ICP-MS Inductively coupled plasma-mass spectrometry
LLE Liquid-liquid extraction
DLLM dispersive liquid–liquid microextraction
IL Ionic liquids
RTILs Room temperature ionic liquids
CPE Cloud point extraction
d-CPE Dual cloud point extraction
CMC Critical micellar concentration
MDLP-µE Modified dispersive liquid-phase microextraction
TIL-DLLME Temperature controlled ionic liquid-dispersive liquid phase microextraction
UDIL-μE Dual dispersive ionic liquid based on ultrasound assisted microextraction
DES Deep eutectic solvent
SS-E Switchable solvents extraction
PAN 1-(2-pyridylazo)-2-naphthol
7
Oxine 8-hydroxyquuinoline
Morin 3,5,7,2ʹ-4ʹ pentahydroxy flavone
DBU 1, 8-diazabicyclo [5.4.0] undec-7-ene
MWD Microwave-assisted acid digestion
CDM Conventional wet acid digestion method
CNS Central nervous system
BBB Blood brain barrier
ROS Reactive oxygen species
SN Substantia nigra
SRM Surface response methodology
CCD Central composite design
PBD Plackett–Burman designs
ANOVA Analysis of variance
[C4MIM][PF6] 1-butyl-3-methylimidazolium hexafluorophosphate
Triton X-100 Polyoxyethylene octyl phenyl ether
Triton X-114 Octylphenoxypolyethoxyethanol
CRM Certified reference material
WHO World health organization
(%) Percentage
°C Degree Celsius
EF Enhancement factor
ER Extraction Recovery
Kg Kilogram
gm Gram
HCl Hydrochloric acid
HNO3 Nitric acid
H2O2 Hydrogen peroxide
8
LOD Limit of detection
LOQ Limit of quantification
r Correlation coefficient
mg Milligram
mL Milliliter
pH Negative logarithm of hydrogen ion concentration
ppb Part per billion
ppm Part per million
L Liter
M Molar
µg Micro gram
RPM Rounds Per Minute
RSD Relative Standard Deviation
9
CHAPTER 1
INTRODUCTION
In this chapter we provide motivation and contributions of this thesis and at the end of
the chapter, we present structure of the rest of the thesis.
1.1 MOTIVATION
Neurodegeneration is a complex and multifaceted process leading to many chronic
diseased states. Neurodegenerative disorders include a number of different pathological
conditions, like Alzheimer's and Parkinson's diseases, and psychiatric disorders, which
share similar critical metabolic processes, such as protein aggregation, which could be
affected by some metal ions. A huge number of reports indicate that, among putative
aggravating factors, metal ions (aluminum, copper, iron, manganese and zinc) could
specifically impair physiological system such as protein aggregation of Aβ, prion
protein, ataxin, huntingtin, etc. and their oligomeric toxicity [1].
In human body metals having major role to maintain structure of cell & regulating
gene expression, neurotransmiting and antioxidant response. Though, increased level of
metal in neuro system may be toxic, inducing oxidative stress, disrupting mitochondrial
function, and impairing the activity of numerous enzymes . Damage caused by metal
accumulation may result in permanent injuries, including severe neuro disorders.
Epidemiological and clinical studies have shown a strong correlation between aberrant
metal exposure and a number of neurological diseases, including Alzheimer’s disease,
multiple sclerosis, Parkinson’s disease, and physiological disorders [2]. In brain metal
stored mitochondrial dysfunction, oxidative stress & protein misfolding are the most
commonly deficits related with metal-induced toxicity [3, 4]. By the increase of lifetime
between the general population, greater exposure of metal for long time leads to neuro
diseased. So, there is a growing demand to determine the neurotoxicity results from
10
metal exposure. Future studies need to focus more on the joint effect of metal mixture
exposure, identifying specific transporters of each metal as well as developing target-
specific therapeutics for patients with metal poisoning. Although additional research is
necessary to investigate molecular mechanisms of metals on neurosystem [5].
It is a challenging task for a chemist to assess level of under studied metal in
biological samples (scalp hair, blood, and serum) and developing new methods which
are environmentally benign and green. In order to meet this challenge an analyst should
use toxic organic chemicals as less as possible. Keeping in view importance of the issue
the present study was carried out to evaluate levels of elements, Al, Mn, Fe, Cu and Zn
after their enrichment and extraction from biological samples by simple and easy
miniaturized preconcentration methods
1.2 CONTRIBUTIONS OF THE THESIS
The present study design to inform about obtained the consequence and
alteration of Al, Mn, Fe, Cu and Zn in biological samples of Psychiatric
and neurological disorders patients with related to age matched
healthy/referent subjects, to share information with scientific community
and general public.
The determination of very low levels of studied elements in scalp hair
and blood serum samples is a difficult job to use cost effective
instruments such Flame/electrothermal atomic absorption spectrometry.
But due to very low concentration of analytes, presence of complex
matrices and other interfering ions, it is hard to determination them
directly in biological/environmental samples. For that purposes different
advance, innovative and green methodologies were developed for
separation and preconcentration of studied analytes. This work has been
designed to eliminate the use toxic organic solvents being employed for
classic extraction methods, a step towards greener chemistry.
The thesis encourages the reader to replace classical extraction methods
by advance miniaturized extraction/preconcentration methods.
11
1.3 STRUCTURE OF THE THESIS
The current dissertation is divided into five chapters. Each chapter contributes to the
whole study in different perspectives.
1.3.1 CHAPTER 1
This chapter includes a general introduction about neurological/psychiatric
disorders and possible role of metals in the development of these disorders have been
mentioned in this section. Chapter 1 has also been attributed to the importance of
advance extraction methods for the determination of trace elements in biological
samples. More over optimization of the newly developed techniques by multivariate
strategy has been discussed. The aims and objectives of the present study have also been
mentioned.
1.3.2 CHAPTER 2
A brief review of the neurological/psychiatric disorders and possible adverse
effects of element are discussed in this chapter. All the aspects of the present work has
been visualized in light of the work already published. Various methodologies along
with their background, advantages and drawback has documented, which are used for
the analysis of selected trace elements and also the importance of multivariate
optimization.
1.3.3 CHAPTER 3
All the chemicals, reagents and instruments used throughout the current study are
mentioned in this chapter. Here it also includes biological sample preparation in
laboratory along with the extraction and preconcentration procedures of selected metals.
In addition, analytical figure of merits for the developed methods have also been
presented in this section.
1.3.4 CHAPTER 4
In this part of the thesis the optimization study done for the developed
methodologies for studied elements has been given. Comparison study has also been
12
presented here. Different types of experimental designs have been discussed. Finally,
application of the proposed methods to the real samples of neurological/psychiatric
disorders has been mentioned. The results have been given and discussed.
1.3.5 CHAPTER 5
At the end the thesis have been concluded. This chapter also contains
socioeconomic impacts of the present study and recommendation suggested the
researcher.
1.4 NEUROLOGICAL DISORDERS
Neurological disorders are considered by active inflammation processes and inside
brain parenchyma protein deposits accumulate, which leads to neuron loss and damage. The
aging is the main cause of neurological disorders [6]. Such as Parkinson’s (PD), Alzheimer’s
(AD) and multiple sclerosis (MS) disease. Oxidative stress is the main feature which may be
responsible of neuron cell damage or dys-functioning that leads to disease pathogenesis. [7,
8].
PD is another common neurological disorder after AD. The PD have the few mutual
pathologies, distressing dopaminergic and non-dopaminergic neurons in substantia nigra
(SN), extra-nigral prognosis bundles that regulate routes for premotor, sensory, associative,
and motor pathways. Experimental, biochemical, clinical and microanatomic suggestions
showed that several factors are included in PD that causes oxidative neurodegeneration and
oxidative stress due to levodopa treatment. The SN is distinctively susceptible to oxidative
stress, have higher content of neuromelanin, oxidized dopamine, fatty acids polyunsaturated,
Fe, and comparatively lower antioxidant complement with higher metabolic level. Oxidative
phosphorylation abnormality impair energetics in the SN mitochondria, also increasing
oxygen free radical producer [9-12].
Alzheimer’s disease (AD) is the type of senile dementia. The problem of AD is
mostly common in America approximately 4.5 million people are affected with this disorder
and in a year minimum $100 billion is spent for the prevention only. By 2050, if no cure
measure is developed risk of AD patients increased ranging from 11.3 million to 16 million in
United States [13, 14].
13
AD is complex genetic disorder whereas 5-10 % cases were reported familial by autosomal
dominating heredity array. From many years AD symptoms increases due to progressive
damage of neuron functions and this indicates disease etiology [15]. The micro-environment
of neuron and, in specific, the constancy of the physico-chemistry of brain interstitial fluid
(BIF) are essential to the optimize function of neuron [16]. AD revealed by progressively
starts with irreversible and increased cognitive decline. In the earlier stage of AD memory
loss appeared whereas sensory and motor function are normally not effected till the later
stage [17, 18].
MS is the demyelinating disease of central nervous system (CNS) having long-term
weakening disease that on average decreased lifetime 7 to 8 years. 50% MS patients are not
capable to perform their responsibility in house and employment after 10 years of disease and
after 25 years 50% are incapable to changing their position. MS patients and clinicians are
more effective by two features [19].
Firstly, variation in the course of neuro disease. Secondly, most of the clinically silent
disease process. For that reason 20s and 30s are effected by MS disease increasing number of
patients due to the uncertainty professional and personal decisions. When compared healthy
referents of same age group with MS patients that attempting 7-8 times more suicides and
greater than fifty percent suffering to depression. Variation of disease increases effects MS
clinical trials, to prove treatment efficiency that needs hundreds of patients that adequate for
statistical power.
As initiative of disease progress clinical silent, primary indication of MS clinical trials
based on brain image, which have greater trial costs and arises queries about the changing of
brain image for long-term prediction [20, 21]. Connection of brain image study to central
nervous system inflammatory and brain blood barrier with neuro disability is the initial
demonstration and consequent degenerations that mostly MS patients suffer. Anti-
inflammatory therapy are effective in reducing the progress of MS, whereas aim to prevent or
stop disease process was not achieved successfully [22].
1.5 PSYCHOLOGICAL DISORDERS
Number of disorders are due to mental health problems, however the common factor
is that the all effects the affected personality peoples, social links or thinking process. Several
diverse psychological disorders have been recognized and classify, include eating disorders,
14
i.e. mood disorders (depression), anorexia nervosa; personality disorders (as antisocial);
psychotic disorders, such as schizophrenia [23].
Psychological disorders are not particularly identified but contributing factors may
include brain chemical imbalances, childhood experience, inheritance, stress, illnesses,
prenatal exposures. Some disorders, such as depression and borderline personality occur
more often in females. However intermittent explosive disorder and substance abuse, are
more common in men [24-27].
The rates of psychological disorders are mostly common in women than men, while it
was reported that women have a higher rate of depression. Symptoms of some of the
psychiatric disorders create a task to the physicians to distinguish or identify precise mental
illness. The symptoms of Psychiatric disorders are mainly depression, anxiety, and
personality disorders [28]. Schizophrenia is chronic mental illness psychiatric disorder that
effect behavior, intellectual and emotions.
The word schizophrenia arises from the Greek words schizo means divided and
phrenos means mind. This rarely known as psychotic disorder affects teen age groups and
young adults, leads to severe psychological disability during the potentially productive and
creative life [29, 30].
Damaged relations, poor job or school routine and suicide are the signs of bipolar disorder
which could be treated with medications and therapies. Psychosis is one of the less discussed
elements of bipolar disorder. Which could be the disturbing part of bipolar I. It is a
neurological disorder that causes mood swing, energy levels and decrease in task
performance ability and it is also termed as manic-depressive illness [31, 32].
1.6 ROLE OF METALS IN PATHOGENESIS OF NEUROLOGICAL/PSYCHIATRIC
DISORDERS
Metals and metalloids are the important component of several functions in living
system. 23 elements have been identified to participate in several physiological functions
where as trace amount of 11 elements is present in living beings [33].
For normal physiological functions in human body about one-third of all proteins,
needs metal ions to perform their functions. There are specific binding cites for these metals
which are complex enough for specific monomeric/polynuclear metal centers. The selectivity
of metal for the specific protein is uncertain, from the mixture of metals present in the cell
15
using even the most exact set of amino acid donor systems [34] . The coordination geometry
and binding affinity as a result of set of donor atoms in not enough for the exact selection of
metal ion. However, the level of required metal could be too high or low so the exportation
and restoration of these elements would be necessary. Adverse management of even vital
elements such as copper could result toxic effects. More effectively cell disrupt the metal–
protein systems speciation and exactly approach to control over the metal attainment,
spreading and regulating process [35].
Limited data is available about the quantity of element in serum during pathological
conditions of PD. According to few studies about transition metals in serum such as Fe, Cu &
Zn suggest that in PD these metal are not indicated as risk factor [36, 37]. The role of
essential and toxic metals in the neuroscience has established slowly in the past years with
discovery of their related to main neurodegenerative diseases like AD, PD [38, 39]. Several
neuro disorders pathogenesis generated by metabolic unbalanced of essential metals such as
Fe, Cu.
Metal homeostasis affects variations in brain function in neurological disorders
however, it is reported that changing in the homeostasis, transition metals localization, redox-
activity is too significant to recognize that alterations in definite Cu and Fe-containing
metallo-enzymes appeared to have main cause of neuro disorders [40].
1.6.1 ALUMINUM
Aluminium (Al) in the earth’s crust is the third abundantly present element and it is
not an essential trace metal for living beings. Although the level present in the body enough
to modified the various important enzymes and 2nd messenger pathway [41]. Al is termed as
heavily neurotoxic at higher exposure, it is responsible to inhibited the post-natal and prenatal
brain growth [42, 43].
It might be specific to impaired protein aggregate and oligomeric toxic effect in AD
and produces neurotoxicity through different mechanisms. The metal-induced (direct) and
metal-amyloid-β (indirect) link to neuron cell degeneration by the development of reactive
oxygen species (ROS) being difficult to understand the mechanisms which metal-induced cell
death [44].
Fetus brain chronically exposed by Al. The prenatal exposure to Al is unidentified in
the development of children and account for changes especially in development stage of
16
neuronal function constancy of BIF is pressured through Al [45, 46]. The distribution of Al
increases in our environment and their compound use to prepare from decades in glass, clays
& alum [16].
Living beings exposed to Al is not avoidable, but neither cases of Al insufficiency nor
any physiological function for Al have been defined until now [47]. It is stated in few studies
that Al accumulate in the brain by diverse ways (water intake, medications & foods) and also
interfere normal activity of nervous system. Living persons easily exposed by Al because it is
widely use in our everyday routine. It have been proposed that through water intake higher
concentration of Al in regions leads to increase mortality by several neurological disorders
[48-50]. However many suggestion on processes by which brain tissue effected through Al
includes synthesis of protein, axonal transport and neurotransmitter-linked actions for
instance intraneuronal calcium homeostasis disruption, inhabit of catechol-O-methyl,
transferase, cholinesterase, choline acetyltransferase, Mg-adenosine triphosphatase,
glycerokinase and calmodulin, and activation of adenylate cyclase and daminolevulinic acid
dehydratase. Al interact with ATP to form an Al–ATP complex, which is a competitive
inhibitor of hexokinase and dihydropteridine reductase.
Ease in the accumulation of Al in brain and slow elimination is a concern. In a study
on rats shown higher permeability of blood brain barrier to specific small peptide such as
endorphin due to high level of Al [43, 51]. As the permeability of blood–brain barrier
increases might be due to changing in biochemistry of brain, results functionality and
behavior abnormality eventually manifesting CNS and dementia disorders. As a consequence
of higher permeability, the CNS also becoming susceptible to the harmful effects of other
xenobiotics [52].
17
1.6.2 MANGANESE
Manganese (Mn) is an vital element, important for the normal functioning of
physiological process include amino acid, protein, lipid, and carbohydrate metabolism, and
normal immune system functioning .With normal dietary intake, systemic homeostasis of Mn
is maintain by both its rate of transport across enterocytes lining the intestinal wall and by its
effective elimination within the liver. Clinically higher Mn deficiency is rare among human.
As compared to heavy exposure of Mn is more prevalent and associated with a variety of
psychiatric and motor disturbances [53].
Higher Mn consumption can arise from higher diet ingestion and also exposed by
environmentally & occupationally. Professionally exposed to Mn has been the main reason of
human Mn toxicity in persons working in such industries as mining and the manufacture of
dry batteries, aluminum, steel, welding metals, and organochemical fungicides [54].
Additionally, persons taking completely parenteral diet [55], and patients with chronic liver
failure have high risk of Mn toxicity [56]. Generally people exposed to Mn by drinking well
water having higher concentration of metal [57], from soy-based child formulations [58, 59],
and may be from Mn release into the atmosphere resulting by the addition of
methylcyclopentadienyl manganese tricarbonyl (MMT) to gasoline as an anti-knock agent
[60, 61].
This problem develop increases concerned in light of possible adverse effects from long-
term exposure to increasing ambient levels of Mn in the environment. Exposed to Mn from
mining, working in alloy smelters and Mn metal, dry cell battery making, working with
fertilizers and fungicides having this element, and welding are examples of industrial work
which may causes damage to the peripheral and center nervous systems and psychological
disorders which can be progressive and irreversible [62, 63].
High exposure to Mn causes it to accumulate in the brain, creating an intoxication called
manganism, a condition whose symptoms (movement disorder, firmness, characterized by
tremor, dystonia and/or ataxia and psychiatric disturbance, include reducing learning
capability and reduced mental flexibility) [64, 65], might be indistinct from idiopathic
Parkinson's disease [66]. In brain parts subcortical and cortical, specifically the basal ganglia
loss of neuron leads to manganism [67].
The base for the related neurotoxic effect of Mn still not completely understood however
an increases number of study are revealing underlying mechanisms by characterization of the
18
passage of Mn into the brain, the influence on function of neuron, synaptic transmission and
the inflammation response of populations of glial cells in affected brain areas [68].
1.6.3 IRON
Iron (Fe) is necessary for life, and is one of the essential metal in biological and
environmental systems [69]. But both severe deficiency and excess leads to significant
serious health risks [70]. The Fe play important role in biological activity and active centre
for protein to transfer electron and oxygen in metalloenzymes such as dehydratases and
oxidases [71]. The brain uses Fe for many essential processes as either haem iron
(including the iron transport oxygen in haemoglobin), or non-haem iron [72, 73]. Which is
responsible for the synthesis of neurotransmitters and cellular aerobic metabolism [74].The
accumulation of Fe in brain region SN causes degeneration of dopaminergic neuron and
form complex with neuromelanin inducing oxidative stress which leads to different
nervous system disorders such as PD and AD [75-77].
Fe is also multifunctional for the CNS, involve DNA formation, myelination, gene
expression, mitochondrial electron transport, and neurotransmission. Various proteins involve
in brain Fe homeostasis leads to disorders with abnormal Fe metabolism. To understand basis
of Fe homeostatic mechanism is clinically relevant, as either depletion or accumulate
intracellular Fe might be impaired normal function and increases cell death [78]. The
accumulation of Fe in specific brain areas during aging, causes neurological disorders such as
PD, AD and in genetic disorders degeneration of neuron occur.
In AD, Fe is primarily complexed with ferritin and concentrated in the neurotic
processes associated with amyloid plaques. Fe may have directly impact on plaque formation
through its effects on amyloid precursor protein processing by α-secretase, deposition of
amyloid-β, and oxidative stress [79, 80].
1.6.4 COPPER
Copper (Cu) have essential role in all living systems being an active center in proteins
involves in the oxygenase and oxidase activities, regulating the level of oxygen radicals and
electron transfer. It also plays important role in many disease such as neurological disorders
i.e. AD and Cu metabolic disorders. Due to its excessive intracellular accumulation cause
toxic effects such as apoptotic mechanism and ROS generation [35].
19
The adverse effects of Cu appears due to its elevated levels from its normal
concentration in soft tissues. It build connective tissues and stores calcium in bones. Elevated
level of Cu results depression, fatigue and stress that leads to chronic sinus infections.
Thyroid gland and intestinal yeast are also sensitive to the concentration of Cu present in the
body. Cu has intense role in human’s CNS. Neurological and psychological conditions such
as anxiety, stress, depression and schizophrenia are mainly arise due to disturbed level of Cu
[81, 82].
The Welding and plumbing are the main environmental exposure sources of Cu. whereas
increased concentration in blood are due to inhaled Cu which may leads to disorder such as
liver damage, allergies, anemia and anxiety [83].
Cu is an important constituent for dopamine synthesis in various metalloenzymes by
biochemical pathways involving either antagonism of dopamine formation or catalysis of its
breakdown. In schizophrenia dopamine is involved, dopamine dys-regulation and excess
leads to Cu homeostasis. For the normal development of CNS, Cu is a cofactor for many
enzymes [84-86]. The formation of free radical might be produced by high levels of Cu ions
through Haber-Weiss reaction .The Cu is injurious at excess exposure while incomplete
development, is due to the deficiency of Cu resulted into mitochondrial destruction, DNA
breaking, and injury of neuronal cells [87-89].
1.6.5 ZINC
The divalent cation Zinc (Zn) is essential for normal physiological functions of human
beings, especially for brain and other important functions of body. It is also required for
cellular development and survival [90]. The Zn plays important role as cofactor of different
enzymes, to assist various biochemical functions, enzymes regulating a wide variety of
cellular and signaling pathways as well as synthesis of DNA and transcription of RNA [91].
Zn status affects basic processes of cell growth, division, development, differentiation,
aging and performance by its requirement not only for repair and synthesis of protein,
RNA, and DNA but also for many other metabolic activities. Zn is irregularly spread within
the brain mainly at higher level in the hippocampal mossy fiber system where it functions
as a neuromodulator [92]. Interactions with excitatory and inhibitory amino acid
neurotransmitter are well recognized with Zn [93]. The severe deficiency of Zn causes
developmental abnormalities in humans and animals [94]. It was reported in literature that
20
the human subjects in depression had considerably lower levels of Zn in biological samples
(blood, serum), than healthy persons, these adverse consequences might be clinically
improve on providing Zn supplements [95, 96].
The deficiency of Zn at initial stage creates different disorders such as diarrhea,
dermatitis, alopecia and loss of appetite. The deficiency of Zn for long time might be
creates growth impairment, especially in children and neuropsychological disorders such
as cognitive development (children), concentration, depression, emotional flux, and
irritability [97, 98]. The deficiency of Zn creates adverse impact on central nervous system
such as schizophrenia, bipolar, depression and distorted or absent sensory function
involving taste, smell, and vision [99, 100].
At recommended level of Zn for normal metabolism of human, have neuroprotective
activity, though higher level of Zn are neurotoxic [101, 102].
Zn ion availability in the learning, memory functions, brain aging, neurogenesis process
and neurological disorders have been reported in numerous reports [103-106].
1.7 BIOLOGICAL SAMPLES
The analytical study of elemental concentration in biological samples (mainly human
biological samples) has become very important in the past few years. The importance of these
investigations is linked with the fact that there are various trace elements in the body that
plays an important role in the biochemical processes. Insufficiency or excess of essential
elements leads to severe physiological effects in human. In addition to essential elements,
some toxic elements may be present in human bodies which are severely poisonous even in
low concentration [107].
The quantification of elements in human body fluids and tissues is significant in
forensic medicine, in the treatment and diagnosis of a range of disorders and in valuation of
the internal exposure of individual [108]. The human biological analyses are performed on
different biological specimens, among them, the most widely used are blood [109, 110],
serum or plasma [111-114], hair [115] and other tissues [116]. Intracellular elements have
specific role in circulating blood cells. Extracellular elements functions are transported in
plasma/serum [117].
Whole blood analysis assess the total level of toxic elements that circulate
extracellularly (plasma/serum) [118]. It is the medium of transport of trace metals and offers
21
direct confirmation of metabolism about their levels. Hence, serum, plasma and whole blood
are suitable samples for the assessment of trace metal status of an individual [119, 120]. The
capability of the blood to count changing in elemental status keeps nutritional and many toxic
concentration within limited range, unless under heavily exposed. Mechanism of clarity in
blood effectively shown by homeostasis reaction and mainly describes the short term utility
of blood analysis. The significance of discovering the depot-storage capability of several
elements, specifically the toxic ones, remains main feature in elemental analysis largely met
by hair and urine tests [121, 122].
Even though urine and blood analyses are the most traditional methods, they alter in
response to any change in environmental or physiological conditions. Hair can give a more
permanent record of trace elements related with normal and abnormal metabolism and
assimilation from the environment. In addition, easy to collect hair, convenient to store, and
can be readily analyzed. Human hair analysis having an important approach to understand the
quantitative changing in certain elements inside the body [123, 124]. Another advantage is
that hair provides information on the trace element level of the intracellular space (blood
provides information on the extracellular space). In addition, trace elements present in the
body are incorporated into the hair during its growth; the exogenous trace elements are fixed
on its surface [125].
1.8 ANALYTICAL TECHNIQUES AND EXTRACTION METHOD
To analyze the trace elements in biological and environmental samples diverse analytical
techniques are used. However, flame atomic absorption spectrometry (FAAS) is commonly
used instruments to the analysis of several elements with significant accuracy and precision.
This analytical technique is remarkable for its speed, selectivity and fairly lower operating
cost [126]. However, in some cases there are many difficulties in determining traces of metals
in environmental samples due to insufficient sensitivity or matrix interferences [127, 128].
In analytical chemistry accurate analysis of trace elements is challenging task. Directly
analysis of trace elements appeared to be difficult work as the level of them is near to or
under the limits of detection of most of the analytical techniques in addition to the real
sample matrixes might be creating serious interfering effect for the analysis. Although,
preconcentration methods uses to overcome these problems by simultaneously eliminate the
sample matrixes and increases the quantity of element [129]. The analysis directly of trace
22
metals by spectroscopy techniques, is challenging task because of inadequate selectivity and
sensitivity. Due to this, initially separation and enrichment of trace elements require from
different matrix.
Sample preparation has directly effect on precision, accuracy and limit of quantitation
in analytical processes is rate determination step, particularly when trace analysis is the
objective. Processing the aqueous sample to separate and concentrate analyte from matrixes
prior to analyzed by instrument. However prominence of preparing sample is important step
in an analytical process. Analytical chemist searching easy, fast and low cost methods by
giving authentic data with reasonable limits of quantitation [130, 131]. The analysis of trace
elemental level in human fluids and tissue was using to evaluate about nutrition level,
identify disease, indicates toxicity of the system and obtaining information of living beings
expose to environment [132].
Atomic absorption spectrometry is selective and sensitive instrument to the trace elemental
analysis in biological specimens [133, 134]. This instrument require to solubilize analyte and
completely or partially decompose matrixes uses either microwave ovens or convective
systems and dry ashing. In microwave assisted digestion pretreatment of sample require less
quantity of mineral acids reduce the formation of nitrous vapors is one of the main advantage.
Lower concentration of blank in microwave systems require only less volume of reagent and
greater number of samples runs per hour than conventional digestion method [135, 136].
Recently, several analytical instruments commonly uses to determine lower
concentration of metals are flame atomic absorption spectrometry (FAAS), and
electrothermal atomic absorption spectrometry (ETAAS), inductively coupled plasma-atomic
emission spectrometry (ICP-AES), [137-139]. FAAS is most widely used technique for the
analysis of element, for the direct determination it is not enough sensitive for the trace
quantity of metal in biological/environmental specimens, possibly may be complexity of
matrix and lower amount of element, which required sensitive instruments and often an
enrichment step. So, it is important to developed sensitive and economical procedure for the
determination of trace quantity of analyte by FAAS [140, 141].
Extraction–spectrophotometric procedures has been applied to analyze metal ions in
different environmental and biological samples such as water samples; however, the organic
solvents used are hazardous, causing damage to environment and human health. The micellar
systems used such as cloud point extraction (CPE) for preconcentration and separation attract
significant consideration in the last few years, due to agreement with “green chemistry”
23
principles. Green chemistry is define as those methods that decrease or eliminate the uses or
formation of substances toxic to health of human and environment [142].
CPE is one of the easiest and simple separation approach to inhanced sensitivity and
selectivity toword the metal determination with FAAS [143]. In CPE mode phase separation
occurred between two medium (aqueous and non-ionic surfactants). In the result a cloudy
solution is formed having critical micellar concentration (CMC) of surfactant, which can be
easily achieved by heating to specific temperature called cloud point temperature (CPT) [144,
145]. The surfactant rich part using as a separating medium because of their ability to
solubilize or entrapped metal complex.
Although, cloud point strategy has expand uses of FAAS , due to depends on the
enrichment factors achieved, it is important to increases their sensitivity, so this makes
procedure with greater advantage by comparing with directly analysed by ETAAS and ICP-
AES techniques [146].
Numerous organic reagents have been used for the CPE of Mn and Zn. It was reported
that the Mn and Zn forms a highly stable complex with 1-(2-pyridylazo)-2-naphthol (PAN),
with a high stability constant as compared to other ligands [147, 148]. As CPE is mainly
based on the hydrophobic link among the solutes and surfactant, in surfactant-rich part the
extraction of other hydrophobic species also occur which might be creates inferring effect to
the determination of element of interest [149].
The dual-cloud point extraction (d-CPE) overcome the disadvantages of CPE
procedure. The d-CPE method is carrying two times in a single sample pretreatment process.
The 1st step of d-CPE method is same as conventional CPE. The hydrophobic surfactant is
adding into the solution having the elements that formed hydrophobic complexes with
suitable ligands. The elements of interest with other hydrophobic interfering species are
extracted in surfactant- rich part, after heated in a thermostatic bath and centrifugation. To
perform 2nd round of CPE method before directly analysed, by treating surfactant-rich part
with aqueous acid solution to back extract element in aqueous acidic media, after heating in
thermostatic water bath and centrifugation [150, 151]. To enhanced the selectivity and
sensitivity of the developed d-CPE procedure by removing interference species and making
more compatible for FAAS by extracting in back extractant. Although so far some studies
suggests that d-CPE applied for the extraction and enrichment of inorganic elements in
environmental/biological matrices [152]. To analyze Al by FAAS the existence of
interference cations like Fe, Cr, Cu and Zn in environmental samples or having Al lower the
24
limit of detection, it is difficult to directly analysis of Al in biological/environmental samples.
Thus it requires before preconcentration and separation [153].
For this purpose, several techniques have been suggested to the separation and
preconcentration of Al was carried out by liquid–liquid extraction (LLE) [154], ion
exchange [155, 156], solid-phase extraction [157], and single drop microextraction [158]
etc. From many years LLE is applying, however this technique is usually time consuming
and needs quite greater quantity of highly purifying solvents. Additionally, causes adverse
effect on environment by the discard of these solvents uses.
In this sense, considerable interest to be taking on the use of room temperature ionic
liquids (RTILs) as the greener solvent to substitute the conventional organic solvents have
greater application, mainly in LLE of pollutants and heavier metal ions [159, 160].
Ionic liquids (ILs) is a type of lower melted ionic compounds, having various
properties makes greener solvents to prepare sample. It is largely apply to refer wide class of
salts which have significant liquid ranges. Room temperature ionic liquids (RTILs) defines a
subclass of ILs that are liquid at room temperature [161]. It chiefly consists of organic
cations (ammonium quaternary, imidazolium or pyridinium) and inorganic/organic anions
(Cl−, Br−, I−, AlCl4, BF4
, PF6−, ROSO−3, trifluoromethane sulfonate) [162].
In addition to their lower melting points, ILs have several other exclusive
physicochemical properties, like wide liquid ranging, neglecting vapor pressures, not
flammable, good thermally stabile and good extraction for many organic compounds and
metal ions as charged or neutral complexes, and also adjustable viscosity and miscible with
water and organic solvents, which making them very attractive in separation processes [163-
165], Recently, ILs has gained more interest in many fields such as separation technology,
organic synthesis [166] and electrochemistry [167] .
Recently a miniaturized solvent extraction procedure for different
enrichment/extraction methods such as co-precipitation method [168, 169], solid-phase
extraction (SPE) [170, 171], hollow fiber membrane and cloud point extraction (CPE) [172,
173], has been used for the separation and enrichment of iron in biological matrices. The some
other simple, rapid and in expensive methods and high extraction capability i.e. dispersive
liquid-liquid microextraction (DLLME) have grabbed a great deal of space in the recent
literature. The main limitation of the DLLME based procedure has need to be a long time to
reach equilibrium. This fact create a negative effect on the extraction capability of the desired
method that might be due the small contact between both medium (aqueous and the extractant)
25
[174]. The developed method modified dispersive liquid-phase microextraction (MDLP-µE)
not only eliminate the solubility effect of organic solvent in aqueous media, but also reduce
the matrix effect of the organic solvent on the target analyte [127].
The drawbacks of dispersive solvents in DLLME are studied and eliminated, to
enhance the extraction efficiency of analytes [175-177]. A fast and simple dual dispersive
ionic liquid based on ultrasound assisted microextraction (UDIL-μE) method was developed
to optimize dispersive process of IL, whereas ultrasonic radiation accelerates the migration of
analytes into fine droplets of IL and also increasing the extraction yields [178-180]. Recycled
many time without losses extracting efficiency is the main advantage. For this procedure of
extraction and separation the ultrasound energy is provide effective assist in the acceleration
of several steps, like emulsion forming, homogenizing, and transferring of mass among
immiscible phases, [181, 182]. The ultrasound extraction is used to attain equilibrium in a
short time [183, 184]. Though the waves of ultrasound makes organic solvent volatile.
Subsequently taking the advantage of IL the low-priced, fast, simple, efficient & sensitive
ultrasound assisted IL microextraction methods was established and used [164, 185].
Analytical chemistry chiefly follow the purpose of substituting toxic reagents, and
reducing and automating analytical methodology, thus to decrease human and environmental
risks by substituting polluting procedures with clean ones. As concerns to prepare sample,
developing and enhancing of newly acceptable analytical methods is a fast increasing trend in
analytical chemistry.
In this section, microextraction techniques have developed from the more traditional
sample-pretreatment techniques [186]. Nowadays, a new class of solvents such as switchable
solvents introduced due their tunable property having diverse polarity, which can facilitate
the need of solvent separation by diverse processes such as centrifugation [187, 188].
In the past decades among industrialist and academicians the application of switching
solvent gaining more consideration. The main effect of switchable solvent that no loss of
extraction ability as well as to reused several times. For this purposes low polarity solvents,
amine base and alcohol, upon exposure to an atmosphere of carbon dioxide switches to high
polar salt in liquid form. The salt in liquid form are converted into low polar upon heating in
the presence of nitrogen or argon gas [189-192]. The solvents have switchable characteristics
should aid the organic syntheses and isolations to avoids the steps for separating the solvents
after every reaction step. Among other benefits of the switchable solvents, they have the
capability to reuse/ recycle without lacking of extraction efficacy [193-196].
26
A newly developed types of solvents include eutectic mixtures prepared from zinc
salts and acetamide or mixtures having metal cations and anions in comparison to other ionic
liquids with stable physical properties. These new solvents are acquired by simply mixing
two constituents by self-association, often through hydrogen bond interactions. In DES
formation, hydrogen bonding leads to charge delocalization where, for example, the hydrogen
donor and halide ion moiety are responsible for lowering the melting point of the mixture
relative to the melting points of the discrete components [197-199]. The distinguishing
factors of DES solvents in comparison to ionic liquids are that they contain only discrete
anions. The resulting DES becomes liquid at < 100 °C, whereas the individual components
have high melting points. Abbott et al. [200] investigated that the eutectic combination of
metal chloride and donor molecules, such as acetamide and urea, result in a eutectics mixture
with melting points of < 150ºC. Conversion of solid zinc chloride and acetamide into a
solvent has low conductivity and viscosity in comparison to other solvents with different
composition [201, 202]. The physico-chemical properties of DES are similar to the usual
ionic liquids, except they are low cost and greener. The combination of eutectic has a low
freezing point of about 150 °C. Due to these extraordinary benefits, DES is now gaining more
attention in the different fields of research [203, 204]. DESs have been extensively used for
the extraction of organic solvents, dissolution of metal oxides, synthesis of nanoparticles,
electrodeposition of metals, digestion of inorganic compounds, drug dissolution, CO2
absorption and purification of biodiesel, drug dissolution, and refinement of biodiesel [205-
208].
The complexing/chelating agent has recently received significant attention due to their
complexity and selectivity towards different metal ions, which can be using to observe
environmental exposure of these toxicant to the environment [209, 210]. Diverse feature
effects the properties of sorption and selectivity of complexing agent like chelating agent
chemical activity, type of metal ion, solution pH, naturally and chemically stable & ionic
strength [211]. 8-Hydroxyquinoline (8-HQ) and its compounds have been extensively
employed as complexing or enrichment reagent in analytical chemistry. Various CPE
extractions procedures for Mn and other elements (Al & Fe, Cu) analysis have been
describing in the literature. Ligands such as 1-(2-pyridylazo)-2-naphthol (PAN) [152, 146],
3,5,7,2’-4’ pentahydroxy flavone (morin), [212], 8-hydrooxyquinoline (oxine), has been used
as a chelating agent in various methods for under studied elements [213],
27
1.9 MULTIVARIATE STUDY
In analytical science the use of multivariate experimental strategy increases by at
once studied the various control variables, fastly implemented and less expensive
comparing with classical univariate approach [214]. Various experimental design
models present that reducing the number of experiments and uses in diverse cases [215,
216]. Therefore, effect of variables identified, employing the experimental strategies for
1st -order models (Plackett–Burman designs or factorial designs). Moreover to
estimated a response function or for optimization, experimental designs for second-
order models should be uses.
The PBD was employed as a screening method with the objective of achieving
the significant factors that effects the suggested methodology [217, 218]. To apply this
experimental design decreases the developing time of the procedure and give less
indefinite extracting conditions, therefore easing data elucidation. For the assessment of
five factors at two levels, a PBD with only sixteen experiments is described instead of
the 25 = 32 needs a full factorial design. Analysis of the variance (ANOVA) and using
p-value investigated the significance effects.
The central 23+ star orthogonal composite design (CCD) reveled the interlinking
among factors and more optimizing of variables have significant effect which are most
extensively employed for 2nd -order RS modeling within k factor experiments [219]
To optimize developed procedure, a CCD strategy with six degree of freedom
and including sixteen experiments were achieved [220].
1.10 AIMS AND OBJECTIVES
The objective of our present work were:
To evaluate the variation in level of Al and Mn, Fe, Cu, Zn in biological specimens
having different types of Psychiatric and neurological disorders and compare the
resulted data with age matched healthy/referent subjects.
Developing advance extraction methodologies for enrichment of trace quantity of
study analytes in acid digested biological specimens.
To established a d-CPE procedure to analyse of lower amount of Mn. 1-(2-
Pyridylazo)-2-naphthol (PAN) was employed as a complexing reagent, and the
resulting Mn complex was entrapped in a nonionic surfactant .Then the enriched
28
analytes was back-extracted with aqueous (HNO3) to reduce the adverse influence of
the surfactant.
The d-CPE procedure was completely characterizing by investigating and
optimization of the related variables effects the extraction of analytes from the
complex organic matrix. The efficiency of d-CPE was compared with those results
achieved by conventional CPE procedure on the same CRM and real samples.
The suggested procedure was successfully apply to the determination of Mn2+ in acid-
digested scalp hair samples of Parkinson's patients and Zn in serum samples of
psychiatric patients with related healthy referents.
To develop Temperature controlled ionic liquid-dispersive liquid phase
microextraction (TIL-DLLME) with FAAS for the analysis of Al3+ in scalp hair
samples of AD patients with related healthy referents. Various parameters affecting
the extraction efficiency includes the volume of [C4mim][PF6], sample pH, and
concentrations of complexing reagents were investigated To compare the efficiency
of complexing reagents (oxine and morin) in proposed method was studied by
multivariate techniques, have been used to optimize many variable simultaneously.
An efficient and reliable dual dispersive ionic liquid based on ultrasound assisted
microextraction (UDIL-μE), for the enrichment of trace levels of Cu2+ in blood serum
samples of patients suffering from different neurological disorders. The important
feature of develop method was to eliminating the adverse effect of ionic liquid via
back extraction in aqueous acid solution, before analysis with FAAS. Various
variables under the optimum experimental values were studied such as (sonication
time, complexing agent concentration, pH, volume of IL, time and rate of
centrifugation).
An innovative, modified dispersive liquid-phase microextraction (MDLP-μE) method
was developed to assess the iron (Fe) concentration in blood serum samples of
different neurological disorders patients. The main objective of this work to disperse
extracting solvent by using air-agitated syringe system to overcome the matrix effect
and avoid the dispersion by using heat, hazardous dispersive organic solvents. The
MDLP-μE consists of two dispersive liquid-phase steps with chloroform as an
extractant solvent. In the first step, Fe form complexes with a chelating reagent, 8-
hydroxyquinoline (oxine) in aqueous phase and extracting into extracting solvent
(chloroform). In the second step, Fe was back-extracted into the acidic aqueous phase
29
and finally determined by flame atomic absorption spectrometry (FAAS).The
variables play a key role on the extraction efficiency and reproducibility such as pH,
first extractant volume, back-extractant volume, concentration of complexing agent
and aspirating/dispensing cycles through a syringe were studied and optimized.
A green switchable solvent extraction procedure has been proposed for the
enrichment and determination of Al in blood samples of different neurological
disorders patients preceding to analyze with FAAS. The combination of decanol and
1, 8-diazabicyclo-[5.4.0]-undec-7-ene (DBU) made switchable solvents (SS), which
convert/switch from hydrophobic to hydrophilic and vice versa, on exposure to CO2
as an antisolvent trigger. To develop switchable solvent extraction (SS-E) method
different factors, pressure and purging time of CO2, pH, concentration of complexing
agent, were optimized. The validation of developed method was carried out on
applying to certified reference material and spiked blood samples. In literature, no
any previous study was reported to analysed Al in blood samples, especially of
neurological disorders patients.
To synthesize DES from zinc chloride and acetamide mixture and using it for the
enrichment of trace level of Mn2+ in blood samples of neuro patients. The PAN was
used as a complex forming agent and the resulting Mn2+ complex was entrapped in a
DES. Decanol was added to enhance the extraction efficiency, then was easily
introduced into the nebulizer of the FAAS by using a self-prepared injection system
made up of a Teflon® funnel and an Eppendorf pipette attached to the capillary tube
of the nebulizer.
1.11 SUMMARY
This part of thesis give brief introduction of neurological/psychiatric
disorders and the central role of metal in the etiopathogenesis and progression of
these disorders such as Alzheimer’s, Parkinson’s, multiple sclerosis, and
schizophrenia bipolar disorder. To determine the trace metal level of Al, Mn and
Fe, Cu, Zn in biological sample (scalp hair, blood serum) different advance
extraction methodologies is established. For optimization of proposed methods
by multivariate experimental design has been uses. The aims and objectives of
the present work have been also discussed.
30
CHAPTER 2
LITERATURE REVIEW/BACKGROUND
In this chapter we present previously reported work about the neurological/psychiatric
disorders. It also includes the general information about preconcentration methods
previously reported for the quantitative analysis of understudy elements.
2.1 NEUROLOGICAL DISORDERS
Neurological disorders, like Parkinson’s disease (PD), Alzheimer’s disease (AD), and
multiple sclerosis (MS), due to the increasing damage of particular neuron cell populations
and are associate with protein aggregate. Number of evidences suggest that oxidative stress,
is the most common factor of these disorders, which may be contribute to the damage or
dysfunction of neuronal cells that leads to disease pathogenesis [221]. Irregular formation of
reactive oxygen species (ROS), results oxidative stress like superoxide, nitric oxide,
hydrogen peroxide and higher reactive hydroxyl radicals. Oxidative loss in brain tissues due
to heavily consuming oxygen comparatively lower antioxidant concentration and lower
regenerative capability.
Andersen et al., 2004 [222] reported that neurological diseases such as PD, AD,
Stroke because of oxidative stress can cause neuron cell damage in a number of conditions
and take place whereas the normally balanced among oxidative activity & antioxidant
defendants is disrupting whether by losses of reducing agents/antioxidant enzymes or by
increases forming of oxidizes species [223-226].
Developing suggestion by several works proposed that oxidative stress could be
commonly last pathway in several types of neuronal cell damages includes an extensive
variations of chronic and acute neurological disorders, also normal aging [227-230].
Li et al., 2008 [231] stated in literature that PD takings a heavier toll in mentally
suffering, losing efficiency, and expenses on health caution. It was immensely investigated
about pathogenesis of neuro degeneration in the pars compacta of substantia nigra (SN) in
31
patients with PD is still not obviously identified. In PD patients having oxidative stress in SN
proposed by various studies [232].
Jellinger et al., 2000 [233] stated that brain uses dopamine as a main neurotransmitter
by various ways. One of them way from SN to the nearby striatum and is sometimes referred to
as the nigrostriatal region or pathway. Dopamine-forming cells degeneration is a consequence of
low levels of dopamine with this pathway [234].
Dopamine transmitter deficiency is dominating factor of PD, and present management is
almost completely dependent on dopamine replacing drugs. Mostly patients are initially effected
by these drugs, underlie degeneration is not slower in SN part of affected brain region. Their
efficiency decreases by the time passes away & their adverse influence grow progressively more
disturbing. Wider choices for continuing managing is instantly required. Various diverse lines of
evidences have converge to stated that PD is initially an oxidative disease, fueled by
endogenous exposure and driven by the cumulative aids of exogenous (environmental) and
endogenous and oxidative stressors [235].
Neurons afflicting with Lewy production remains sustainable for a comparatively
longer time, yet are functionally settled and expire in prematurity. As a rule, projection
neurons with long axons are more susceptible than local circuit and projection neurons with
shorter axons, which tend to be spared [236-238].
Braak et al., 2004 [239] reported that earliest stages of PD might be start from years
or even decades before stiffness and tremor become appeared. Impair smell judgement
constipation, and too much sleepiness are sometimes early signs of PD [240-242]. In later
stages, dementia, psychosis, and depression may appear, however depression possibly will be
an initial indication of the disorder. In a person’s 50s or 60s PD usually stars and with aging
progressive slowly. Early onset of PD before age 30 is rare, but up to 10% of cases start at the
age of 40 [243].
AD is a distressing neuro disorder by not modified -disease treatment accessible so far. The
pathological hallmarks of AD are brain deposits β-amyloid in senile plaques and appeared
neurofibrillary tangles (NFTs) made of hyperphosphorylated tau protein [244, 245].
Curtain et al., 2001; Dedeoglu et al., 2004; Huang et al., 1999 [246-248] have
been investigated that AD neuropathology is considered by the presence of insoluble Aβ
amyloid peptides, NFTs, the misfolded microtubule associated tau protein), neuropil threads,
and neuron loss in AD brains postmortem.
32
Hebert et al., 1995 [249] reported in literature that AD is the most commonly type of
adult onset dementia. The study base on community has proposed that about 4 million peoples
have AD in United States. This observed by study that AD is occur in 3 percent of people with
age 65-74 years, 18.5 percent with age 75-84 years and 47.2 percent above 85 years older. In
this country it is the 4th and 5th leading reason of death. It is estimate that in society with aging
about nine million peoples having AD by the year 2040, unless prevention strategy are found
[250, 251].
Walton et al., 2006 [252] have been stated that regio-specific accelerate loss of neurons
which is the feature of AD are both a cause and results of extensive neuronal dys-function. From
many years by the loss of neuron symptoms of AD increases and this indicates disease aetiology
which is founded upon increasing changing of neuron function. The neuronal micro-
environment and in specific, the steadiness of the physico-chemistry of brain interstitial fluid
(BIF) are important to the optimization of neuron functioning [253-255].
It was reported that Multiple sclerosis (MS) is an immunologically mediated disease
with a genetic predisposition [256]. These factors may be environmental, virus mediated, and/or
arise from metabolic changes resulting from excessive production of nitric oxide (NO). Active
nitrogen species are overproduced in inflammatory brain lesions in MS [257, 258]. NO has been
shown to mediate the death of oligodendrocytes, the myelin-producing cells that are primary
targets of damage in MS [259-261].
MS is an inflammatory-mediated demyelinating disease of the human central nervous system
(CNS). The clinical disease course is variable, normally begins with reversible episodes of
neurological disability in the third or fourth decade of life, and transforming into a disease of
irreversible and continuous neurological decline by the 6th or 7th years [219, 262, 263].
A greater number of studies have been carried out for histological demonstrations of axonal
transaction and loss in postmortem MS brains [264-268], progressive brain atrophy in MS
patients [269-271] and reductions in the neuronal specific marker, N-acetyl aspartic acid
[272-274] are abundant and unequivocal. The main reason of MS is non-traumatic neuro
disability in young adults in North America and Europe, however greater than 2.5 million
peoples are effected [275-277].
Ho et al., 1995 [278] have been stated that MS disease-causing agent is a
glycoconjugate shared by the bacteria and motor axons of the peripheral nervous system.
33
2.2 PSYCHOLOGICAL DISORDERS
In developed and developing countries, increasing rate of mental distress occurs, which is not
frequently reported. . Hansen et al., 2005 ;Owiredu et al., 2012 [279, 280] has been investigated
that, a mental illness or disorder is a behavioral pattern or psychologically related with disability or
distress that occurs in an individual and is not a part of normal development or culture. A number of
studies evidence that developing impairments in childhood precedes schizophrenia in the region of
language, cognition, motor performance, emotional, social and behavioral growth [26]. Alternatively,
the pathophysiology is not clearly understand schizophrenia, and their diagnosis also remains
difficult. It has been criticized as lacking in scientific reliability or validity [93]. Few developmental
and epidemiological factors similar in bipolar disorder and Schizophrenia [281].
By comparing both these disorders show different neuroimaging results, such as in
schizophrenia reduction of brain size [282] and severely enlargement of lateral and third ventricles
than bipolar patients [283].
Hoge et al.,1999; Wright & Brown., 2000 [284, 285] investigated seven works that there
were no dissimilarities in brain size of controls and bipolar disorder patients. Ventricular
enlargement is though, stated in the general category of affective psychosis, although the effect size
is smaller than in schizophrenia [286]. It was investigated that examination for specifically bipolar
disorder, there is some indication that ventricular enlargement is present in these patients [287] who
have some other severe illness [288, 289].
Hafner et al.,1999; Koreen et al.,1993 [290, 291] have been reported that most of the
schizophrenia patients also have depression. It was also reported that more than 75% schizophrenic
patients have also depression even in initial stage [292].
2.3 ROLE OF METALS IN PATHOGENESIS OF NEUROLOGICAL DISORDERS
Adair., 2002 [293] reported that greater number of essential trace elements function
as main components proteins or in enzyme systems that have important roles in the human
body. As most of the trace element can be bound to a part of the enzyme molecule as a metal
ion. If the metal ion is bound firmly the enzyme is known as a metalloprotein or
metalloenzyme and if they are replaced with certain non-essential metal ions, and bound
loosely, the enzyme become inactivated. In enzymes system metal ion contribute in catalytic
process and stable the protein’s structure, in addition to induce the binding of the substrate to
the protein [294].
34
Furthermore, the metal ions are important in membrane permeability, transport
processes and redox reactions as well as in muscle contraction, respiration, nerve conduction,
growth, reproduction and on sub-cellular level in mitochondria and more. So imbalance of
trace metal optimized concentration might be severely influence biological processes and are
related various disorders [295].
Bush., 2003 [296] have investigated the role of essential and toxic metals in the
neuroscience has developing progressively in the past years with discovery of their relevancy
to main neurological disorders such as AD, PD. Lovell et al., 1998 [297] indicated that main
organ of brain that normally concentrates some essential trace elements such as Fe, Cu and
Zn in the neocortex, and cerebral homeostasis of Fe, Cu and Zn are closely associated with
AD. Increases evidences suggesting that changing metal homeostasis might be contributed to
neuron damage in neurological disorders such as Al(III), Fe(III) and Mg(II) [298].
Now a days, various reports stated that, among factors, metal ions (Al, Zn, Cu, Fe,
etc) could particularly impair aggregation of protein and their oligomeric toxicity. Also,
metal-induced (direct) and metal-amyloid-β (indirect) associated with neuronal cell damage
by the formation of reactive oxygen species (ROS) it is difficult to understand mechanisms
by which metal-induce cell death, and thus its role in neuro disorders [299].
35
2.3.1 ALUMINUM
Recently, much interest has been increases by the biological and toxic effect of Al
[300]. Few studies purposed that might be Al stored in the brain via various ways
(medicines, water intake & food) and inhibit the regular activity of nervous system [301]. It
have been reported that Al exposure is a risk factor for the development of AD in humans
[302] and it has been detected in the senile plaques and neurofibrillary tangles of AD [303].
The hypothesis of a link between Al and AD has been supported by several biological
findings [304]. In biological fluids, presence of trivalent cation is rare as an ion because it
complexes extensively with biologically available ligands such as phosphate, citrate and
hydroxide [305]. Al is a known environmentally toxic, it has been associated with various
pathological conditions such as stroke, dementia, dialysis, AD and PD [306]. This element
distributed extensively that ensure the potential cause of human exposure and created adverse
effects [307].
Becaria et al., 2002; Yokel et al., 2002 [308, 309] suggests that there is a link
between higher concentration of Al and increases risk of a different neurological diseases.
The Al gains entry to the brain throughout all stages of human development, from the fetus to
old age [310]. The biological reactivity of Al is primarily the bioinorganic chemistry of its
free solvated trivalent cation Al [311, 312].
It is stated in literature that Al can increase the Fe-mediated lipid per-oxidation
processes by which AD is initiated both by interact with lipid membrane conformation and by
competing with Fe3+ for citrate in the cerebrospinal fluid [313]. Increasing suggestions in the
recent years proposed that Al have adverse toxicity effect on the CNS [314].
2.3.2 MANGANESE
It was reported that in USA, the health of between 68,000 and 185,000 workers is
possibly severely affected by Mn and its compounds. Toxic effect of Mn causes damage to
cells in the basal ganglia, indicating an affinity for the globus pallidus in particular, and to a
less extent the, putamen, caudate nucleus, midbrain tegmentum, subthalamus and SN [315].
The exposure of Mn to human can be derived from several sources, the fumes released from
welding rods contain high Mn fumes. After heavy exposure, two types of effects have been
36
found, such as psychiatric symptoms that dominate such as Mn madness with hallucinations,
emotional lability, and compulsive and aberrant behavior [316]
Sjogren et al., 1996 [317] have been indicated that inhalation of Mn from the air,
had more adversely effects people and showed signs of neurological disorders especially in
aged ≥50 years. Levy & Nassetta [318] have been stated that Mn is a naturally present
element, and its organic and inorganic compounds have a several effects on human’s health.
Mn is an important for the normal functioning of various enzymes and is necessary micro-
nutrient for nervous system normal bone growth and brain function. Arain et al., 2015;
Crossgrove & Zheng., 2004 [319, 320] has been stated that Mn in some cases optimized
the membrane transport and enzyme functions. The higher and lower concentration of Mn in
the body can leads to severe impairment of important biochemical and physiological process,
excess consumption can causes headache, lesions, drowsiness, psychotic behavior, and other
related symptoms [321].
Yokel., 2006 [322] have been stated that the heavy exposure, of Mn adversely effects
neuro health such as psychiatric symptoms, emotional lability, compulsive and aberrant
behavior. Persons, who breathe in the atmosphere of Mn, had its higher concentration in their
blood, they indicates symptoms of neuro problems that were similar to those reported in
occupationally exposed persons. Mostly significant in the people aged ≥50 years [323].
Toxic effect of Mn more common in those workers that have chronic exposure of
dusts or aerosols that containing very higher concentration (>1–5 Mn mg/m3) of it [324].
Some reports suggest that higher exposure of Mn linked to developing of neurotoxic effect in
children [325, 326].
2.3.3 IRON
Iron (Fe) is one of the most abundantly present metal in the human body. It is
necessary for several brain functions and it is involved in neuronal communication. Mounting
evidences suggest that Fe is involve in the mechanisms that causes various neurological
disorders [327]. In CNS Fe is important cofactor for different metabolic functions including
transport oxygen, nitric oxide metabolism phosphorylation oxidative [328]. Due to the
increased level of Fe cause imbalance of brain hemostasis creates pathogenesis of
neurodegenerative disorders [329-331].
It was reported in literature that Fe have key role to transport oxygen,
neurotransmitter formation, myelin synthesis, and electron transfers, being an important
37
cofactor in normal CNS metabolism. Fe is also abundant in SN and globus palladium when
comparing with other areas and with aging increases in humans [332]. Usually, under healthy
conditions, these metal ions are bound to ligands (e.g., transferrin), although when they are
found non-bound, Fe are possibly harmful mainly due to their redox activities in the synaptic
cleft [333].
The alterations in free Fe have been involved in a different neurological disorders
such as AD [334] and those characterized by nigral degeneration involves PD [335] multiple
system atrophy and progressive supranuclear palsy [336].
Whereas cellular Fe homeostasis is mainly mediated by the transferrin receptor and
ferritin, it is also under the control of the lacto transferrin receptor [337] ceruloplasmin,
melanotransferrin [338] and divalent cation transporter. Therefore, changing in any of these
proteins contributed to alter in brain Fe metabolism in PD, AD disease [339].
Fe is essential element uses by almost all living beings, often incorporate into the
heme complex, which mediate redox reactions. Imbalance of brain Fe homeostasis have been
associated to severe neuron damage [340, 341]. Additionally, Fe is toxic to neural tissue,
causes neurological diseases. Organic Fe may increases the genotoxic influence of other
compounds when they are joined. Together with aluminum sulfate, at nanomolar level, Fe
activate the release of ROS. At higher concentration, Fe is genotoxic & mutagenic. In AD, Fe
is mainly cause oxidative stress due to it’s over accumulation in the brain and colocalized
with AD lesions, neurofibrillary tangles and senile plaques [342].
2.3.4 COPPER
Copper (Cu) is one of essential metal ion of body functions but are toxic in higher
level [343]. The presence of Cu throughout the brain and is most prominent in the
hippocampus, basal ganglia, numerous synaptic membranes, cerebellum and in the cell
bodies of cortical pyramidal and cerebellar granular neurons [344]. The Cu is also classify as
a biogenic element because of its important role in photosynthesis, metabolism of nitrogen
compounds or regulation of the DNA and RNA transcription processes [345].
Toxicity of Cu might be created by the consumption or inhaling of higher level by
inhabiting metabolic functions in living beings. Cu plays crucial role in various psychiatric,
autoimmune, neurological disorders [346, 347].
Madsen & Gitlin., 2007 [348] had reported that irregular transport of Cu and its
abnormal interactions with protein in several human neurological disorders, which confirm its
38
critical significance for the normal neurological functions and development. The adverse
effect of Cu arises in human body when alteration the level from normal to higher or lower
concentration and accumulated in the soft tissues. Afridi et al., 2010; Tolls., 2000 [118,
349] have been reported that Cu is also deeply involved in CNS of human. Neurological and
Psychological conditions such as anxiety, stress, depression, and schizophrenia, often
disturbed by incorrect concentration of Cu [350, 351].
2.3.5 ZINC
Zinc (Zn) deficiency may alter its homeostasis in the brain created different
dysfunctions. Consequently, for proper brain functioning and vesicular Zn is an essential
nutrient for neuronal signaling factor [352, 353].
In neurodegenerative and psychiatric disorders, the levels of Zn in plasma is varied
than normal values. Zn deficiency is also related with neurological disturbance [354, 355]
which might be main reason to disturb aged peoples [356]. The effect of aging have
considerable psychiatric and neurological disorders such as bipolar and schizophrenia and
AD, PD. It is generally stated that deficit levels of Zn in food is a major dietary problem in
different countries; which might be resulted into impairment of cognitive functions in
addition to delay in growth . Nevertheless, additional investigation must be necessary to
search other nutritional and physiological parameters of any study population for better
interpretation of adverse impacts of Zn deficiency [357].
Vallee et al., 1991; Vallee & Falchuk., 1993 [358, 359] have been stated that Zn is
essential for the function of greater than 200 enzymes; few of them are associated with RNA
and DNA synthesis. It is important for immune system and optimized functioning of a several
of physiological and biochemical processes [360]. The greater number of the spinal cord Zn-
enriched terminals are GABAergic (γ-aminobutyric acid) and the other ones are glycinergic
[361]. The higher Zn level are present in the hippocampus, neocortex and amygdala. The
transportation of Zn into the brain occurs via the brain barrier system: the blood-
cerebrospinal fluid and blood-brain and barriers [362].
In physiological level Zn presents neuroprotective activity, however higher level of
Zn is neurotoxic [101, 363]. It has been stated by various reports indicating the critical role of
Zn ion availability in the learning, memory function, neurogenesis, processes associated to
brain aging and neurodegenerative disorders [105, 364].
39
Zn is important in normal function of the CNS, axonal, synaptic transmission, nucleic
acid metabolism, brain tubulin growth and phosphorylation [365]. Approximately 90 percent
of the whole brain Zn is strongly bind to metalloproteins whereas remaining of it is store in
synaptic vesicles by glutamatergic neurons and can modulate brain excitability [366].
2.4 BIOLOGICAL SPECIMENS
The analysis of trace concentration of particular important elements in biological
mediums is becoming gradually significant as a consequence of the alertness of their part in
few bio-chemical process [367, 368].
Although, the analysis of trace elemental level is progressively important as they have vital
parts in both normal biological functioning & toxic effect stated by [369]. Human exposed to
trace elements were investigated by using diverse biological samples such as whole blood,
serum or plasma, hair, milk, kidney or teeth, depends on the element analysed [370].
Serum & Blood are commonly employed to determining of absorbing dosage of an element
in relating to disease and health particularly for the toxicity of elements.
It was suggested that the inorganic elements in a human entity i.e. the hair, serum,
blood and inner organs have an important role in the physiological and biochemical
processing of the human body and give the information according to health [371]. Authors
have been conduct a study to recognize the association among the concentration of the trace
elemental level in serum & human disorders [372, 373].
The characteristic features of hair can also be used to determine the exposure of populations
or individuals to pollutants and toxins such as toxic metals [374, 375]. The profile of metals
toxicity in hair samples has been used to detect the source of exposure and the physiological
behavior disorders [141]. The stability of hair also presents unique opportunity which allow
us to analyze the anthropological effect [376].
2.5 ANALYTICAL TECHNIQUES AND EXTRACTION METHODS
The greater number of literature on using several analytical instruments to analyze trace
elemental level in environmental and biological media, such as atomic absorption spectrometry
has been stated by many researchers [377, 378].
Few advanced analytical techniques including inductively coupled plasma optical
emission spectrometry, inductively coupled plasma mass spectrometry & neutron activation
40
analysis and are present to for the analysis of trace elements by enough sensitivity for many
applications [379-382]. More complex instruments required for the methodology, which isn’t
accessible mostly in analytical labs.
It was observed that enrichment and microextraction methods can resolve the sensitivity
limitation with higher confidential level and it is convenient to determine easy trace elements by
lower sensitivity, but simpler techniques such as GFAAS & FAAS [175, 383]. FAAS is
considered to be an excellent tool to quantify metal, coupling with several
preconcentration/extraction steps [384-386]. This instrument is reliable and simple and cost
effective then highly complex and expensive atomic based techniques. The FAAS is
comparatively less sensitive, which could be easily removed by coupling it with enrichment
procedures to quantify trace metal determination [387, 388].
Researchers suggested several methodology to the enrichment of trace elemental level,
i.e., cloud point extraction (CPE), co-precipitation, liquid–liquid extraction (LLE), ion exchange,
[389, 390] and dispersive liquid-liquid microextraction in different biological and environmental
matrices. Tavakoli et al., 2008 [391] have been reported that LLE has consumed comparatively
greater quantity of highly purify solvents with longer long time is needs and their discard
contaminating the environment.
It is stated in literature CPE is based on the phase behavior of non-ionic surfactants in
aqueous solutions results in phase separating after an increases in temperature or by adding of a
complexing agent [378, 392]. The diverse non-ionic surfactants are employed in CPE for the
enrichment of metal, (TX-100), and octylphenoxypolyethoxyethanol (Triton X-114) has been
used by diverse researchers [393-395].
It is an excellent technique that lessens the use of organic solvents and hence exposure to
them, and eventually paves the road to green chemistry. Furthermore it responsible for low
disposal costs and short extraction time [152, 396, 397].
In conventional CPE other hydrophobic species could be extracted and might be
interfering with the analysis of the analytes of concern [398]. So it is modified by back extracting
of analyte in acidic media possibly will enhance the results, known as d-CPE [147, 399].
Currently, scientific analytical community shown a keen interest to reduced uses and
exposure of toxic solvents and chemicals in such separation procedures through miniaturizing of
classical extraction techniques. Researchers have stated that nowadays scientific community
have proposed several scientific methodology to miniaturize the classical LLE technique. [154,
400].
41
Aguillera-Herrador et al., 2008; Han et al., 2007 [401, 402] suggested a significant
concern on the uses of room temperature ionic liquids (RTILs) as the green solvent to substitute
the conventional organic solvents in a wide ranging of application, specifically in LLME of
heavier metal ions and other pollutants. To define RTILs as organic salts which liquefying at
room temperature [403].
It is suggested that this specific behavior observed due to the less coordinating exist in
their components, as there is atleast one delocalized charge present which prevents the stable
pattern of crystal lattice [404]. The IL has ability to create intermolecular interactions as
compared to other volatile organic media. This include strong and weak ionic type
interactions, dispersive, n–π and π – π interactions, van der Waals and hydrogen bonding
[405]. Several works have been suggested which indicates that IL is effectively utilized for
enrichment of metalloids and metal [406, 407].
Panhwar et al., 2014; Naeemullah et al., 2016 [164, 408] has been stated over the
RTILs successfully applied as a RTIL-based microextraction of metals. Arain et al., 2015
[153] proposed temperature-controlled IL-DLLME, alike to DLLME, yet dispersing
surfactant by thermal assisted not using chemical. Several ILs have been employed for the
enrichment of metal, i.e., 1-butyl-3-methylimidazolium hexafluorophosphate [C4MIM][PF6],
1-Hexyl-3-methylimidazolium hexafluorophosphate [C6MIM][PF6] [205, 409].
In dispersive liquid–liquid microextraction (DLLME), a dispersive solvent in small
volume having miscibility with both the extracting solvent and water, to disrupting the
extracting solvent at a volume level of ml into the aqueous phase. Then it is normally lowers
the partition coefficient of the analytes in the extracting solvent. Arain et al., 2017 [127] have
been purposed that modified dispersive liquid-phase microextraction (MDLP-µE) not only
eliminate the solubility effect of organic solvent in aqueous media, but also reduce the matrix
effect of the organic solvent on the target analyte .
Ultrasound-assisted dispersive liquid– liquid microextraction (USA-DLLME) was
proposed very recently [410-412]. To disperse extracting solvent in this established procedure
into the aqueous phase by ultrasound is an alternative of dispersing solvent, results in a more
environmental benign technique with a higher coefficient of partition of the sample in the
extraction solvent and good extraction efficiency. It is taking greater interest and effectively
apply for the analysis of trace inorganic & organic compounds in several fields [413, 414].
It has been suggested a new type of solvents called ‘switchable or tunable solvents’ to
achieved improved absorption medium for CO2 elimination. Also their higher CO2 capture
42
efficacy, additional benefit is that no unwieldy organic synthesis procedures are requisite
[193, 415]. Switchable solvents have lower polarity till they are expose in the steam of CO2,
which changing them into highly polar solvents [187]. Several study have revealed the
capability of ionic compounds to captured CO2 and act as scrub agents [416, 417]. By
exposing to CO2 polarity of liquids changes from non-polar to polar & heated in the presence
of nitrogen or argon gas [194, 195]. Such types of solvents could be used in separations and
organic syntheses by replacing solvents during each reaction process. Therefore, their use
covers a wide range of applications in different field such as organic, analytical chemistry
and biotechnology [418].
Abbott and his group made an excellent discovery by simply mixing salts in different
ratios with varieties of hydrogen bond donors compounds which produced a deep eutectic
solvent (DES) with low melting point [197, 419]. The low melting point is a result of
mixing an organic salt (zinc chloride, choline chloride) with hydrogen bond donor (HBD)
compound such as an amine, alcohol, amide or carboxylic acid [420, 421]. DES are
observed as ionic liquids similarities because they sharing many of their intrinsic favorable
properties like their bio-degradability, non-flammability due to their lower or none
measurable vapor pressure and less toxic [201, 422].
It has been reported that DESs have been extensively used in extraction of organic
solvents, synthesis of nanoparticles, electrodeposition of metals, digestion of inorganic
compounds, drug dissolution, CO2 absorption and purification of biodiesel, drug dissolution,
and refinement of biodiesel [205-208, 423]. It has been proposed DES as effective
extraction media for Cd-ammonium pyrolidine dithiocarbmate complex in aqueous samples
[424].
2.6 MULTIVARIATE STUDY
The multivariate studies are using to optimize several variables throughout diverse steps
in the establishment of methodologies. It has been proposed that multivariate calibration as
different from univariate calibration in that the experimental data depend on various variables
[425]. It is stated that multivariate strategies emphasis on the development and application of
mathematical models that relating the multivariate techniques signals with analyte quantity or
sample properties, therefore decreasing the number of experiments needs [426].
43
Zougagh et al., 2000; Dinc et al., 2009 [218, 427] using factorial design as a screening
procedure in order to selecting the variables effects the system. It has been suggested that
application of factorial and central composite designs to optimizes sample preparing steps [428].
Plackett-Burman designs as experimental method is using & stated in wet acid digestion by
[429]. Factorial design involvement in optimizing of experimental parameters has been stated by
several authors also [430-432]. For the enrichment methods, optimizing step involves by using
experimental design has been stated by various researchers [433, 434].
De Amorim et al., 2006; Baranda et al., 2005 [214, 435] suggested the application
of factorial design involved the variables in LLE. A Plackett-Burman experimental design
has been employed and suggests as an approach for assessment of the influence of various
factors and central composite design for optimizing in instrumental determination steps by
[436].
2.7 SUMMARY
This chapter includes reported information about different
neurological/psychiatric disorders and adverse effects of metal. Moreover, the
biological role of the understudied elements i.e. Al, Mn and Fe, Cu, Zn was also
presented to understand the possible biological mechanism of these elements
producing the adverse effects. To determine the trace elemental level of neuro
patients, importance of collecting biological samples was necessary. For the
analysis of elements different preconcentration methods have been reported.
44
CHAPTER 3
RESEARCH METHODOLOGY
In this chapter, plane of work, chemical reagents and procedures of developed methods
has been given.
3.1 PLAN OF WORK
The experimental portion of current work was attained in diverse steps, which includes:
i. Biological specimens were collecting (Blood, scalp hair & serum) of patients
having different neurological disorders (Alzheimer’s, Parkinson’s, Dementia,
Multiple sclerosis, Brain tumor, Brain hemorrhage, Stroke,) and psychiatric
disorders (Schizophrenia, Bipolar disorder, Depression).
ii. To comparing the biological samples were collecting from healthy controls/
referents of similar age groups, socioeconomic status of both genders.
iii. Development of advance extraction methodologies to the determination of trace
levels of Aluminium (Al), Manganese (Mn), Copper (Cu) and Iron (Fe), Zinc (Zn)
in acid digested biological specimens of neurological & psychiatric disorders
patients and healthy referents.
iv. The developed methodology authenticity checked by certified reference materials
& standard addition procedure to the real biological samples.
v. The chemometrics (multivariate techniques), involving Plackett-Burman
experimental, Central 23+ star orthogonal composite design were used to optimize
the experimental variables of developed method.
vi. The preconcentrated analytes (Al, Cu, Fe, Mn and Zn) in different biological
specimens was analyzed with Flame Atomic Absorption Spectrophotometry.
45
3.2 STUDY POPULATION
The study population was selected on a random basis from urban and rural areas
of Sindh, Pakistan. The collection of biological samples of neuro & psychiatric patients,
admitted and attained as outdoor patients, in the neurological wards of Civil Hospital,
Hyderabad, Liaquat National Hospital, Karachi, Cowasji Jehangir Institute of Psychiatry.
This work was approved by ethical committee of University of Sindh, operating under
Higher Education Commission, Pakistan auspices.
Prior to starting the work, all control and the relatives of neurogical and
psychiatric disorders patients, age ranging 45–70 years, were informing by performa,
about the objective of work, and all approved to participating and signed the performa.
The scalp Hair, blood and serum specimen was collecting from
neurological/psychiatric patients and age matched healthy subjects (control/referents)
were selected as given below:
The Al & Mn in scalp hair samples of 102 patients have diverse types of psychiatric
disorders, Schizophrenia (52) and Bipolar disorder (50), in the age-matched of 45–60
yrs.
The Al in scalp hair specimens of 110 Alzheimer’s disorder male patients, together with
90 referent subjects of age-matched 60–70 years.
The concentration of Al in blood samples of healthy referents (60) and different neuro
disorders male patients, Alzheimer’s (45), Stroke (20) and Dementia (25), in the age
group of 50–70 years.
Mn in scalp hair samples of 102 Parkinson’s patients and referent subjects (n=95) of both
genders with age-matched control subjects (60–70 years).
The amount of Mn in blood samples of 100 male patients leading different neuro
disorders, Parkinson's (50), Dementia (30), Multiple sclerosis (20), and healthy referents
(60) of age ranged (50–70).
To determine Fe in serum specimens of male patients have neuro disorders, Alzheimer’s
(20), Parkinson’s (20), multiple sclerosis (15) and normal referents (60) of similar age
matched (40–70) years.
The level of Cu in serum samples of different neurological disorders male patients,
Alzheimer’s (20), Depression (20), Dementia (20) and normal referent (40) of age
ranged (50–70) years.
46
The Zn in blood serum samples of 55 male patients having diverse types of psychiatric
disorders, Schizophrenia (20), Depression (20) and Bipolar disorder (15), together with
60 referent subjects age-matched 50–70 years.
Selection criteria of study subjects were that they don’t have diabetes, failure of kidney,
or other disorders nor have been treating with medicines, which can affect nutritious
status of the elements (mineral supplements or antihypertensive drugs, diuretics etc.). To
excluding from the studies those who were mentally suffering & retarded because of
addicted to drugs.
The controls were belonged to the similar age group, socioeconomic position, and diet
ways, not suffered to any disease & not takes any mineral supplement. They are
commonly the healthier family members of the patients. Before to collect biological
sample, they have undertaken a standardize routinely medical examining.
47
3.2.1 QUESTIONNAIRE EMPLOYED IN SAMPLING CAMPAIGN
Serial No………………… Patients File Case No. ………………
(1) Demographic/Personal Information:
Full Name …………………………………Place of Birth ………….… Age………
Address: ………………………………….. Profession: ……………..……
Sex (male/female)…………………… Weight (kg/Pounds)………..…..
Height (cm) ……………
(2) Disease and Treatment Information:
Blood group….…… Family history ……… Neuro/psychiatric disorder………...
Diagnosis Date …… Treatment Starting Date ………………
(3) Food intake and Life Style (Brief description):
Diet / Food intake (Fill through a to d)
(a) Regularly (b) Moderately (c) Rarely (d) Never
1. Meat ………. 2. Fish …………. 3. Chicken ……………4.Vegetables ……………………… 5. Fruit intake ………….. (Frequently used fruit(s) ……………) 6. Smoking ……………………7. Alcohol drinking …………. 8. Chewing tobacco ………. 9. Snuff & Betel quench …………10. Shampoo ………….… (Brand name …………) 11. Soap …………
3.2.2 SAMPLING
SCALP HAIR
The specimens were takings from nape of the neck. Which then stores in a labelled
plastic bags and attach to a questionnaire. Hair samples was cut into about 0.2 to 0.3-cm parts
in labs & also washing several times by Triton X-100, acetone and distilled water. After that
samples were become dry at 80–85 °C [135, 437].
48
WHOLE BLOOD
Venous blood specimens was collecting by studied subjects was taken by into metal-
free blood-collecting tubes (Becton, Dickinson and Company, Rutherford, NJ, USA) having
K2EDTA (>1.5 mg L-1) [118, 438]. By using standardized procedures for biochemical testing
approximately 2mL of blood samples sending to the pathology laboratory. For the analysis of
element 2 mL blood samples was stores at −20 °C, although residual 3 mL was employed to
isolating the sera. Blood is allow for clotting for 15 to 30 min at room temperature than
centrifuged at 2500 rpm for 5-10 min. By using Pasteur pipette separating the supernatant
fluid than store at −20°C till analysis.
3.3 CHEMICALS AND REAGENTS
The (PAN) 1- (2-pyridylazo)-2-naphthol, were taken by (Fluka) & 8-hydroxyquuinoline
(Oxine) was acquired from Merck, dissolve specific quantity to prepare the reagent in 10
mL C2H5OH (Merck) and diluting to 100 mL with 0.01 mol L-1 CH3COOH.
(Morin) 3, 5,7,2,4 pentahydroxy flavone was acquired by Fluka & its 0.01 percent
solution was making by solubilize 0.01 g in 100 mL of C2H5OH.
HNO3 65% and 30% H2O2, chloroform, Acetamide of analytical reagent-grade (Merck,
Darmstadt, Germany) were employed.
Certified reference material (CRM) of human hair NCS ZC81002 – from China National
Analysis Center, human blood (Seronorm Trace Elements Whole Blood ( LOT 1103128)
from Sero AS and Bio-Rad (Milan,Italy), and human serum from Clincheck control
lyophilized ® human serum Recipe (Munich, Germany) were uses as CRM.
[C4MIM][PF6] 1-Butyl-3-methylimidazolium hexafluorophosphate, Triton X-114, Triton
X-100 non-ionic surfactants was obtained from Sigma-Aldrich (St. Louis, MO, USA).
1, 8-diazabicyclo [5.4.0] undec-7-ene (DBU), 1-Decanol, Zinc chloride, and hexanol were
purchased from sigma Aldrich.
The running standardized solutions were made by successive dilution of the stock Al
standardize solution with 0.2 mol L-1 HNO3 prior to analysis, purchased from Fluka
Kamica (Buchs, Switzerland).
Acetate and phosphate buffers were employed to monitor the pH in the range of 2-6 and
7-11 respectively, adjustments being made by uses 0.1 mol L-1 NaOH/HCl
49
3.4 INSTRUMENTATION
A PEL domestic microwave oven (Osaka, Japan), programmed for time & power
from 100 to 900 W, was employed to digest samples.
WIROWKA Laboratory jna type WE-1, nr-6933 centrifugation; speeds ranging 0-
6000 rpm, timer 0-60 min, 220/50Hz, Mechanika Phecyzyjna, Poland was using to
isolation.
Metals was analysed by employing flame atomic absorption spectrometers, Perkin
Elmer Model “A Analyst 700” (Norwalk, CT, USA) Table 3-1.
For pH measurements a pH meter (Ecoscan Ion 6, Malaysia) was employed.
A programmed ultrasonic water bath, model no. SC-121TH (Sonicator, Deep Park,
NY, USA) was employ to incubate with temperature ranging from 0 to 80 °C at
intensification frequency of 35 kHz.
Self-made microsample injection system connected to the nebulizer tip, uses a PTFE
capillary tube (12 cm in length) attaching with a micropipette tip was using for micro
sample nebulization.
50
Table 3-1 Instrumental conditions for Perkin Elmer Model 700 operations
key: aOxidant (Nitrous oxide), D2 deuterium lamp, mA milliampere, nm nanometer, C centigrade, s second, mm millimeter, L min-1 liter min-1
3.5 STATISTICAL ANALYSIS
Statisticaly determination were made by employing the computer programing Excel
(Microsoft Corp., Redmond, WA, USA) and Minitab 13.2 (Minitab Inc., State College, PA,
USA). The Shapiro–Wilk test for normality is applied to checking the data distribution of
both elements to each studied group.
Possibility link among Al & Mn level in scalp hair specimen of control and neuro
patients were distinctly observed by employing Spearman correlation analysis.
Nonparametric Mann–Whitney U tests were apply to test for significant differences in metal
level among patients & control. All interactions were significant at 95 percent CI (p<0.05),
other than this checked it.
51
Flame conditions
Eleme
nts
Wave
length
(nm)
Slit width
(nm)
Lamp current
(mA)
Oxidant
(Air/Nitrous-
oxide) L min-1
Fuel
(acetylene)
L min-1
Al 309.5 0.7 7.5 17.0a 2.0
Mn 279.5 0.2 2.0 17.0 2.0
Fe 248.5 0.2 7.5 17.0 2.0
Cu 324.8 0.7 7.0 17.0 2.0
Zn 213.9 0.7 7.5 17.0 2.0
3.6 SAMPLE DIGESTION METHODS
The scalp hair, serum and blood specimens was prepared by CDM and MDM method
in order to remove matrixes effects [439].
3.6.1 CONVENTIONAL WET ACID DIGESTION METHOD
For conventional digestion method (CDM) , duplicate of every biological specimens,
200 mg of scalp hair, blood 0.5 mL and 0.2 mL of serum of patients, referents and CRM were
digesting with (2:1 v/v) of nitric acid-hydrogen peroxide. Weighing or estimated volume of
all samples in separate Pyrex flask treat with mixtures of acids and set aside for 10 min at
room temperature, than at electric hot plate 60–70 ºC heated by covering the it until the
cleared matrix appeared repeated the treatment. Evaporate the excessive acid and dilute the
semidried masses with 1 mol L-1 HNO3 and centrifuge or filtered by whatman No. 42, volume
make upto 10 mL of filtered solution with 0.1 mol L-1 HNO3. For every experiment blanks are
prepared by using acids mixture without standards or samples. Same acid matrixes use for
standard & blank [182].
3.6.2 MICROWAVE-ASSISTED ACID DIGESTION
Microwave assisted sample pretreatment is advantageous because the requisite of little
quantity of mineral acids, reducing the formation of nitrous vapors and to attain a smaller
digestion time [369].
Duplicate samples, 0.5 mL blood, 200 mg of scalp hair and 0.2 mL of serum specimens of
every patients, controls and replicate six matrix matched CRM were directly takes into Teflon
PTFE flasks (capability 25 mL). Mixed solution of nitric acid-hydrogen peroxide (2:1, v/v),
prepare fresh and add 2 mL to every flask than set aside for 10 min at room temperature and
place in a covering PTFE container. Heating the flask at eighty percent of entire power (900
W) followed by one-stage digestion program. The biological samples needs 4–5 min for
complete digestion. Allow to cool the resulted solution and excessive acid were evaporate.
Then in digested samples add five mL of 0.1 mol L-1 HNO3 and filtered solution dilution
made upto ten mL
52
The authenticity & efficacy of the MWD procedure were check by CRMs values of
biological samples and comparing with CDM [319]. The %R of all metals in CRM samples
achieved by MWD and CDM was calculating by eq:
%Recovery=metals obtained by MWD∧CDM /Certified value×100
98.17–99.95 % for all CRMs studied. Not significantly different are observing (p > 0.05)
by compared the results achieved MWD and CDM (paired t-test) are given in (Table 3-2).
Table 3-2 Analysis of Al3+ & Mn2+, certified human hair NCSZC81002 samples
both procedures (n = 10).
Elements Certified values CDM
% Recoverya MWD
% Recover
ya
T value^
Al 13.3 ± 2.3 13.2 ± 0.62 (7.09)
99.24 13.26 ± 0.94(4.62)
99.69 0.145
Mn 3.83 ± 0.39
3.76 ± 0.28 (8.97)
98.17 3.79 ± 0.34(7.45)
98.95 0.727
Key: ^Paired t-test among CDM and MWD DF = 9, T (critical) at 95 % CI =2.262, p = 0.05, Values in ( ) are RSD, a%R=metals obtained by MWD∧CDM /Certified value× 100
53
3.7 DEVELOPED ADVANCED EXTRACTION METHODOLOGIES
3.7.1 DUAL-CLOUD POINT EXTRACTION (d-CPE)
PROCEDURE
The d-CPE procedure was performed in two steps, based on the conventional-CPE
method. 1st step of CPE, 10 mL aqueous standard solution contains Mn2+ ranging of 10–50 µg
L-1 were transfers into centrifuged tubes with glass stopper (capacity 25 mL). Then, PAN (1–5
× 10-5 mol L-1) 0.5 mL, Triton X-114 (0.1–0.5%, v/v) 2 mL and phosphate buffer 2 mL was
adding, and the pH was maintained ranging of 7–11 with 0.1 mol L-1 NaOH/HNO3. The flask
were placed in a thermostatic bath for 2–20 min at 30–60 °C. By centrifuged for 5 min at 3500
rpm phase separation was obtained, the extraction efficiency of Mn2+ not effected by increase
of centrifugation time. Kept the tube in an ice water to increasing the viscosity of the
surfactant-rich part. Then, decant aqueous part. As an alternative of adding of diluents or
analyzed, the surfactant-rich part having the metal complexes are treating with two mL of 0.5
to 2.0 mol L-1 of HCl/HNO3 and retained in thermostatic bath at 30–60 °C for 5–20 min as the
2nd round of CPE. Centrifugation was carried out at 3500 rpm at five min after diverse time
intervals. Consequently, the supernatant was proceed into FAAS for determination. Similar
method is used to prepared blanks. The suggested d-CPE procedure was applied to acid-
digested scalp hair (n=2) of every PD patient.
ANALYTICAL FIGURES OF MERIT
The linearity to the enrichment of Mn2+ by the established d-CPE method with
coefficients of determination 0.991–0.998 in the ranging of 10–50 µg L-1. The LOQ & LOD
were found for Mn2+ as 0.324 & 0.097 µg L-1, respectively, as shown in Table 3-3. The
validity of the developed procedure was obtained with a CRM of human hair and spike
recovery test in real samples Table 3-4. The e enhancement factors (EF) of Mn2+ was 46. The
higher sensitivity and lower limits of detection of the suggested d-CPE procedure stated that
it is sensitive and efficient for the analysis of very lower level of Mn2+ in scalp hair
specimens.
54
Table 3-3 Performance characteristics of the suggested d-CPE procedure.
Concentration range 10–50 µg L−1
LODa 0.097 µg L−1
R2 (coefficient of determination) 0.998
Repeatability (RSD%)b (n=10) 3.2
Enhancement factorc 46
Key: a Detection limit (3σ/m), b Mn2+ level 10 µg L-1, to achieve RSD c Calculating as enriched samples/without enrichment.
Table 3-4 Analysis of Mn2+ in CRM (µg g-1) by d-CPE (n =10)a
CRM of human
hair NCS
ZC81002
Observed value
(x ± s)
%Recoveryb Certified value
Without preconcentration
3.72 ± 1.23 97.1 3.83 ± 0.39
CPE 3.78 ± 0.15 98.6 3.83 ± 0.39
d-CPE 3.80 ± 0.09 99.2 3.83 ± 0.39
Spiked recovery test of Mn2+ in scalp hair sample of Parkinson's patients
Added Measured (x ± s) %Recovery
0.0 9.83 ± 0.67 ---
5.0 14.8 ± 0.70 99.4
10.0 19.76 ± 0.76 99.3
Key: a Mean ± S.D, b %Recovery = measure values/certified value×100
3.7.2 DUAL CLOUD POINT EXTRACTION (d-CPE) METHODOLOGY TO
DETERMINE ZINC IN SERUM SAMPLES
PROCEDURE
The d-CPE was carried out in two steps, based on the straight CPE procedure. At
the start of methodology of CPE, 10 mL solution of Zn2+ ions ranging of 20 – 400 µg L-1 was
55
taking in centrifugation flask (capacity 25 mL). Then 4 × 10−5 mol L-1 PAN level in ranging
of (0.2 – 0.7 mL), Triton X-114 (0.1–0.5%, v/v) 2 mL and phosphate buffer 2 mL of were
adding, then the pH was maintained ranging of 5–11 with 0.1 mol L -1 HNO3/NaOH. The
tubes contents was heated at 40–60 °C for 2–20 min in a thermostatic water bath. The
separated of two phase by centrifuged for 5 min at 3500 rpm, it was observed that
enhancement of time for centrifugation had not any major effects on the extraction recovery
of Zn2+. Then cool in an ice water to enhance viscosity of solution. The surfactant become
viscous & separated from aqueous phase. The surfactant rich part was isolating from aqueous
part carefully. In second step of CPE called as d-CPE, a instead of addition of diluents or
analysis, added two mL of 0.5 to 2.0 mol L -1 of HCl/HNO3, and heated at 40–60 °C in
thermostatic water bath for 5–20 min. The contents of tubes after each time intervals, were
centrifuged for 5 min at 3500 rpm. To isolated upper layer and subsequently analysed by
FAAS. For each step of methodology blanks were prepared simultaneously. The proposed d-
CPE method was applied to serum samples digest in acid of psychiatric patients and healthy
controls subjects. The graphical diagram of suggested method is show in Fig. 3-1.
Figure 3-1 Graphical abstract of d-CPE method
ANALYTICAL FIGURES OF MERIT
The linearity of Zn2+ in the ranging of 20–400 µg L-1, after preconcentration by the
developed d-CPE method with correlation coefficients 0.991–0.998 was prepared. The LOQ
56
and LOD was calculating for proposed method as, and 3.63 and 1.09 µg L-1 respectively
Table 3-5. The validity of the developed d-CPE and conventional CPE procedures was
verified by the analysis of certified sample of serum have certified value for Zn2+ Table 3-6.
The enhancement factors 40 was obtained for proposed methods measured by slopes
obtained with & without pre-concentration of Zn2+. The d-CPE method have good sensitivity
and low detection limits which recommend to analyze trace amount of Zn2+ in serum samples.
Table 3-5 Characteristics performance of the stated d-CPE procedure.
Concentration range 20 – 400 µg L-1
LODa 1.09 µg L-1
R2 0.998
Repeatability (RSD%)b (n=10) 3.4
Enhancement factorc 40
Key: a Limit of detection (3σ/m). b Zn2+ level 20 µg L-1 to obtaining R.S.D. cCalculated as enriched/without enrichment.
Table 3-6 Preconcentration of Zn2+, in certified reference material (mg L-1) by conventional
CPE and d-CPE methods (n=10).
CRM of Clincheck control
lyophilized ® human serum
x±sa %Recoveryb Certified value
CPE 0.778±0.019 97.40.798±0.014d-CPE 0.791±0.016 99.1
Key: aMean±S.D, b % Recovery=measure valuescertified value
×100
57
3.7.3 TEMPERATURE CONTROLLED IL-BASED DISPERSIVE MICRO-EXTRACTION (TIL-
DLLME) USING TWO COMPLEXING AGENTS, TO ANALYZE Al IN SCALP HAIR
SPECIMENS OF AD PATIENTS: A MULTIVARIATE STUDY
PROCEDURE
Replicate six of each 10 mL standard solutions contains 50 – 200 µg L-1 of Al3+ and
duplicate acid digested scalp hair samples were occupied in a conical tube with screw cap
glass. For (TIL-DLLME) of Al3+ 0.1–0.5 mL of oxine (0.113 percent); and 0.1–0.5 mL of
morin (0.125 percent) two complexing agents were using individually and pH was
maintained in the ranging of (4–8) by adding of 0.1 mol L-1 NaOH/HCl solution in acetate
buffer. Heating on a ultrasonic water-bath at 45 °C by the adding 75 µL of [C4MIM][PF6],
mixed solution. By the formation of cloudy solution the test tube was immersed in ice water
for 10 min. Throughout this step, the extraction of hydrophobic chelate of Al3+ into the fine
droplets of [C4MIM][PF6]. Now centrifuged mixed solution for 10 min at 3500 rpm to attain
phase isolation. Then IL part was sediment at the bottom tube. Acidic methanol of 0.5 mL
was adding into the viscous IL phase before analysis by FAAS. The graphical presentation of
proposed method in Fig. 3-2.
Figure 3-2 Graphical diagram of TIL-DLLME method
58
EXPERIMENTAL DESIGN
The PBD being uses for selection purpose by the objective to develop important
parameters that effects the (TIL-DLLME) of Al3+ in aqueous extracts of scalp hair samples,
employing 2 chelating agents. Experimental design apply to reducing the developing time of
the procedure and give lesser uncertain extracting conditions, therefore help to interpret data.
To estimate 5 parameters at 2 levels, a PBD with only 16 experiments is describes as an
alternative of the 25 = 32 require for a full factorial design. The lower (−) & higher (+) levels
of all parameters were shown in Table 3-7. The PBD matrix show in Table 4-5. However
significant influence was check by ANOVA and employing p-value. The central 23+ star
orthogonal composite design (CCD), studied to interlinking among parameters and more
optimizing variables that have major influence, the CCD widely uses 2nd -order RS modeling
within k factor experiments [219]. To optimized proposed method, CCD with 6° of freedom
and involves sixteen experiments were achieved. In current study the statistically significant
variables IL, P and both ligands (L1 and L2) were observed as factors for optimized
experiments Table 4-6.
Table 3-7 Variables and levels uses in the factorial design to extracting Al3+
Variables Symbol Lower (−) Higher (+)
Ionic liquid (μL) IL 40 1008-hydroxyquinoline
(Oxine) mL R1 0.1 0.5
3,5,7,2′-4′pentahydroxyflavones
(morin) mLR2 0.1 0.5
pH P 4 8
Incubation time (min) It 1 5
CALIBRATION AND SENSITIVITY
Enhancement factor (EF), Extraction recoveries (ER), and consumptive index (CIn)
were 3 major factors, used to obtain the performance of our microextraction method. ER is
the % whole concentration of Al3+ extraction into viscous IL part. Mathematically:
59
ER=mIL phase
maq=
C IL Phase× V IL Phase
Caq× V aq× 100
In equation mILphase is the level of Al3+ in final IL rich part and maq is the Al3+ initial quantity in the
sample media. CILphase and Caq are the amounts of Al3+ in IL phase and aqueous phase,
correspondingly. Similarly, VILphase and Vaq are the volumes of these 2 parts [440]. An extracting
recoveries of 99.0% and 98% were obtained under the optimized experimental conditions for L1
(oxine) and L2 (morin), respectively. Calibration graph using for the enrichment of Al3+ with L1
and L2 were linear with a correlating coefficient (R2) of 0.997–0.981 correspondingly, in the
ranging of 50– 200 µg L-1. The analytical features, methods precision, express as the percentage
RSD of atleast 10 independent determination of CRM, then TIL-DLME of Al3+ employing L1 and
L2 was achieved to be 3.88% and 4.74%, and LOD were achieved to be 0.56 µg L-1 and 0.64 µg
L-1 for complexing reagents L1 and L2, correspondingly. It indicates the oxine is more efficient as
compared to morin. The ‘‘enhancement factor (EF)’’ was observe to be 85 and 73 respectively.
The consumptive index (CIn) can be defined as:
C∈¿V s
EF
Where Vs is the volume of sample (in mL) uses to attain the EF value. The CIn achieved for the
developed procedure was 0.117 and 0.136 correspondingly. A higher EF was found with a
reducing sample volume, results a lower CIn. Thus, CIn show effectiveness of sample used, and
it is meaningful to select a microextraction procedure while the sample level is limited, when the
determination of body fluid [441]. Quantitatively precise consequences are achieved by using
matrixes-matching calibration of certified standards and CRM of scalp hair, and also adding
certified standard into a real sample (Table 3-8). The recovery of Al3+ complexing with both
ligands in CRM samples as well as real sample obtained by TIL-DLME were found to be in the
range of 96.8–99.0%. The preconcentration factor (CF) of 30 was obtained, after optimization.
To use greater concentration of initial solution then CF and LOD can be enhanced. The
procedure was effectively apply to the analysis of Al3+ in biological specimens.
60
Table 3-8 Analysis of Al3+ in certified reference material and spiked sample of scalp hair using
TIL-DLLME method.
Certified sample of Hair (µg g-1)
Certified values(µg g-1)
Without TIL-DLLME With TIL-DLLME
Oxine Morin Morin Oxine
CRMs a13.6 ±2.8 13.2 ±0.14 13.05±0.07 13.5±0.15 13.3±0.13
%Recoveryb 97 96 99.3 97.7
Paired t testc tExperimental
0.07 0.016 0.112 0.145
Sample Added calculatedvalue
Experimental values
Without TIL-DLLME
With TIL-DLLME
Oxine Morin Oxine MorinAD Patients 0 24.4 24.4 ----- ----- -----
50 74.4 a72.3±0.36 72±0.42 73.5±0.16 73.2±0.21
75 99.4 96.6±0.49 96.3±0.7
98.2±0.21 97.8±0.28
100 124.4 121±0.62 120±0.69
123±0.26 122±0.35
%Recoveryb 97.1-97.3 96.5-96.2
98.7-99.0 98.2-98.6
Paired t testc tExperimental
0.018 0.03 0.015 0.039
Key: aMean±S.D b%Recovery= Measured value
Certified∧added values , cPaired t-test among certified/added values and
experimental values. tCritical at 95% CL=2.57 at DF 5=(n-1).
61
3.7.4 PRECONCENTRATION OF TRACE LEVEL OF CU IN SERUM SPECIMENS OF
PATIENTS HAVING NEURO DISEASED USING ULTRASOUND ENERGY
For experimental series of standard (10 μg L-1) of Cu ions (10 mL) were taking
individually in centrifuge tubes (capacity 25 mL).The complexing agent PAN 0.5 mL ranging
of (1–5× 10-5 mol L-1) and (2 mili litre) Acetate/phosphate buffer was added to maintain the
pH 2 to 10 with 0.1 mol L-1 NaOH/HCl. Then added 100 μL of IL [C4MIM][PF6]. The flask
were reserved in an ultrasonic bath for 10–60 sec at < 40 °C. By centrifugation at 3500 rpm
for 5 min separation of phases occur. The aqueous part was discarded. The resulting Cu
enriched organic part was shifted into another glass tube. Then for 2nd part of microextraction,
added 0.5 mili litre of the back extracting solution (1.5 mol L−1 of nitric acid) to the
sedimented enrich portion and kept in ultrasonic bath for 10–60 sec at < 40° C. To
centrifuged at 2500 rpm for 1 min. Lastly, aqueous portion was separating & analysed by
FAAS. Similarly also prepare blank. The proposed UDIL-μE method was applied to serum
samples of each neurological disorders patients and healthy controls subjects. The graphical
presentation of developed method is show in Fig. 3-3.
Figure 3-3 Graphical diagram of UDIL-µE method
ANALYTICAL FIGURES OF MERIT
To achieve linearity for Cu ion in the ranging of 10–100 μg L-1, after by the developed
UDIL-μE methodology with coefficients of correlation 0.998. The LOD and LOQ were
quantified for proposed method as 0.36 μg L-1 and 1.22, respectively Table 3-9. The accuracy
of the proposed UDIL-μE procedures was checked by the determination of certified sample of
serum has certified value for Cu ion Table 3-10. The enhancement factors 31 was obtained for
62
proposed methods calculated on the basis of ratio of slopes obtained before and after
enrichment of Cu ion. The UDIL-μE method have good sensitivity and low detection limits
which recommend to analyze the trace concentration of Cu ion in blood serum using FAAS.
Table 3-9 Characteristics performance of the developed UDIL-µE procedure.
Concentration range 10 –100 µg/L
LODa 0.36 µg L-1
R2 (coefficient of determination) 0.998
(RSD%)b (n=10) 3.3
EF 31
Key: a Limit of detection (3σ/m), b Cu ion level 10 µg L-1 to obtained R.S.D.,
cCalculated as preconcentrated samples & without preconcentration.
Table 3-10 Preconcentration of Cu ion in certified reference material (µg L-1) by UDIL-µE
(n=4).
CRM of Clincheck control
lyophilized ® human serum
x±sa %Recoveryb Certified value
(UDIL-µE) 795±0.054 99.7 797±0.051
Key: aMean±S.D, b % Recovery=Measure valuesCertified value
× 100
3.7.5 AN INNOVATIVE MODIFIED DISPERSIVE LIQUID-PHASE EXTRACTION OF IRON IN
SERUM SPECIMENS OF NEURO DISEASED PATIENTS
DESIGN OF MODIFIED DISPERSIVE LIQUID-PHASE MICROEXTRACTION METHOD
(MDLP-µE)
The MDLP-µE method is required a glass test tube with a syringe system. In the first
extracting MDLP-µE step, 10 mL standard (10-100 µg L-1) were taken into glass test tube.
Then 0.2 mL of desire buffer and 0.1–0.5 mL of oxine (0.113%); added and pH value was
adjusted to pH 6. The extracting solvent chloroform (80 μL) was added. The syringe system
63
(10 mL) was used to aspirated and dispersed back the portion of each standard and sample
solution. This aspirating/dispensing cycle made the sample solution more cloudy. The mixed
solution was centrifuged at 2500 for 4 min to extract the analyte into finely-dispersed droplets
of the extractant to settle down at the bottom of the centrifugation tube. In the second step of
this method, the resulted Fe enriched organic phase was transfer into other tube. Followed by
the addition of 0.5 mL of the (1.5 mol L−1 of HNO3) by using syringe system the centrifuged
at 2500 rpm for 1 min. In the final aqueous part was separating then analysed by FAAS. For
each step of methodology blanks were prepared simultaneously. The proposed MDLP-µE
method was applied on acid-digesting serum specimens of neurological disorders patients and
healthy controls subjects. The graphical presentation of developed method is shown in Fig. 3-
4.
Figure 3-4 Graphical representation of MDLP-µE method
ANALYTICAL CAPABILITY OF MDLP-ΜE METHOD
The linearity of the developed method for the preconcentration of Fe was studied in
ranging of 10-100 μg L−1 as shown in Tables 3-11.The EF 47 was achieved from slope of
calibration curves for the purposed MDLP-µE method. The LOD and LOQ which was
obtained to be 0.44 μg L-1 and 1.47 correspondingly. The validity of the purposed procedure is
verified to determine certified sample of serum have certified value for Fe Table 3-12. The
MDLP-µE method have good sensitivity and low detection limits which recommend to
analyze the concentration of Fe in blood serum.
64
Table 3-11 Characteristics performance of the developed MDLP-µE procedure.
Concentration range 10 – 100 µg L-1
LODa 0.44 µg L-1
R2 (coefficient of determination) 0.998
Repeatability (RSD%)b (n=10) 3.4
Enhancement factorc 47
Key: a Limit of detection (3σ/m), b Fe level 10 µg L-1 for R.S.D. was found. cCalculated as enriched samples/without enrichment.
Table 3-12 Preconcentration of Fe in certified reference material (µg L-1) by
MDLP-µE method (n=10).
CRM of Clincheck control lyophilized ® human serum
x±sa % Recoveryb Certified value
MDLP-µE 739±0.016 99.5 742±0.014
Key: aMean±S.D b % Recovery=measure values
certified value×100
3.7.6 DEVELOPMENT OF GREEN, SWITCHABLE SOLVENT EXTRACTION METHOD FOR
ENRICHMENT OF ALUMINUM IN BLOOD SAMPLES OF DIFFERENT NEUROLOGICAL
DISORDERS PATIENT
PROCEDURE OF SS-E
A three-necked flask was used for the synthesis of switchable solvent (SS) on a
stirring apparatus. The DBU/decanol equimolar mixtures at 1:1 ratio was taken in flask with
ultrapure water. The resulted mixture named as SS, have not miscible with water and non-
polar in nature, made a biphasic system Figure.3-5a. To the biphasic system added replicate
six Al standards (10 mL), in the ranging of 10-50 µg L-1, separately. Then added 0.1– 0.5 mL
of 0.125% morin. The pH of the solutions were made in the range of 4–8 with
acetate/phosphate buffers and further set by using 0.1 mol L-1 HCl/NaOH medium. The
65
stream of CO2 were bubbled slowly into the contents of three neck flask, connect with gas
diffuser. The biphasic system [DBUH]/[decanol]:water exposed to CO2 for 3-7 min, with
continued stirring in the range of 200-600 rpm at a magnetic stirring bar. The resulted
solution become homogenous as monophasic Figure.3-5b. The monophasic system
(DBUH/decanol: aqueous system), function as hydrophilic ionic liquid, considerably enhance
the extractive recovery of organo-metallic complexes from aqueous phase to polar SS micro-
emulsion, which have switchable characteristic. The polar SS/aqueous single phasic system
was converted into its individual aqueous and SS layers by heating in a water bath at 55°C,
and bubbling of N2 gas continued till the biphasic system was appear and heating till 2 layers
of biphasic system appeared Figure. 3-5c. After this step switching process was completed,
then SS was separating from the aqueous medium cautiously by a syringe, as shown
graphical abstract. To extract back the enriched Al bound with ligand (morin) in SS, was
extravagance via treating with 0.5 mL of 1.0 mol L-1 of HNO3 medium. For this purpose CO2
at pressure of 2 to 6 MPa was bubbled in acidic aqueous - SS phase with continual stirring,
till the biphasic system convert in to single homogenous phase. On treating with acidic
solution the metals can be leached out from SS, because most of the metal complexes are
unstable at low pH. Then again bubbling the N2 and heating the polar SS/water single phasic
system convert into its respective SS and aqueous layers. The SS was further used for the
subsequent experiment. While enriched Al in acidic solution was analysed by FAAS.
For validation replicate six samples of acid digested certified reference material (SRM
3101a) were subjected to proposed method. The developed procedure was applying to acid-
digested blood samples of every patients of neurological disorders (n=2) and referent male
subjects. The graphical presentation of developed method is shown in Figure 3-6.
66
Figure 3-5 Visual diagram of SS of [DBUH][decanol] in aqueous media (a) upper immiscible SS and
lower aqueous phase (b) convert to a cleared homogenous monophasic solution of SS in aqueous
medium by exposing to 4 MPa of CO2 while stirrer for 5 min at 500 rpm (C) The polar SS/water
monophasic system was separated into its biphasic layer by heating at 55°C.
Figure 3-6 Graphical representation of SS-E method
METHOD VALIDATION
The repeatability, precision, linearity, accuracy and detection limits developed SS-E
were investigated at optimum experimental conditions for its capability and efficiency. A
linear graph Al was obtained at concentration range of 10 – 50 µg L-1 with 0.9987 correlation
coefficient. The accuracy and precision of the developed procedure was estimate to analyze
replicate six standards of Al (10 µg L-1) as (%RSD), was observed to be >5%, indicate the
good reproducibility. The LOD of Al was obtained to be 0.47 µg L-1. The EF was achieved
to be 25. The authenticity of the developed SS-E procedure were check by the determination
of (SRM 3101a), applying standard addition method. The recovery of Al was found to be >
95% as shown in Table 3-13.
67
Table 3-13 Analysis of Al in certified reference material and spiked sample of blood using
(SS-E) method.
Certified sample of Blood
(µg L-1)
Certified values (µg L-1) With SS-E
(SRM 3101a) 9.6±3.15a 9.4±2.14
%Recovery --- 98.1
Sample Added µg L-1 With SS-E
AD patients 0 21.2±0.18
25 45.8±0.18
50 70.8±0.18
%Recovery 98.6 99.2
aMean±Standard deviation (x±s)
3.7.7 PRECONCENTRATION OF TRACE LEVEL MANGANESE IN BLOOD SAMPLES USING
A DEEP EUTECTIC SOLVENT EXTRACTION (DES) METHOD
PREPARATION OF DES
DES was prepared in 50-mL flasks and ZnCl2 and acetamide at different ratios (1:1,
1:2, 1:5) was added. The flasks were placed on a hot plate with magnetic stirrer at the
temperature and stirring rate of 75-85 ºC and 600 rpm respectively, until a homogeneous
colorless liquid was obtained, then kept standing at room temperature. The resulting DES is a
biphasic system which is insoluble in water as compared with individual components see
Figure 3-7. The characteristics of DES are described in the literature [197, 203, 442].
DES-ASSISTED EXTRACTION METHOD
Six replicates of each standardize solution (10 mL) having 10–50 µg L-1 of Mn2+ were
placed into individual conical flask (capacity of 25 mL). Then, (1–5 × 10 -5 mol L-1) of PAN
0.5 mL and 2 mL phosphate/acetate buffer were adding, and a pH ranging of 4–11 was
maintained with 0.1 mol L-1 HCl/NaOH. To the contents of the flask, DES was added in
ranging of 0.5 -2.0 mL and shaken in the mechanical shaker for 2 minutes to ensure the
transfer of the hydrophobic Mn-PAN into the DES-rich top phase. Then decanol was added
68
in the range of 0.2-1.0 mL. The enriched metal complex in DES was separated carefully
using a syringe and subjected to FAAS analysis.
For validation, six replicate samples of acid-digested certified human blood samples
(SRM 3132) were subjected to the proposed method. The developed method at optimized
parameters were applied to acid-digested duplicate samples of blood from each neuro patient
and healthy male subject. The graphical diagram of suggested procedure is shown in Figure
3-8.
Figure 3-7 Prepration and their checked miscibilty of DES in water
Figure 3-8 Graphical representation of DES-E method
69
ANALYTICAL FIGURES OF MERIT
The linearity of proposed DES process for the enrich Mn2+ with correlation
coefficients of 0.991–0.998, with ranging of (10 − 50 µg L-1) of standards. The LOQ and
LOD were achieved for Mn2+ as 0.98 and 0.29 µg L-1 correspondingly, in Table 3-14. The
validity of the developed procedure was verified by applying the procedure on CRM SRM
3132 Human Blood and spike recovery tests in a real sample of blood Table 3-15. The EF
was 42. The low detection limits of the proposed DES-E procedure stated that it is sensitive
and efficient for the analysis of lower level of Mn2+ in blood specimens.
Table 3-14 Characteristic Performance of the Proposed DES-E procedure.
Concentration Range 10–50 µg L-1
LODa 0.29 µg L-1
R2 0.998
Repeatability (RSD%)b (n=10) 3.4
Enhancement Factor 42
Key: a Detection Limit (3σ/m), bMn2+ level was 10 µg L-1 for R.S.D. was achieved,
Table 3-15 Determination of Mn2+ in CRM and Spiked Blood Sample using DES-E method.
CRM of Blood(µg L-1) Certified Values (µg L-1) DES-E Method
SRM 3132 Human
Blood)
20.7±3.15a 20.4±2.16
(%) Recoveryb --- 98.7
Sample Added µg L-1 DES-E Method
Parkinson’s Patients 0 23.4
10 34.5
15 38.1
20 43.4
%Recoveryb 98.1-98.6
aMean±Standard deviation (x±s) b%Recovery= Measured Value
Certified∧added values
70
3.8 SUMMARY
This section comprises the details of chemicals reagents along with
instrumentations were used during the research work was given. To collect the
Blood, scalp hair & serum specimens of patients having different
neurological/psychiatric disorders to determine the trace level of Al, Mn, & Cu,
Fe Zn in acid digested biological specimens. To assess the level of under studied
element different advance extraction methodologies were developed prior to
analyze by FAAS. The proposed procedure are authenticated by using CRM and
standard addition procedure to the real biological samples.
71
CHAPTER 4
RESULTS AND DISSCUSSION
The present investigation found that neurodegenerative and psychiatric individuals
had significant disturbances in metabolism and homeostasis of (Al, Cu, Fe, Mn and Zn). The
consequences of this work observed that excess & deficiency of these metals correlated well
with the consequences of neuro and psychiatric disorders. It is suggested that imbalances in
trace elemental level, as an effect or cause of the neuropathology, have been found.
Furthermore, the occurrence of a certain degree of oxidative damage in these patients
confirmed the idea that oxidative injury is the main factor in neurodegenerative status. To
analyze trace levels in biological specimens might be difficult, due to lower level in
complicated nature of real specimens, mostly requiring enrichment step prior to analyze by
sensitive instrumental techniques [150].
4.1 ANALYSIS OF MANGANESE IN SCALP HAIR SPECIMENS OF PD
PATIENTS.
GENERAL REMARKS
The Work has been publishing as:
M. S. Arain, T. G. Kazi, H. I. Afridi, et al., "Enrichment & analysis of Mn in biological specimens by d-CPE coupled with FAAS," Journal of analytical atomic spectrometry, vol. 29, pp. 2349-2355, 2014.
4.1.1 RESULTS
OPTIMIZING OF d-CPE METHOD
The key factors influence the extracting process, i.e. pH, level of surfactant, chelating
reagent, back-extractant HNO3, equilibrium time temperature & were optimizing.
72
EFFECT OF PH.
The pH play key role to form complex and extraction of metal. The influence of pH to
extract Mn2+ was studied by employing 6 replicate standardize media of Mn2+ (10 µg L-1) at
the pH ranging of 5–11, whereas other factors were at their optimized levels. Each required
working pH was acquired by adding of 0.1 mol L-1 of HNO3/NaOH in the manifestation of
phosphate buffer. PAN is choose to form complexes with Mn2+ at the pH range of 7–11. The
greater extracting efficiency of Mn2+ was achieved at pH 10, as shown in Fig. 4-1. The signal
for Mn2+ increasing after pH 9 and decreases after pH 10. Thus, pH 10 was selected for Mn2+
extraction in subsequent experimental work.
4 5 6 7 8 9 10 11 120
20
40
60
80
100
%R
ecov
ery
pH
Fig 4-1 Influence of pH on the %recovery of Mn2+ using d-CPE method
EFFECT OF PAN CONCENTRATION
To extract metal/metalloid PAN is employed as a chelating regent because of greater
hydrophobicity. The extracting efficacy of Mn2+ as a function of PAN amount ranging from
1–5 × 10-5 mol L-1 (w/v) Fig. 4-2. The efficacy of Mn2+ is enhanced up to 4 × 10-5 mol L-1;
more increase of PAN level causes no variations in the signals. Therefore, 4 × 10-5 mol L-1 of
PAN was select Mn2+ extraction of further work.
73
1 2 3 4 5 60
20
40
60
80
100
PAN (1-5×10-5 mol L-1 ) (w/v)
%R
ecov ery
Figure 4-2 Influence of PAN level on the %R of Mn2+ using d-CPE method
TRITON X-114
This surfactant was selecting due to it is commercially obtained in a highly purifying,
homogeneous form, lower toxicological property & greater density, which assists phase
isolation by centrifugation. Their level influence on percentage recovery of Mn2+ in Fig. 4-3.
The variant in extracting efficacies within the Triton X-114 ranges 0.1–0.5 percent v/v was
investigated. The optimum quantity of Mn2+ complex was extracting at 0.2% (v/v).
0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.550
20
40
60
80
100
%R
ecov
ery
Triton X-114 % (v/v)
Fig 4-3 The effects of the surfactant Triton X-114 on the %recovery of Mn2+ using d-CPE method
74
INCUBATION DURATION & TEMPERATURE
The extracting efficacy of the complexed element is achieved while the CPE
procedure is occurred at the equilibrium temperature above the CPT of the (Triton X-114)
surfactant. In the current work, the equilibrium temperature of the ultrasonic bath was
studied in ranging of 35–60 °C and observing that 45°C was sufficient. The dependency of
extracting efficacy upon equilibrium time was study for a time duration of 2–20 min. An
equilibrium time of 10 min was selection to extract Mn2+. In the 2nd CPE part, the time &
ultrasonic bath temperature were also 10 min and 45 °C correspondingly.
BACK-EXTRACTANT
In the 2nd CPE part, the influence of different extracting acidic was observed. For
this work, HCl and HNO3 of 0.5 to 2.0 mol L-1 were employed for the back-extracting Mn2+
in aqueous part from its hydrophobic form entrapped in micellar media (Triton X-114). The
greater recovery was achieved at 1.0 mol L-1 of HNO3; a lower recovery of Mn2+ was
obtained in 1.0 mol L-1 HCl at 10–20%. In 2nd part of d-CPE, nitric acid at 1.0 mol L-1 was
choose for back-extractant of Mn2+ ions into the aqueous part.
INTERFERING IONS
The influence of matrixes ions were analyzed in the efficient extraction of Mn2+ by d-
CPE. To achieve this work, 10 mL solutions having 10 µg L-1 of Mn2+ by added co-
existing ions (K+, Na+, Ag+, Mg2+, Ca2+, Co2+, Zn2+, Ni2+, Cu2+, Al3+, Fe3+) at diverse
interferent-to-metal ratios were used to the established method. The amounts of interfering
ions were analysed according to the element-to-interferent ratios (w/w) of 1 : 1000 for K+,
Na+ ; 1 : 800 for Ca2+, Mg2+; 1 : 25 for Co2+, Zn2+, Ni2+; 1 : 20 for Ag+, Cu2+; 1 : 30 for Fe3+
and 1 : 30 for Al3+. Table 4-1 shows the tolerance limits for each interfering ion. Usually
encountered matrix components, includes IA and IIA elements, normally do not forming
stable complexes with PAN. Thus, the established procedure has good selectivity for Mn2+.
75
Table 4-1 Influence of selected foreign ions on the % recoveries of the Mn2+
analysed by applying the d-CPE procedure.
Ion Tolerance limit (mg L-1)
Na+ 1000
K+ 1000
Ca2+ 800
Mg2+ 800
Co2+ 25
Ag+ 20
Zn2+ 25
Al3+ 30
Fe3+ 30
Ni2+ 25
Cu2+ 20
4.1.2 APPLICATION
The developed method was employed for Mn2+ analysis in scalp hair
specimens of healthy referents and Parkinson's patients of the same age group. The
resulting data indicate that the levels of Mn2+ in scalp hair samples of Parkinson's
patients are significantly greater than in controls age-matched Table 4-2. The amount of
Mn2+ in scalp hair specimens of male & female patients was observed to be significantly
greater at confidence intervals 95% CI (9.64–10.5) µg g-1 versus referents CI (3.65–4.09)
µg g-1. It was also found that the Mn2+ content in scalp hair samples of females was
greater than in male PD patients, however difference was not significant (p > 0.05). The
neuro toxic effect of Mn2+ has been well-known since the last century as manganism; this
has been describes & characterized by extra-pyramidal dys-function and
neuropsychiatric symptoms. Since then, this disease has been observing during the world
in 100 of cases between industrial, miners employers who had been exposed to greater
concentration of Mn2+ [443].
Mn entering in the human system mainly through inhalation and damaging the
CNS and respiratory system and also have severe influence on neural health.
Occupationally exposing to Mn2+ happens largely in alloy producing, mining, &
processing, ferro-Mn operations, welding, and working with agrochemicals; between the
76
neurologic influences is an irreversible, Parkinsonian disorder [63]. In PD, death of
neuron which produce dopamine in SN causes to decreases dopamine supply &
compromises the capability of brain effected the movement. PD is clearly age-dependent
and progressive, possibly due to its growing oxidative destruction and steady decreasing
in antioxidant capability. An assessed 500, 000 to 1.5 million people in the US have PD,
and physicians required to consider Mn exposure in its differential diagnosis [100].
The analytical performance of the suggested procedure applied the complexing agent
PAN for the analysis of Mn2+ in scalp hair specimens of PD patients & Normal controls
were compared to previously suggested methodology, with and without preconcentration of
Mn2+ in different matrices and using different spectrophotometric techniques Table 4-3. The
enhancement factor of the present study is comparable with literature-reported work using
ICP-OES and ICP-MS [115, 142, 146, 383, 444-447]. The stated data illustrate that the
diverse analytical factor, LOD and EF are superior to those of instrumental techniques. The
obtained LOD using the ligand was adequately lower as to be valuable for identifying Mn2+
in diverse samples.
Table 4-2 Quantity of Mn2+ in scalp hair specimens of PD patients & healthy control subjects (µg g-1).
Subjects Male Female
Healthy controls 3.68±0.52 3.89±0.43
PD patients 9.83±0.67 9.98±0.56
P value <0.001 <0.001
77
Table 4-3 Comparative data of analytical parameters for Mn2+ with and without
preconcentration methods coupled with different Instrumental techniques
Preconcentration
Methods
Element Technique Reagent Sample LOD EF Ref:
CPE Mn ICP-OES TTA Water 0.1-2.2
µg L-1
42-
92[142]
CPE ICP-OES PAN Materials rich
in calcium
0.3 µg L-
1
….. [445]
….. ICPMS Multivitamin 9.0 ng L-1 …
….[446]
….. ICPMS Serum and
saliva
0.07
ng mL-1
…
….[447]
…… ICPMS Human hair
and nails
0.006
µg g-1
…
…[115]
….. ICPMS Biological
samples
0.003
µg g-1
…
…[444]
CPE FAAS PAN Natural water 5 µg L-1 ….. [146]CPE GFAAS PMBP Water 0.02
ng mL-1
31 [383]
d-CPE FAAS PAN Scalp hair 0.097 µg
L-1
46 Present
work
Key: a1-(2-thenoyl)-3,3, 3-trifluoraceton reagent (TTA), 1-(2-pyridylazo) - 2-naphthol (PAN), inductively coupled plasma optical emission spectrometry. (ICP-OES), inductively coupled plasma mass spectrometry (ICPMS), 1-phenyl-3-methyl-4-benzoyl-5-pyrazolone (PMBP). Cloud point extraction (CPE), Dual-Cloud point extraction (d-CPE), Flame atomic absorption spectrometry (FAAS), Graphite furnace absorption spectrometry (GFAAS)
78
4.2 ZINC LEVELS IN SERUM SAMPLES OF PSYCHIATRIC PATIENTS
GENERAL REMARKS
The work has been accepted in “Journal of Industrial and Engineering Chemistry” as:
M. S. Arain, T. G. Kazi, H. I. Afridi, et al., "Variation in zinc levels in serum samples of
psychiatric patients using dual cloud point extraction.
4.2.1. OPTIMIZATION OF d-CPE PROCEDURE
The parameters that mainly affects the extracting procedure, including level of
surfactant pH, complexing agent, equilibrium temperature, time and nitric acid as back-
extractant were studied.
EFFECT OF PH
The extent of %R of studied analytes based on pH values where the complexation of
metals formed. The outcome of pH effects on the extracting efficiency of Zn2+ ions was
working in ranging of 5−11 applying a replicate standard solution of analyte (n=6) for 20 μg
L-1. Whereas other factors were set at their optimal values. The pH range of solutions were
made by acid/base solutions such as 0.1 mol L-1 of NaOH/HNO3 in phosphate buffer. The
PAN is efficient to formed complexes with Zn2+ at the pH range of 5–11. The greater
extracting efficiency of Zn2+ was observed at pH 8, as shown in Fig. 4-4. The signal for Zn2+
enhance at pH 8 and reduced after pH 8. Thus, pH 8 was selected for Zn 2+ extraction in
subsequent experimental work.
4 6 8 10 120
20
40
60
80
100
%R
ecov
ery
pH
79
Fig 4-4 Influence of pH on preconcentration of Zn2+ by d-CPE
PAN CONCENTRATION
Ligand (PAN) was used for complexation with studied analyte Zn2+ using proposed d-
CPE method. The selected ligand have beneficial characteristic such as made metal/metalloid
complex with highly hydrophobic in nature. The extraction efficiency of Zn2+ based on the
concentration of PAN ranging from 0.2−0.7 mL of 4× 10-5 mol L-1 (w/v) is presented in Fig.
4-5. The recovery of Zn2+ is enhanced up to 0.5 mL, further increase of PAN concentration no
any changes in the signals was observed. Hence, 0.5 mL of PAN was chosen for quantitative
extraction of Zn2+ for subsequent experimental work.
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.80
20
40
60
80
100
PAN 4×10-5 mol L-1 (0.2-0.7 ml)
% R e c o v e r y
Figure 4-5 Influence of PAN level on % recovery of Zn2+ by d-CPE
EFFECT OF SURFACTANT VOLUME
The non-ionic surfactant (Triton X-114) was selecting due to its accessibility in a well
purifying form, lower toxicity & high density, which made possible part separating by
centrifugation. The effects of Triton X-114 level on % recovery of Zn2+ in Fig. 4-6. The
variant in extracting efficacies within the Triton X-114 ranges 0.1–0.5 percent v/v was
investigated. Therefore 0.2% of surfactants was used for optimum recovery of Zn2+.
80
0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.550
20
40
60
80
100
%R
ecov
ery
Triton X-114 % (v/v)
Fig. 4-6 Influence of surfactant level on %recovery Zn2+ by d-CPE
DURATION AND TEMPERATURE OF INCUBATION
The efficacy of proposed method was studied at the equilibrium temperature,
greater than CPT of the Triton X-114 to optimized these parameters ultrasonic bath was,
where equilibrium temperature and time were studies at 40 to 60 °C and 2 to 20 min
respectively. It was found that 50°C and 10 min were sufficient for optimized recovery of
studied analyte.
BACK-EXTRACTING REAGENTS
The effect of different extracting acidic reagents were worked for back extraction of
analytes from surfactant phase termed as d-CPE phase. For this purpose, HNO3 and HCl in
the ranged of 0.5 -2.0 mol L-1 were using to extracting back the analytes from its hydrophobic
form present in micellar medium. It was observed that maximum extraction of Zn2+ was
achieved at 1.5 mol L-1 of HNO3, whereas at same concentration of HCl, the recovery was
reduced 10 to 20%.It was observed that % recovery of zinc was decreased for conventional
CPE as compared with d-CPE, might be due to the effect of matrix contain Triton X-114
Table 3-6.
EFFECTS OF IONS IN MATRIX
The matrix ions effect were studied for the competent extraction recovery of Zn+2 by
proposed, d-CPE. To carry out this study, 20 μg L-1 Zn2+ in (10 mL) were added with co-
existing (Ca2+, Co2+, Fe3+, K+, Na+, Ag+, Mg2+, Ni2+, Cu2+, Al3+) at diverse element to
interferent ratios and applying the established procedure. The ratios (w/w) of studied analyte
and matrices ions were set according to 1 : 1000 for K+, Na+ ; 1 : 800 for Ca2+, Mg2+; 1 : 25 ,
Ni2+; 1 : 20 for Ag+, Cu2+; 1 : 30 for Fe3+ and 1 : 30 for Al3+.The acceptance limits for recovery
81
of Zn−PAN complex with various foreign ions was found to be <5%. The alkali and alkaline
earth elements are not counted as matrix components, because they forms unstable complexes
with PAN. Therefore, the proposed procedure is better selectivity for trace levels of Zn2+.
4.2.2 APPLICATION
The proposed procedure at optimum values of different variables was employed to
analyze Zn2+ trace levels in serum specimens of PSD patients (schizophrenia, depression,
bipolar disorder) and healthy referents of age-matched. The resulting data indicate that the
Zn2+ levels in serum samples of PSD male patients are significantly lower than the controls
Table 4-4. At 95% confidence intervals the ranges of Zn2+in the serum samples of male PSD
patients were observed to be (CI) for schizophrenia (CI 0.292–0.310 mg L -1), depression (CI
0.255–0.281 mg L-1), bipolar disorder (CI 0.217–0.239 mg L-1) versus controls (CI 0.338 –
0.489 mg L-1). It was reported that due to insufficiency of Zn2+ causes mental action, learning
behavior, and the susceptibility to different patients have psychiatric disorders. Zinc
deficiency may influence alter its homeostasis in the brain created different dysfunctions.
Consequently, for proper brain functioning and vesicular Zn2+ is an essential nutrient for
neuronal signaling factor [448]. In neurodegenerative and psychiatric disorders, the levels of
Zn in plasma is varied than normal values. Zinc deficiency is also related with neurological
disturbance [354, 355] which might be main reason to disturb aged peoples [449].
The effect of aging have considerable psychiatric diseases i.e. Alzheimer's, bipolar
Parkinson’s and schizophrenia disorder. It is generally stated that deficit levels of Zn2+ in
food is a major dietary problem in different countries; which might be resulted into
impairment of cognitive functions in addition to delay in growth [450]. Whereas the all
disabilities due to only Zn2+ deficiency are not true because may be due to variation in
nutritional habits and environments of study population. Nevertheless, additional
investigation must be necessary to search other nutritional and physiological parameters of
any study population for better interpretation of adverse impacts of Zn2+ deficiency.
82
Table 4-4 The quantity of Zn2+ in serum samples of PSD male patients & healthy control
subjects (mg L-1).
Element Healthy control
(n =60)
Schizophrenia
(n= 20)
Depression
(n= 20)
Bipolar disorder (n=15)
Zn+2 (mg L-1) 0.423±0.08 0.318±0.02 0.273±0.014 0.234±0.012
P= 0.01 – 0.001
4.3 ANALYSIS OF AI IN SCALP HAIR SPECIMENS OF AD PATIENTS BY
ADVANCE EXTRACTION METHODOLOGY: A MULTIVARIATE STUDY
GENERAL REMARKS
The work has been published as:
M. S. Arain, S. A. Arain, T. G. Kazi, H. I. Afridi, et al., "Temperature controlled IL-based dispersive micro-extraction using two ligands, to determine Al in scalp hair samples of AD patients: A multivariate study," Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, vol. 137, pp. 877-885, 2015.
4.3.1 OPTIMIZATION OF EXPERIMENTAL FACTORS
Considering the TIL-DLLME procedure for the analysis of Al3+ in aqueous extracts
of scalp hair specimens employing 2 chelating agents, five factors, volume of ionic liquid
(IL), ligands concentration (L1), (L2), and incubating time (It), pH (P) were choose to
optimizing the percentage recovery of Al3+ by multivariate method. The PBD matrixes in
Table 4-5. Where the lower (−) and higher (+) levels are those specifying in Table 3-7. The
influence of altering the parameters from lower to higher level values was examining on a
response selecting i.e. % recovery, base on one point standardize adding procedure (50–100
µg L-1). The resulted data of both ligands were estimated by analysis of variance
(ANOVA), in Table 4-5, Table 4-7.
Where the %R of Al+3 was described as the dependent factor and the 5 selection of
factors as independent. The significant influence related to ionic liquid volume (factor A),
83
pH (factor C) and concentration of both ligands (L1 and L2). These effects show p<0.05,
represents that they are statistically significant at a 95.0 percent confidence level.
Therefore, the influence of these parameters were study more carefully. From the
results of PBD Table 4-5, it is obviously found that, for Al3+ recovery, more significance
influence were observing for variables includes ionic liquid, chelating agent (oxine and
morin) level and pH Table 4-5 showing that the greater recovery of Al3+ was obtained at (+)
level of L1, L2 and IL and (−) level of pH. These variables have significant influence on the
recovery of Al3+ (p < 0.01). It can be seen in experiment two and nine of Table 4-5 that at
(−) level of pH, while (+) levels of IL, L1 and L2, the percentage recovery of Al3+ was 80%
and 76%, respectively. The high pH value have negatively effecting on the %recovery of
Al3+, as found in experiment 2 and 5, while the other significant factors IL and L1 were at (+)
levels, the % recovery of Al3+ was only 25%. The 2 order interactions among variables after
multivariate optimized results in Table 4-7 showed that L2 (morin) and P (pH) have high
effects on the recovery of Al3+, but this interaction effects is not significant (p = 0.07). The
estimated influence and interacting parameters, is shown in Table 4. For both ligands, the
greater significant estimated effect was obtained for variable IL. The smallest main effects
were observe for variable (R2, It) and (R1, It) for L1 and L2 respectively. The most relevant
interaction between two variables was seen for (R1, P) and (R2, P), while the least interaction
was achieved in IL and P for both ligands. Therefore, it can be concluded that the ionic
liquid, ligands concentrations and pH of exhibited significant influence on extraction
efficiency of Al3+. Though, the effect of incubation time (It) is less important in order to
achieve quantitative recovery of Al3+.
84
Table 4-5 Plackett–Burman design for the significant variable analysis
(n=5)
Experimen
tIL R1 R2 P It %Recovery
R1 R2
1 + _ + _ _ 43±1.3 64±2.1
2 + + _ + _ 40±2.9 36±2.6
3 _ + + _ + 43±1.4 30±2.2
4 + _ + + _ 25±2.3 34±2.5
5 + + _ + + 47±1.2 25±0.9
6 + + + _ + 80±0.5 76±0.7
7 _ + + + _ 26±2.5 24±1.3
8 _ _ + + + 18±1.9 19±1.6
9 _ _ _ + + 10±0.85 24±1.2
10 + _ _ _ + 48±1.6 36±2.2
11 + _ _ _ 45±2.4 20±0.9
12 + _ + _ _ 10±2.7 16±2.3
OPTIMIZING BY CENTRAL 23+ STAR ORTHOGONAL COMPOSITE DESIGN (CCD).
After screening the parameters those did not have important influence on the %
recovery of Al, the remaining factors were optimized to give its optimum results. A CCD
with 6 DF and includes sixteen experiments was carry out to optimizing the variables i.e.
both ligands (L1 and L2), which have significant influence on the percentage recovery of Al3+,
and have a strongly interacting with ionic liquid (IL) and pH (P) as indicated in Table 4-7. So
variable of both reagents, IL and pH were optimized to give the higher recovery of Al3+. The
85
variable have insignificant influence as shown Tables 4-5 and 4-7, was fixed at optimum
level i.e., incubation time (3 min). The experimental field definition for this design is given in
Table 3-7, while Table 4-6 shows the CCD together with the % response acquired for Al3+
with both ligands. It was found that at lower level of L1 (−) the recovery of Al3+ was <35%
(experiments 2, 4 and 6), while it was 39–43% at (+) level of IL (experiment 7). The higher
recovery of Al3+ was achieved at average levels of all 3 variables (IL0, R10 /R20 and P0)
(experiments one & sixteen). The ionic liquid was also have significant effects (p < 0.01), on
entrapping the metal complexed with both ligands. The less volume of ionic liquid has -ve
influence on the % recoveries of Al3+ in (experiments 4, 6 and 8). The optimum entrapping of
Al3+ complex was observed at 70 µL of IL (experiments 1 and 16). The pH is also consider as
another main parameter for metal-complex forming. The results shows that higher recovery
of Al was achieved at P0 i.e. pH 6 (experiments 1 and 16), while, at (−) levels <45%
recoveries was found with the combinations of different values of other variables. It is
widely recognized that pH play vital part to form stable metal-complexing reagent &
extracting. So it is important to analyze pH that will give higher complex formation. In this
experiment, the influence of pH upon the extraction of Al3+ ions from the solution was
worked within the ranged of 4–8 by adding accurate volumes of 0.1 mol L -1 HCl /NaOH
media with acetate buffer to the samples. As indicated in Table 4-6 at optimum pH value the
maximum extraction efficiency was obtained for both ligands. The decreased analytical
signal at higher pH values because of hydroxide forming of aluminium ions, consequential in
reduced quantity of free Al3+ ions in sample media.
So, in order to maintaining a constant working pH that allows complex formation and
stability, pH was maintained at six in following experiments. The study of estimated three
dimension (3D) surfaces response for variables ([L1–IL], [L1–P]) and ([L2–IL], [L2–P]) was
assessed by quadratic equation, indicated that the 100% recovery of Al3+ will be obtained at
concentration of complexing reagent L1 (0.280 mL/4.35 × 10-5 mol L-1), pH (6.5) and IL
volume (64.5 µL) Figs. 4-7a and 4-7b. While for L2 (0.288 mL/3 × 10-6 mol L-1), pH (6.0) and
IL volume (69.1 µL) were required in Figs. 4-8a & 4-8b.
86
Table 4-6 Central 23 +star orthogonal composite design (n = 16) for the
set of (IL), (L1) and (P)
Experiments A (IL) B (R1/R2) C (P) % Recovery
R1 R2
1 ILo R1o/R2
o Po 96.8±1.4 95.2±0.9
2 _ _ _ 25±2.3 22±2.5
3 + _ _ 35±2.7 32±1.8
4 _ + _ 32±0.8 30±1.2
5 + + _ 42±1.3 38±2.4
6 _ _ + 28±3.2 25±2.8
7 + _ + 43±2.6 39±2.9
8 _ + + 38±1.1 34±1.4
9 + + + 40±2.4 38±2.1
10 IL1 R1o/R2
o Po 16±2.2 14.5±2.4
11 IL2 R1o/R2
o Po 72±1.6 65±1.2
12 ILo R11/R2
1 Po 8±0.9 8±0.7
13 ILo R12/R2
2 Po 75±2.1 69.4±2.6
14 ILo R1o/R2
o P1 18±3.2 15±3.5
15 ILo R1o/R2
o P2 14±1.6 10±1.2
16 ILo R1o/R2
o Po 98.7±0.8 97.6±1.1
IL1 = 19.5 µL, IL2 = 120 µL, ILo = 70 µL, R11/R2
1= 0.0363 mol L-1, R22/R1
2 = 0.636 mol L-1, R1
o/R2o= 0.3 mol L-1, P1 = 2.64, P2 = 9.36, Po= 6
87
Table 4-7 The estimated Effects and Interaction of variables by ANOVA for
recovery test
Oxine Morin
Sources aD bSS cMS dF p SS MS F P
IL 1 145 172 59.7
0.00
5
128 670 16.5 0.027
R1 1 867 118 40.9
0.00
8
133 86.
8
2.13 0.24
R2 1 75 16.2 0.56
0.50
8
104 663 16.3 0.027
P 1 936 864 30
0.01
2
867 565 13.9 0.034
IT 1 133 12.8 0.44
0.55
3
120 3.4
7
0.09 0.789
IL*R1 1 238 8 0.28
0.63
5
220 66.
7
1.64 0.291
IL*p 1 187 336 11.7
0.04
2
123 123 3.01 0.181
1 267 267 9.27
0.05
6
306.3 306 7.52 0.071
88
Residual
Error3 86.3 28.8 122
40.
7
Total 11 424 422
Fig. 4-7a 3D surface response for %recovery of Al3+ by TIL-DLLME.
Interaction among ionic liquid [IL (µL)] and oxine [L1 (mol L-1)]
89
Fig. 4-7b Interaction between IL (µL) and pH for L1.
Fig. 4-8a 3D surface response for %recovery of Al3+ by TIL-DLLME.
Interacting among IL (µL) and morin [L2 (mol L-1)].
90
Fig. 4-8b Interaction between IL (µL) and pH for L2.
INTERFERENCES
To assess the selectivity of the established procedure for the analysis of trace amount
of Al3+ the influence of some IA, IIA and transition elements on the recovery of Al3+ ions was
examined. The results are given in Table 4-8. The interference work were those relating to
the enrichment step, i.e. cations that can reacting with (oxine) and (morin) and may formed
chelate with Al3+ and decreasing extracting efficacy. An ion was measured as interferent
which leads to varying the absorbance of the sample greater than ±5%. The tolerance limits
of several foreign ions in the recovery of Al3+ with L1 (oxine) was <5%. In the case of Al-
morin complexes the %recovery was <95% due to interferences of Fe3+ and Mn2+.
Table 4-8 Effects of the matrix ions on the recoveries of the Al3+
Foreign ion Concentration (mg L-1) % Recovery
L1 L2
Na+ 5000 100 99
91
K+ 1000 102 100
Ca2+ 500 99 99
Mg2+ 500 99 98
Cd2+ 50 98 94
Fe3+ 10 94 92
Ni2+ 10 97 95
Cu2+ 5 97 94
Mn2+ 5 99 94
Cr3+ 1 100 99
CH3COO− 1 99 99
PO4−3 1 99 98
4.3.2 APPLICATION
The optimized developed methodologies were applied to the analysis of Al3+ in
triplicates acid digested scalp hair specimens of AD patients and controls. The achieved
results indicates that the metabolism & accumulation of Al3+ are changed in AD patients
which can be an important feature in the pathogenesis of AD. The mean concentrations with
standard deviations of Al3+ in scalp hair are shown in Table 4-9. In referents the Al3+ levels
found in the range of (9.4–15.5 µg g-1), while AD patients have (23.4–33.6 µg g-1). The
multiple logistic regression analysis was applied to evaluate the significant different levels of
Al3+ in AD patients with related to non-diseased subjects of age-matched. The odds ratio for
AD patients to referents was higher at 95% confidence interval, 0.375 (Cl: 0.174–0.807) with
p < 0.01. The distribution of Al3+ resulted data of referents and AD patients was checked by
the Shapiro–Wilk test for normality. It was identified that difference was found among
normal and log normal distribution but it was not significant (p > 0.05). The unpaired
Student’s t-test between referents and AD patients at diverse DF and probabilities was
calculated. Our calculated tvalue exceeds that of tcritical value (2.12 ± 0.1) at the 95% confidence
intervals, which shows that the difference among values Al3+ in referents and patients, have
significant differences (p < 0.01). The unpaired student t-test at different DF among AD
Patients and referents were calculated at different probabilities. Our calculated tvalue exceeds
that of tcritical value at 95% confidence intervals, which shows that the difference among means
values Al3+ in referents and AD patients showed significant differences (p < 0.001). There are
obvious difficulties in a case-control study of Alzheimer’s patients that relies on people’s
92
memory to establish exposures, and there was no ideal control group for such a study. So in
present study we compare the level of Al3+ in scalp hair samples of AD patients and normal
referents have no any neurological disorders of same age and socioeconomic status. Few
prior epidemiologic work have stated and link among Al3+ from dementia and drinking water
[451]. Though, there is higher disagreement about these findings and their analysis, in
specific owing to in recent time’s published epidemiologic work that failed to find a link
[452, 453]. Other causes of exposed to Al3+ have been investigated [454] showing an
association among exposure to Al3+ powder and intellectual loss, however more recently
[455] failed to finding a link among occupational exposed to Al3+ and AD.
Due to the abundance of Al that highly contaminate environment it is not exist in
pure form always present in combine with other elements i.e. silicate, hydroxide, phosphate
and sulphate. The extensive spreading of this element confirms the possible for causing
human exposure and harm [456]. It has been purpose that there is a connection among higher
quantity of Al3+ and increases risk of a number of neurological diseases i.e. dialysis
encephalopathy, PD & AD [457].
Table 4-9 The concentration of Al3+ in scalp hair samples of referents and
AD patients using TIL-DLLME Method
Element Referents (90) Diseased (110) p-Value
Al3+ (µg g-1) 11.3 ± 2.03a 24.5 ± 3.02 <0.001
aMean ± standard deviation (x ± s).
COMPARING WITH OTHER ENRICHEMENT PROCEDURES
The analytical characteristics of developed method employing 2 chelating agents
(oxine and morin) for the analysis of Al3+ in scalp hair samples of AD patients and healthier
referents was comparing with prior reported enrichment procedures of Al3+ in diverse
matrixes Table 4-10. The EF obtained in this study are comparable with literature reported
works [154, 377, 406, 458-461]. The reported data demonstrated that the different analytical
93
factors, LOD and EF are superior to those of instrumental techniques. The achieved LODs
employing 2 chelating agents were adequately lower as to be valuable for detecting Al3+ in
diverse samples.
94
Table 4-10 Comparative data of analytical characteristics of TIL-DLLME for Al+3
with previous reported preconcentration techniques
Method Reagent Surfactant/ solvent
Technique Sample EFa LODb Ref
DLLME Morin 1-undecanol ICP-OES Water 128 0.8 µg L−1 [154]
DLLME Oxine chloroform + acetonitrile GFAAS Urine 0.3 µg L−1 [458]
CPE PMBP Triton X-114. GFAAS
biological and water
sample37 0.09 ng
mL−1,
[377]
CPE Xylidyl Blue
Triton X-114. FAAS water 50 1.43 μg L−1 [461]
CPE PAN Triton X-114 GFAAS human albumin 34.8 0.06 ng
mL−1[460]
CPE ECR Triton X-114
ETAAS,UV-visible
spectrophotometry
Water sample
0.03 ng mL-
1
0.01 mg mL-1
[459]
(IL- DLLME) Oxine [Hpy][PF6]
ionic liquid SFS
water, fruit juice and food samples
100 0.05 µg L−1
[406]
TIL-DLLME
Oxine [C4MIM][PF6]
FAAS Scalp hair 85 0.56 μg L−1 This work
Morin 73 0.64 µg L-1
Keys: aEnhancement factor, bLimit of detection, Dispersive liquid–liquid microextraction (DLLME), Inductively coupled plasma-optical emission spectrometry (ICP-OES), Eriochrome Cyanine R (ECR) ,cloud point extraction(CPE), flame atomic absorption spectrometry (FAAS), electrothermal atomic absorption spectrometry (ETAAS), 1-phenyl-3-methyl-4-benzoyl-5-pyrazolone (PMBP), 1-(2-pyridylazo)-2-naphthol (PAN), Ionic liquid-based dispersive liquid–liquid micro-extraction (IL-based DLLME), stopped-flow spectrofluorometry (SFS), 8-hydroxyquinoline (oxine), 1-butyl-3-methylimidazolium hexafluorophosphate, [C4MIM][PF6].
4.4 DETERMINATION OF TRACE LEVEL OF COPPER IN SERUM SAMPLES OF
PATIENTS HAVING NEUROLOGICAL DISORDERS
GENERAL REMARKS
The work has been published in the journal of “Ultrasonics Sonochemistry” as:
M. S. Arain, T. G. Kazi, H. I. Afridi, et al., "Ultrasound energy is used to extract trace level of copper in serum samples of patients having neuro disorders," Ultrasonics Sonochemistry, vol. 37, pp. 23-28, 2017.
95
4.4.1 OPTIMIZATION OF EXPERIMENTAL FACTORS
Various variables that affect the developed microextraction method, were optimizing
i.e. (complexing agent concentration, pH, and volume of IL, sonication time, and, time and
rate of centrifugation).
EFFECT OF pH
Replicate six standard solution for the extraction of Cu ion (10 μg L-1) was used to
worked the pH influence in the ranged of 4 –10. Whereas optimized values were used for
all other variables. Each desired working pH was maintained by adding of 0.1 mol L -1 of
NaOH/HCl. Cu ion form complex with PAN selectively at the pH range of 4–10. The
highest recovery of Cu ion was observed at pH 6, whereas at higher pH the signal for Cu
ion was decreases. Therefore pH 6 was preferred for the quantitative extraction Cu ion as
shown in Fig. 4-9.
2 3 4 5 6 7 8 9 100
20
40
60
80
100
pH
% Re
cov
ery
Fig. 4-9 Influence of pH on preconcentration of Cu ion by UDIL-μE
PAN CONCENTRATION
For the developed UDIL-μE methodology, PAN was used for the complex formation
of analyte (Cu2+). The concentration ranging from 1 - 5 × 10-5 mol L-1 of PAN was studied
for the recovery of Cu ion in Fig. 4-10. It was found that quantitative recovery was obtained
at 4 × 10-5 mol L-1 of complexing agent and by further increase in the concentration of PAN
didn’t show any significant effect. The PAN is an effective reagent for enrichment of Cu
due to its higher hydrophobic and amphiphilic properties and form stabile complex with Cu
ion.
96
0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.50
20
40
60
80
100
PAN ( 1×10-5 mol L-1 )
% R ec o v er y
Figure 4-10 Effect of PAN concentration on %recovery of Cu ion
by UDIL-μE
AMOUNT OF IL
For the efficiency of developed procedure amount of IL is significant .The lowest
possible IL volume required for maximum enrichment factor of the proposed UDIL-μE
methodology. The variation in extraction recovery of analyte using IL as extractant was
studied ranging from 50 –200 μL. The figure indicates the maximum recovery of Cu ion was
achieved at 100 μL of IL as shown in Fig. 4-11.
0 100 200 3000
20
40
60
80
100
Amount of IL (µL)
% Re
co ve ry
Fig. 4-11 Effect of quantity of IL on % recovery Cu ion by UDIL-μE
97
SONICATION TIME
To optimized the impact of sonication time in the range of 10-60 sec at <40 °C. It was found that the highest recovery of Cu ion achieved at 20 sec as indicates in Fig. 4-12.
0 10 20 30 40 50 60 700
20
40
60
80
100
Sonication time (sec)
% R ec o ve ry
Fig. 4-12 Effect of sonication time on %recovery Cu ion by UDIL-μE
CENTRIFUGATION TIME AND RATE
These factors are significantly effects the extraction recovery of analyte by developed methods. In present study the centrifugation time and rate were selected in the range of 5-25 min and 500-4000 rpm respectively. The higher recovery of Cu ion was observed at 15 min and 3500 rpm of centrifugation time and rate respectively.
EFFECT OF MATRIX ION
The interference study of cations and anions on developed method was carried out by addition of different cations in 10 μg L-1 of Cu ion (10 mL) at different ratios (w/w) of 1: 1000 for K+, Na+; 1: 800 for Ca2+, Mg2+; 1: 25 for Co2+, Zn2+, Ni2+; 1: 20 for Ag+; 1: 30 for Fe3+ and 1: 30 for Al3+. The acceptance limits for recovery of Cu−PAN complex with various foreign ions was found to be <5% for adding interfering metals ions.COMPARISON WITH OTHER PRECONCENTRATION METHODS
98
The analytical characteristics of the established method employing the chelating agent
PAN for the analysis of Cu ion in serum samples of neurological disorders patients &
healthier referents were compared to previously reported methods as shown in Table 4-11
[175, 388, 410, 413, 462-468]. The suggested methodology is easy and fast emulsifying, to
use the ultrasound radiation which accelerates to migrate analytes IL and also increasing the
extraction yields. The reported data illustrate that the different analytical parameters, LOD
and EF are superior to those of instrumental techniques. The obtained LOD using the ligand
was adequately lower as to be valuable for noticing Cu ion in diverse specimens.
Table 4-11 Comparative data of analytical characteristics of UDIL-µE for Cu ion
with previous reported preconcentration techniques
Method Reagent Surfactant/solvent
Technique Sample EFa LODb
(µg L-1)RSDc Refs:
SMF-mSPE
APDC [C4MIM][PF6]
FAAS serum 0.304 <5 [462]
USAE–SFODME
PAN 1-dodecanol
FAAS water 12.5 0.76 3.83 [413]
IUSADLLME
DDTC UV-visible water 222 0.05 ng mL–1
3.3 [410]
Solid phase preconcentration method
methylthymolblue
FAAS River or waste water
0.54 ng mL–1
1.4[464]
CPE TAN Non-ionic (Triton X-114).
FAAS water 64.3 0.27 ng mL–1 [175]
SDME Spectrophotometry
Food and water
33 0.15ng ml-1
3.4 [468]
LLE Spectrophotometry
Water and soil
5 2.0-4.0 2.0 [465]
DLLME FAAS water 42–48 3.0 5.1 [388]Co-precipitation
FAAS water 20 1.32 2.5[467]
SPE FAAS food 33 1.9 2.1 [463]DLLME FAAS Cereals
vegetables55 0.05 1.5-
3.5[466]
UDIL-µE PAN [C4mim][PF6]
FAAS Serum 31 0.36 µg L-1
3.3 Present work
Key: Syringe membrane filter solid phase microextraction (SMF-mSPE), Ultrasound-assisted emulsification solidified floating organic drop microextraction (USAE–SFODME), Injection-ultrasound assisted dispersive liquid–liquid microextraction (IUSADLLME),Cloud
99
point extraction (CPE), single drop microextraction (SDME), liquid–liquid microextraction (LLE), Dispersive liquid–liquid microextraction (DLLME), Solid phase extraction (SPE), 1-(2-pyridylazo)-2-naphthol (PAN), ionic liquid 1-butyl-3-methylimidazolium, [C4MIM][PF6], flame atomic absorption spectrometry (FAAS), 1-(2-thiazolylazo)-2-naphthol (TAN), 1-phenyl-3-methyl-4-benzoyl-5-pyrazolone (PMBP), Ammonium Pyrrolidinedithiocarbamate (APDC), Diethyldithiocarbamate (DDTC), ionic liquid based on ultrasound assisted microextraction (UDIL-μE) aEnhancement factor, bLimit of detection, cRelative standard deviation.
4.4.2 APPLICATION
The presented method was used for Cu ion analysis in blood serum of neuro patients
and healthy age matched referents. The resulted data indicated the level of Cu ions in blood
serum of patients having different neurological disorders have greater values than age
matched subjects have no any neurological disorders Table 4-12. The concentration of Cu ion
in blood serum of different neurological disorders was achieved to be greater level at 95
percent confidence intervals (CI) for Alzheimer’s (CI: 1600–1669), depression (CI: 1400–
1459) , dementia (CI: 1510–1540) μg L-1 versus normal referents (CI: 650–885) μg L-1. Cu ion
is significant component of metallo-proteinase for redox reactions due to the presence of Cu
assisted enzymes directly bind with molecular oxygen [469]. The various neurotic disorders
are known to be caused due to reduced activity of Cu-enzyme [470, 471]. Excess brain Cu
ion is a common finding in neurodegenerative diseases such as Alzheimer’s, depression,
dementia. Higher concentration of Cu ion varies the level of neurotransmitter which leads to
dyes functioning of brain and chronic mental disorder. In depressed patients concentration Cu
ion is mostly greater than normal individual [472].
Table 4-12 The concentration of Cu ion in serum samples of neurological
disorders male patients and normal referent (μg L-1)
Element Normal referent
(n=40)
Alzheimer’s
(n=20)
Depression
(n=20)
Dementia
(n=20)
Cu+2 (μg L-1) 801±54.6 1650±21.4 1430±10.9 1530±8.38
P= 0.01 – 0.001
100
4.5 A DISPERSIVE LIQUID-PHASE MICRO-EXTRACTION METHODOLOGY FOR TRACE LEVEL OF IRON IN SERUM SAMPLES OF NEURO DISORDERS PATIENTS
GENERAL REMARKS
The work has been published in the journal of “International Journal of Scientific & Engineering Research” as:
Mariam. S. Arain, et al., “A modified dispersive liquid-phase microextraction methodology for the analysis Fe in serum specimens of neurogical disorders patients ” International Journal of Scientific & Engineering Research, Vol. 8, pp.171-190, 2017.
4.5.1 OPTIMIZED EXPERIMENTAL FACTORS
The variables play a key role on the extraction efficiency and reproducibility such as
pH, first extractant volume, back-extractant volume, concentration of complexing agent and
aspirating/dispensing cycles through a syringe were studied and optimized.
EFFECT OF PH
The pH is considered to be the important variable in the extracting efficacy of
established MDLP-µE procedure. The role of pH on the proposed method for Fe was carried
out in the range of 3 to 8. The maximum extraction efficiency was achieved at pH 5 as shown
in Fig. 4-13.Where as hydrolysis occur at higher pH.
3 4 5 6 7 8 90
20
40
60
80
100
% R e c o v e r y
pH
Fig. 4-13 Influence of pH on the %recovery of Fe by MDLP-µE
101
OXINE CONCENTRATION
For the purposed MDLP-µE methodology, oxine was used for the complex formation
of analyte (Fe). The concentration of complexing agent ranging from 0.1−0.5 mL (0.113%)
was studied for the recovery of Fe as shown in Fig. 4-14. Quantitative recovery was achieved
at 0.3 mL of complexing agent and further increase in the concentration didn’t show any
significant effect.
0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.550
20
40
60
80
100
% R e c o v e r y
Oxine 0.113 % (0.1- 0.5 mL)
Fig. 4-14 Oxine quantity influence on % recovery of Fe by MDLP-µE
VOLUME OF EXTRACTING SOLVENT
The extracting solvent has a key role on the first step of the MDLP-µE method. The
extracting solvent should have the ability to extract the target metal complex due to low
solubility in aqueous medium, and the cloudy solution was formed with tiny droplets. Hence,
chloroform was selected due to higher extraction efficiency. The volume of extracting solvent
was studied in the range of 50 to 200 µL. Thus, 80 µL chloroform selected for the rest of the
work.
BACK EXTRACTING SOLVENT
In current study, we also studied the effect of the back extracting solvent in the second
step of MDLP-µE. For this purpose, nitric acid of 0.5 to 2.0 mol L-1 was used to back extract
Fe in aqueous media from analyte enriched organic phase. The optimum extraction of Fe was
102
observing on 1.0 mol L−1of HNO3. So, HNO3 solution (1.0 mol L-1) of 0.5 mL was used for
back extraction of the target analyte into the aqueous phase.
EFFECTS OF ASPIRATING/DISPENSING CYCLES
The dispersion of the extracting solvent has a major role to achieve the maximum
extraction efficiency of the developed procedure. To disperse the organic solvent in aqueous
phase, number of triggers have been used, which are mostly create a negative effect on the
nature of the solvent and extraction efficiency. In the current study, we used dual-syringe
based MDLP-µE coupled with FAAS as the dispersive medium for organic solvent. The
effect of aspirating/dispensing cycles on the proposed method was carried out ranging from
of 2 to 10 cycles Fig. 4-15. It was found that maximum recovery of Fe was achieved by
increasing the number of aspirating/dispensing cycles, due to higher dispersion and increased
contact with the aqueous phase. Therefore, 8 aspirating/dispensing cycles were selected for
further study. In the back extracting process, the Fe enriched organic solvent aspirated to
aqueous phase (1.0 mol L-1, HNO3) of 0.5 mL by 5 times aspirating/dispensing cycles.
2 3 4 5 6 7 8 9 100
20
40
60
80
100
% R ec o v er y
Number of aspirating /despensing cycles
Fig. 4-15 influence of aspirating/dispensing cycles on the %recovery of Fe by MDLP-µE
CENTRIFUGATION TIME AND RATE
The extracting efficiency of the proposed method was attained at different
centrifugation rate (1500 to 3000 rpm) for 5 min. It was observed that 2500 rpm was
103
adequate for Fe enriched phase. In the second phase of MDLP-µE, the centrifugation rate and
time was also 2500 rpm and 5 min respectively.
THE SELECTIVITY
The matrix ions effect were studied for the competent extraction recovery of Fe by
proposed, MDLP-µE. To carry out this study, 10 µg L-1 Fe in (10 mL) were added with co-
existing (Ca2+, Co2+ , K+, Na+, Ag+, Mg2+, Ni2+, Cu2+, Al3+) at diverse analyte to interferent
ratios, and used for the developed procedure. The ratios (w/w) of studied analyte and
matrices ions were set according to 1 : 1000 for K+, Na+ ; 1 : 800 for Ca2+, Mg2+; 1 : 25 , Ni2+;
1 : 20 for Ag+, Cu2+ and 1 : 30 for Al3+.The acceptance limits for recovery of Fe−Oxine
complex with various foreign ions was found to be <5%. The alkali and alkaline earth
elements are not counted as matrix components, because they forms unstable complexes with
oxine. Therefore, the proposed procedure is better selectivity for trace levels of Fe.
4.5.2 APPLICATION
The developed procedure at optimum values of different variables was used to
determine Fe trace levels in serum samples of different neurological disorders patients
(Alzheimer’s, Parkinson’s, multiple sclerosis) and age matched healthy controls The resulting
data indicate that the Fe levels in serum samples of neurological disorders male patients are
significantly greater than the controls age-matched Table 4-13. At 95% confidence intervals
the ranges of Fe in the serum samples of male neurological disorders patients were observed
to be (CI) for Alzheimer’s (CI 1403–1445 µg L-1), Parkinson’s (CI 1535–1575 µg L-1),
multiple sclerosis (CI 1350–1378 µg L-1) versus controls (CI 600–795 µg L-1) .It was
reported that due to excess of Fe causes mental action, learning behavior, and the
susceptibility to different patients have neurological disorders. Fe is important for normal
neuronal metabolism. The level of Fe increased in many chronic neurological disorders
including AD, PD, and MS leads to deposition of iron in the brain due to the formation free
radical [40, 473, 474]. Excess of Fe cause cellular damage where as deficiency impair cell
growth. Fe is important cofactor for enzymes involved in the neurotransmitters synthesis,
neural function and development [475].
Table 4-13 The concentration of Fe in serum samples of neurological disorders
male patients and healthy control (μg L-1)
104
Element Healthy control (n =60)
Alzheimer’s (n= 20)
Parkinson’s (n= 20)
Multiple sclerosis (n=15)
Fe (µg L-1) 660±50.5 1417±10.9 1562±11.5 1359±16.8
P= 0.01 – 0.001
4.6 A INNOVATIVE SWITCHABLE POLARITY SOLVENT, WAS PREPARED FOR ENRICHMENT OF Al IN BIOLOGICAL SPECIMENS
GENERAL REMARKS
The work has been accepted in the “journal of Applied organometallic chemistry” as:
M. S. Arain, T. G. Kazi, H. I. Afridi, et al., “A innovative switchable polarity solvent, based on 1,8-diazabicyclo-[5.4.0]- undec-7-ene and decanol was prepared for enrichment of aluminum in biological sample prior to analysis by FAAS” Online https://doi.org/10.1002/aoc.4157.
4.6.1 CHARACTERIZATION OF SS
DESCRIPTION OF SS
Was used to characterized the exchange phenomenon of SS system in SS-E
methodology from neutral phase (DBUH – decanol), to polar form after CO2 exposure
(DBUH-decanol-CO2), have been studied by infrared spectrophotometer (IR). To
characterized the SS system by IR, frequency was selected range in the of 4000 cm-1to 500
cm-1. Whereas the region in the range of 2250 cm-1 and 1950 cm-1 not included in study,
which is due to absorbance of CO2. The peaks at 1639 cm-1 was allocated to the ν(C=O),
[DBUH][decanol-CO2 as stretching vibrations which is newly observed (Fig. 4-16 B). As the
SS exposed to CO2, a new peaks was observed at 1639 cm-1, which is ascribed to the ν(C=O)
called carbonate stretching. After exposing of SS to carbon dioxide a broad peak came into
view at 3344 cm-1 indicates (O-H) stretching, might be due to presence of aqueous medium
(H2O). (Fig 4-16 B), and after the removal of CO2 all peaks are reappear in Fig. 4-16 C, as
shown in Fig. 4-16 A [476].
105
Figure 4-16 In-situ IR spectra of the SPS system of (a) [DBUH][decanol] (b) formed [DBUH]
[decanoCO2] by CO2 bubbling into the mixture and (c) recycling [DBUH][decanol] by CO 2
removal from the mixture by bubbling with N2 and heating at 55°C.
CONDUCTIVITY MEASUREMENT
The initial conductivity of SS (vial containing ~4 mL) was measured by platinum
conductivity probe. Then after exposure to CO2, the conductivity was quantified at different
time intervals (1 min) till a stable value was obtained. It was monitored that the conductance
of each step was increased from (10 µS/cm), to (420 µS/cm) of SS. The resulted data
indicates that the SS was changed from low to elevated polarity.
4.6.2 OPTIMIZATION OF FACTORS
Different factor effects the efficiency and practicability of proposed SS-E method, to
accomplish the enrichment/extraction of Al from aqueous medium of samples/standards to
SS system. The pH is main factor to effects the stability of complex (Al-morin), in addition
to have a very important function on interface of SS-aqueous medium, change from polar
(hydrophilic) to nonpolar (hydrophobic) phases. Fig 4-17 indicates the effects of pH in the
range of 4 to 8 on the extracting efficacy of SS-E. It shows that extraction efficiency of Al-
ligand was optimum at pH 6.0, whereas declined slightly > pH 6, indicates that SS have
hydrophobic interface among Al-morin complex at pH 6.0. So, pH 6.0 was selected for
successive experimental work.
106
3 4 5 6 7 8 90
20
40
60
80
100
pH
% R e c o v e r y
Fig. 4-17 Effect of pH on the recovery (%) of Al SS-E
The effects of morin amount on the extraction efficiency was worked in the ranged of
(0.1 mL - 0.5 mL) of 0.125% (m/v). It was observed that up to 0.3 mL of complexing reagent,
the extraction efficiency enhance and attained a plateau, which indicates the selected
concentration of morin is enough for entire complexation of Al and other interferent analytes
in sample solutions. The exposure/Purging time and pressure of carbon dioxide have main
function on changing of nonpolar SS to polar form (SS - CO2) which significantly enhanced
the extraction of studied analyte from samples/stands in aqueous medium. For present study
the 2 to 6 MPa pressure was used as an anti-solvent trigger to exchange the SS system from
nonpolar to polar and vice versa. At high pressure of CO2 pressure the content of was spell
out, although SS-aqueous system (biphasic) switched to more polar monophasic phase. The
influence of time was studied in the ranged of 3 to 7 min for optimum switching phenomena
and extraction efficiency of analyte. It was observing that at 5 min, the highest extracting
efficiency and change of non-polar to polar SS phases, in Fig 4-18. Therefore, CO2 at 4 MPa
pressure and 5 min purging time were selected for further experimental work.
107
3 4 5 6 70
20
40
60
80
100
DBU/DecanolDBU/DecanolCO2
Time (min)
Mol
Fra
ctio
n (%
)
Fig. 4-18 Time % concentration profiles of conversion of DBU/decanol to DBU/decanolCO2 by exposing to 4 MPa of CO2 while stirrer at 500 rpm
EXTRACTION EFFICIENCY OF SS SYSTEM
The enrichment capability of SS-E procedure depends on the partitions of metal
complex among immiscible biphasic stages. The distribution ratio (D) of analytes between
nonpolar and polar phases is expressed by an equation:
D=[ M ]org
[ M ]aq
Where, [M]org (µg/L) and [M]aq (µg/L) are the quantity of analyte in nonpolar (SS) and polar
aqueous phase, correspondingly. To determine the analyte contents in aqueous phase after
enrichment method, then the mass balance is interpreted by equation as:
[ M ]org=[ M ]aq . m[aq]b — [ M ]aq . maq
morg
For approximate selection of different factors, the initial masses of both phases (aqueous and
organic) are employed. So due to mass changes of both phases, the metal concentration
calculations might have some error. The initial and final masses of both phase were also
effect the percentage extractions of analyte and distribution ratios. It is recommending to
employ the measured organic metal concentration. The % recovery of analytes could be
calculated as:
% E= Content of metal extracted by SSTotal levels of metal∈aqueous phase
×100
108
EFFECT OF BACK EXTRACTING ACID SOLUTION
In present study and novel step is that to extract back the analyte from SS enriched
phase to acidic aqueous solution. The concentration of acid is important to completely
extracted back the analyte from its organ-metallic complex (Al- morin). The 0.5 ml of HNO3
at 0.5 to 1.5 mol L-1 concentrations range were used. The high concentration of acid is
required as back extracting (dual step), might be due to strong hydrophobic Al-morin
complex. The optimum back extraction of Al from it complex (99%), might be attained at 1.0
mol L-1 Fig 4-19.
The back extraction phenomena (%S) of Al from SS to acidic aqueous phase may
possibly calculated as:
% S= Amount of analyte extracts∈acidic phaseTotalcontent of analyte∈SS
×100
0.4 0.6 0.8 1 1.2 1.4 1.60
20
40
60
80
100
% Re-
cov
ery
HNO3 mol L-1
Fig. 4-19 (% S) stripping of Al from SS to acidic media
INTERFERENCE STUDIES
As the real samples have complex matrices contains many analytes, which might be
also form complex with morin beside the analyte of interest for proposed SS-E procedure.
The interference of coexisting metals might be effecting the extracting efficacy of the analyte.
To evaluate the specificity of the proposed procedure, the possible interference ions termed as
tolerance limit was carried out. A variation > 5% in the absorbance of the analyte was
consider as an interfering ions. For this purpose the different ratio of coexisting ions to 10 µg
L-1 of Al were preconcentrated by SS-E and determined the effects on % recovery of studied
analyte. It was observed that in the existence of different metals, the % recovery of Al were
109
above 95%. It was reported that the common alkali and alkaline earth elements, which are
occur in most of the environmental and biological samples, not form stable complexes in
working experimental factors. The resulted data indicated that the Cd2+, Cu2+, Zn2+, Fe3+ were
tolerate up to 25 mg L-1 whereas the tolerance levels of Co2+, Ni2+ was > 30 mg L-1.
RECYCLING AND RECOVERY OF SS
One of the green approach of developed method is that the SS was
insoluble/immiscible with aqueous phase. To obtain the SS for further experiment, the back
extraction of the analyte of interest in acidic solution, leave the SS solvent for further
enrichment experiment. The SS can be recycled more than 6 time without loss of extraction
efficiency. After that the SS may lose the extraction about 3 to 5%.
4.6.3 APPLICATION
The optimized proposed SS-E procedure was apply for the analysis of Al in duplicate
blood samples (after acid decomposition) of non-diseased males (controls/referents), patients
have different neurological disorders (dementia, stroke, AD). The resulted data indicated that
the metabolism of Al changed in different neurological disorders which might a key factor
in the pathogenesis [477]. In present study the level of Al in whole blood of different
neurological disorders subjects and age matched controls (have no any neurological
disorders). The average values of Al in blood samples are revealed in Table 4-14. In controls
the Al concentration in the ranged of 8.4–13.5 µg L-1, while AD patients have (18.4–30.6 µg
L-1), stroke (17.2–24.4 µg L-1), dementia (16.5–23.1 µg L-1). It was reported in literature that
the dementia is associated with Al levels in drinking water [478]. However, there is much
debate regarding these findings and their interpretation, in particular due to recent published
epidemiologic studies which indicate the adverse opinion [479].
It is stated the relation among exposure to Al powder and cognitive impairment
[480]. Whereas another study have adverse report about the association of occupational
exposures of Al and different neurological disorders [481]. As the Al does not occur in its
pure form, however it is present in combined forms with other elements such as silicate,
hydroxide, phosphate and sulphate. The extensive applications in industrial and domestic
purposes, it might form severe effects on human health. The resulted data of present study
indicated that there is a relationship among high levels of Al and increases risk of a number
of neurodegenerative disorders including dementia, stroke, and AD [482].
110
Table 4-14 The level of Al in blood samples of referents and different
neuro disorders male patients using SS-E Method
Element Referents
(60)
Alzheimer’s
(45)
Stroke
(20)
Dementia
(25)
Al (µg L-1) 10.3±1.76a 23.4 ± 3.20 21±2.31 19.3±2.49
aMean±Standard deviation (x±s)
4.7 PRECONCENTRATION OF TRACE LEVEL MANGANESE IN BLOOD SAMPLES OF PATIENTS WITH DIFFERENT NEUROLOGICAL DISORDERS USING A DEEP EUTECTIC SOLVENT EXTRACTION
GENERAL REMARKS
The work has been published in the journal of “Atomic Spectroscopy as:
M. S. Arain et al., “Preconcentration of trace level manganese in blood samples of patients with different neuro disorders using a Deep Eutectic Solvent Extraction Method before to analysis by FAAS” vol.38, pp. 92-98, 2017.
4.7.1 OPTIMIZATION OF DES-E METHOD
Some factors affect the efficiency of the Deep Eutectic Solvent Extraction (DES-E) method such as pH, DES volume, chelating agent, volume of decanol and hexanol, and the molar ratio.
EFFECT OF PH
The analyte forms a complex at a specific pH, which is suitable for the complex
formation and maximum extraction efficiency. 6 replicate standardize solutions of Mn2+ (10
111
µg L-1) were prepared to check the effect of pH (4-11) on the extraction of manganese ions.
The desired effective pH value was acquired by adding acetate/phosphate buffer, followed by
adding of 0.1 mol L-1 of HCl/NaOH. The Mn ions form a stable complex in the range of pH
4-11. PAN is used to form a complex with Mn2+ at the pH range of 4–11. The optimum
recovery of Mn2+ was achieved at pH 10 see Fig. 4-20, whereas at higher pH the signal for
Mn2+ decreases. Thus, pH 10 was selected for Mn2+ extraction in subsequent experimental
work.
3 4 5 6 7 8 9 10 11 120
20
40
60
80
100
% Re
co ver
y
pH
Fig 4-20 Influence of pH on preconcentration of Mn2+ by DES-E
EFFECT OF PAN CONCENTRATION
For the DES-E method, PAN was used as the chelating agent. The extracting
efficiency of Mn2+ as a function of PAN amount changing from 1–5 × 10-5 mol L-1 (w/v) in
Fig. 4-21. The recovery of Mn2+ is enhanced up to 4 ×10-5 mol L-1 and the extraction
efficiency of the metal has no significant effect by a further increase of PAN concentration.
Therefore, 4 × 10-5 mol L-1 of PAN was selected for the optimum extraction of Mn2+ from
standards and real samples. The PAN is an effective reagent for enrichment of Mn2+ due to its
higher hydrophobic and amphiphilic properties as well as form stable complex with the study
analyte [146].
112
0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.50
20
40
60
80
100
%
R ec o ve ry
PAN (1.0×10-5 mol L-1)
Fig. 4-21 Influence of PAN amount on % R of Mn2+ by DES-E
EFFECT OF MOLAR RATIO OF EUTECTIC MIXTURES FOR DES
In the present work, a eutectic mixture of ZnCl2-acetamide acid at altered molar ratios
(1:1, 1:2, 1:3 and 1:5) was prepared by stirring at 60-90 ºC until a colorless and uniform
liquid was formed. It was observed that optimum amount of solvent was formed between 75 -
85 ºC, so for subsequent experimental work DES was prepared at 80 ºC. Different molar
ratios of eutectic mixtures of ZnCl2-acetamide were used for the extraction of the
hydrophobic complex of Mn2+ from certified reference standard solutions (10 µg L-1). The
optimum recovery was obtained at the molar ratio of 1:2 of ZnCl2-acetamide. Hence, for the
present study the DES was prepared at 1:2 molar ratio of ZnCl2-acetamide. The
characteristics of the prepared DES were reported elsewhere, while its formation was
checked by the solubility in water. The individual component of the eutectic mixture has a
high melting point, while after mixing it becomes solvent at 80 ºC. Both components are
water soluble, but the resulting DES was immiscible in the aqueous phase [483].
EFFECT OF DEEP EUTECTIC SOLVENT VOLUME
The volume of eutectic combination of ZnCl2–acetamide at the molar ratio of 1:2 is
the chief factor for the extraction capacity of Mn2+ in acid-digested blood samples. The
volume of DES used was in the range of 0.5 – 2.0 mL for the extraction of Mn2+. The
optimum recovery of Mn2+ was achieved at 1.0 mL as shown in Fig. 4-22. Therefore, 1.0 mL
of DES was selected for further study.
113
0 0.5 1 1.5 2 2.50
20
40
60
80
100
% R ec o v er y
DES volume (mL)
Fig. 4-22 Influence of DES volume on % recovery Mn2+ bu using DES-E
EFFECT OF DECANOL AND HEXANOL VOLUME
Decanol and hexanol were used to decrease the dissolution and increase the
hydrophobicity of the DES to entrap the hydrophobic chelate of Mn2+. For the DES-E
method. The extraction efficiency of Mn2+ as a function of decanol and hexanol volumes
ranged from 0.2 −1.0 mL, as shown in Fig. 4-23. The recovery of Mn2+ is enhanced up to 0.5
mL for both solvents. A further increase in their volume caused no changes in the signals. It
was seen that decanol has enhanced the extraction efficiency in comparison to hexanol (15−
20%) and might be due to its higher hydrophobic nature. Hence, 0.5 mL of decanol was
selected for the measurable extraction of Mn2+ for subsequent work.
0 0.2 0.4 0.6 0.80
20
40
60
80
100
%
Re-
cov
ery
Decanol volume (mL)
114
Fig. 4-23 Influence of decanol volume on %recovery Mn2+ using DES-E
INTERFERENCE STUDY
The effect of background ions was also examined for the extraction of Mn ions in the
sample matrix by DES-E method. To accomplish this task, 10 mL solutions comprising 10 µg
L-1 of Mn2+ with additional interfering ions (Na+, K+, Ca2+, Ag+, Mg2+, Co2+, Zn2+, Ni2+, Cu2+,
Fe3+, Al3+) at diverse interferent-to-analyte ratios were exposed to the developed method. The
amounts of coexisting ions were made as element-to-interferent ratios (w/w) of 1 : 1000 for
K+, Na+ ; 1 : 800 for Ca2+, Mg2+; 1 : 25 for Co2+, Zn2+, Ni2+; 1 : 20 for Ag+, Cu2+; 1 : 30 for Fe3+
and 1 : 30 for Al3+. The commonly existing ions, such as alkaline and alkali earth metals,
usually does not make a stable complex with PAN. Thus, the developed extraction method
was found to be selective for Mn ions.
4.7.2 APPLICATION
The optimized proposed DES-E method was practiced on duplicate acid-digested
blood samples from dementia, multiple sclerosis, Parkinson’s patients and healthy
references (mostly relatives of patients) for the determination of Mn2+. The mean level with
standard deviations of Mn2+ in the blood samples are listed in Table 4-15. The level of Mn2+
was found in the range of 15.6–18.7 µg L-1 for the healthy references. Whereas the Mn2+
level for PD, dementia and multiple sclerosis patients was in the range of 28.8–32.1, 23.5–
26.2 and 19.5–22.7 µg L-1 respectively. It was found that the Mn2+ levels in the blood
samples of the PD patients were significantly greater than for the patients with the other
two types of neurological disorders (p<0.01).
Despite the essential role of Mn2+ in several metabolic tasks, unnecessary exposure of Mn2+
causes its storage in the brain which results in various neurological disorders similar to PD
[484]. It is well documented that the nervous system is affected by Mn2+ and is considered to
shows neuropsychiatric signs and extra-pyramidal dysfunction [485, 486].
It has been reported occupational workers and miners can have a high exposure to
Mn2+. Which enters the body through the respiratory tract and can seriously affect the nervous
system [487]. Occupational exposure to Mn2+ arises mostly due to alloy production and
processing, mining, welding, ferro-manganese operations and work with agrochemicals
[488]. In the human brain, excess Mn2+ leads to various neurodegenerative disease i.e.
dementia, PD, and MS [489].
115
The clinical features of Mn2+ neurotoxicity resemble those of idiopathic Parkinsonism
[100]. However, an investigation of cases of Mn2+ poisoning has revealed clinical,
pharmacological and imaging dissimilarities. Yet, numerous interpretations have suggested
the possible role of Mn2+ ions in the several PD patients. Though PD is a very communal
neurological disorder in adult individuals, yet its etiology is still unidentified. The assumption
of a relation between ecological features and different inherited susceptibility, both acting on
normal aging, has been suggested [484, 490].
Table 4-15 Concentration of Mn2+ in blood samples of healthy references and different neuro disorders in male patients using DES-E method.
Element References(n = 60)
Parkinson’s (n = 50)
Dementia(n = 30)
Multiple Sclerosis(n = 20)
Mn2+ (µg L-1) 17.2 ± 1.59a 30.5 ± 1.58 24.8 ±1.32 21.1 ± 1.66
aMean ± Standard deviation
4.8 SUMMARY
In this chapter the optimization study done for the developed methodologies for studied
elements and detailed information of obtained results has been given. The established
methodologies were applied on the real samples of neurological/psychiatric disorders
patients for the enrichment of Al, Mn and Fe, Cu, Zn and also comparing the obtained
results with the reported data and discussed in detail. It was observed that our results are
comparable with previously reported work. The proposed methods provide the good
LOD, EF, LOQ and RSD values. In addition these methods are environmental friendly
and cost effective.
116
CHAPTER 5
CONCLUSION AND FUTURE DIRECTIONS
In this chapter we present conclusion of all the work, general recommendations and
future directions.
5.1 CONCLUSION
Metal ions are necessary for all human beings and taking part in several metabolic
activity in the cells. Though, their level in the body tissues must be strictly consistent
due to their excess & deficiency disturbing the regular functions and might be
responsible for many diseases. The major consequences of metal dys-homeostasis are
mitochondrial dysfunction, oxidative stress and degeneration of neuron in brain. The
present work suggested that imbalance of metal-mediated abnormalities play a important
part in several neurodegenerative/psychiatric disease pathogenesis such as Alzheimer’s,
Parkinson’s, multiple sclerosis, dementia, stroke and schizophrenia, bipolar disorders.
The conclusion is based on: Development of advance extraction methodologies to
preconcentrated the under studied elements in biological samples. To determine the
trace level of Al, Mn and Fe, Cu, Zn in biological samples (scalp hair, blood and serum)
of neurological/psychiatric disorders patients. Due to low concentration of studied
analytes, different advance pre-enrichment methods were developed.
A dual-cloud point extraction, has been proposed for the enrichment of Mn
and Zn ions in acid-digested biological samples prior to coupling with flame
atomic absorption spectrometry (FAAS). The proposed method, eliminated
117
the effects of surfactant by back extracting the analyte in aqueous nitric acid
with good accuracy, efficiency and reproducibility.
To develop Temperature controlled ionic liquid-based dispersive micro-
extraction (TIL-DLLME) method for the preconcentration of Al in acid
digested scalp hair samples. The estimated values of three significant
variables for TIL-DLLME of Al were calculated from 3D surface response by
quadratic equation to obtain the efficiency of two ligands L1 (oxine) and L2
(morin). It was observed that oxine is made complex with Al more efficiently
and extracted with lower amount of IL as compared to morin.
An innovative preconcentration method, dual dispersive ionic liquid based ultrasound assisted microextraction (UDIL-μE), was proposed for the enrichment of Cu ion in acid digested blood serum samples have complex matrixes, before proceeding to FAAS.
An efficient, modified dispersive liquid-phase microextraction method
(MDLP-µE) was developed for the enrichment of Fe level of acid digested
blood serum samples before analysis. The resulted data indicated that the
developed MDLP-µE procedure, having low cost and less time consuming.
Other remarkable features of the developed method was back-extraction step
very simple, achieved in less than 2 min. Modified dispersive liquid-phase
microextraction procedure has some advantages such as good enhancement
factor, low consumption of organic solvent, extracting time short, easy
operation, and low generation of waste.
A switchable solvent extraction (SS-E) method have been first time
introduced for trace levels of Al in blood samples In the proposed procedure
1, 8-diazabicyclo [5.4.0] undec-7-ene (DBU) and decanol made a switchable
solvent (SS), which was reversibly change on and off from nonpolar/
hydrophobic to polar/ hydrophilic, when exposed to CO 2, which is
inexpensive, nonhazardous and easily expelled out. The SS can be reused
many time for enrichment processes.
A deep eutectic solvent (DES) was prepared from two inexpensive and
harmless constituents, with a melting point low than that of each individual
constituent. The DES-E scheme has been established for the enrichment of
118
Mn in acid-digested blood samples before analysis by FAAS. In comparison
with ionic liquids, DES are nontoxic, biodegradable, and easy to synthesize,
to avoid purification of DES and the centrifugation step.
The different advance extraction method were successfully applied to
biological samples of patients have psychiatric and neurological disorders.
The mean level values of Al was obtained to be greater in scalp hair samples of different types of male psychiatric patients,
schizophrenia (13.6±1.02 μg g-1) and bipolar disorder (12.3±1.57 μg g-1) as
compared with normal referent (6.73±1.69 μg g-1). Whereas Mn concentration
was found to be significantly higher (p=0.01–0.001), in schizophrenia
(4.71±0.46 μg g-1) and bipolar disorder (5.83±0.85 μg g-1) normal referent
(3.60±0.47) μg g-1.
The level of Mn2+ in scalp hair samples of Parkinson's male and female
patients was found to be significantly higher (p<0.01) at confidence intervals
95% CI (9.64–10.5) µg g-1 versus referents CI (3.65–4.09) µg g-1. In
Parkinson's, neurons releasing dopamine in the substantia nigra die due to
high exposure of Mn, decreasing the overall supply of dopamine and
compromising the brain's capability to effectuate movement. Whereas level
of Mn in blood samples of Parkinson’s patients, dementia, multiple sclerosis,
was found to be higher 56% , 69% and 81% than normal referent
In Alzheimer’s, stroke and dementia disease patients the concentration of Al
in scalp hair, blood samples was two folds higher than normal referent
(11.3±2.03 µg g-1). The resulted data shown that the accumulation and
metabolism of Al3+ are altered in Alzheimer’s patients.
The mean levels of Fe in serum samples of different neurological disorders
have Alzheimer’s patients are significantly higher (p<0.001) than the controls
(CI 660±50.5 µg L-1) of same age group. Excess of Fe leads to deposition of
iron in the brain due to the formation free radical.
The concentration of Cu ion in blood serum of different neurological
disorders was found to be greater (P<0.001) at 95% confidence intervals (CI)
for Alzheimer’s (CI 1650±21.4), depression (CI 1430±10.9), dementia (CI
1530±8.38) μg L-1 versus normal referents (CI 801±54.6) μg L-1. Higher
119
concentration of Cu ion varies the level of neurotransmitter which leads to
dys-functioning of brain and chronic mental disorder.
The resulting data indicate that the Zn2+ levels are significantly lower
(p<0.001), such as 11%, 15% and 19% in serum samples of schizophrenia,
depression and bipolar disorder respectively than controls of same age group
at 95% confidence intervals (CI 0.423±0.08 mg L-1) . Zinc deficiency may
alter its homeostasis in the brain created different dysfunctions.
Consequently, for proper brain functioning and vesicular Zn 2+ is an essential
nutrient for neuronal signaling factor.
All of the above mentioned advanced preconcentration procedure were
applied for Al, Mn and Fe, Cu, Zn in scalp hair and blood serum samples.
The validity and accuracy of developed procedures were carried out by
analysis of certified reference material of human hair (NCS ZC81002),
human blood (Seronorm Trace Elements Whole Blood (LOT 1103128) and
serum from Clincheck control lyophilized ® human serum. Reliability of the
different proposed procedure was also checked by the standard addition
method in a real sample, which gave satisfactory results.
5.2 SOCIOECONOMIC IMPACT
Neurodegenerative and psychiatric disorders are thought to be
multifactorial, while metals (Al, Mn, Fe, Cu and Zn) can be involved as
cofactors in abnormalities or suspected of being risk factors for this
disorder. Many epidemiological studies have been done worldwide to
investigate the hypothesis of a correlation among trace elemental level
and neurological/psychiatric disorders.
A growing body of suggestion has shown that various essential/toxic
metals play vital part in a number of biological processes by inhabit
enzymes or activating, to compete with other elements and
metalloproteins for binding sites, and by disturbing the permeability of
cell membranes or by other mechanisms.
In developing countries the deficiency of zinc is common, creates adverse
impact on central nervous system such as schizophrenia, bipolar,
120
depression and distorted or absent sensory function involving taste, smell,
and vision.
However, exposure to excessive amounts of manganese is prevalent in,
and associated with, a variety of psychiatric and motor disturbances. High
exposure to Mn causes it to accumulate in the brain, creating an
intoxication called manganism, a condition like Parkinson’s.
Aluminum is known to be extremely neurotoxic and at high exposure, via
different ways (drinking waters, food, and medicines) and interfere with
the normal activities of nervous system.
Inadequate amounts of food causing deficiency of vital micronutrients
such as vitamins, minerals or trace elements) continue to be priority
health problems.
Finally, the evidence concerning the role of trace elements in influencing
neurological disorder risk, are helpful to physicians for diagnosis in
addition to other biochemical test.
5.3 RECOMMENDATIONS
The molecular understanding basis of the metal homeostasis and
regulations in the cells are critical in finding the underlying causes for
neuro pathophysiology, providing proper diagnosis and treatments. It is
also important for the development of new therapeutic agents able to treat
and preventing their occurrence.
To enhance the physicians to understand the importance, alteration of
metabolism of cellular and intracellular regulatory functions of
essential/toxic elements, for causing neurological disorders and utilize
this knowledge for diagnostic and therapeutic purposes.
The deficiency of zinc creates adverse impact on central nervous system
such as schizophrenia, bipolar, depression and distorted or absent sensory
121
function involving taste, smell, and vision. So the supplement of zinc is
necessary during and physiological disorders.
It is especially important for patients to avoid the exposure of toxic
metals especially aluminum and manganese especially in work places for
those patients have initial stage of Alzheimer’s and Parkinson like
syndrome manganism.
Mass awareness program must be introduced to accelerate the knowledge
about the exposure role of toxic elements in addition to overdose of
essential elements such iron and copper, where is deficiency of essential
element (zinc) might be adversely effects the neuro health through
electronic and print media.
5.4 SUMMARY
It can be summarized that imbalance of trace elemental level play a necessary part
in several neurodegenerative/psychiatric disease pathogenesis such as Alzheimer’s,
Parkinson’s, multiple sclerosis, dementia, stroke and schizophrenia, bipolar disorders. To
determine the trace level of Al, Mn and Fe, Cu, Zn in biological samples (scalp hair,
blood and serum) of neurological/psychiatric disorders patients. Due to low
concentration of studied analytes, different advance pre-enrichment methods were
developed with good accuracy and precision.
122
REFRENCES
[1] D. Drago, S. Bolognin, and P. Zatta, "Role of metal ions in the Aβ oligomerization in Alzheimer's disease and in other neurological disorders," Current Alzheimer Research, vol. 5, pp. 500-507, 2008.
[2] R. O. Wright and A. Baccarelli, "Metals and neurotoxicology," The Journal Of Nutrition, vol. 137, pp. 2809-2813, 2007.
[3] S. Angeli, T. Barhydt, R. Jacobs, D. W. Killilea, G. J. Lithgow, and J. K. Andersen, "Manganese disturbs metal and protein homeostasis in Caenorhabditis elegans," Metallomics, vol. 6, pp. 1816-1823, 2014.
[4] G. F. Kwakye, M. Paoliello, S. Mukhopadhyay, A. B. Bowman, and M. Aschner, "Manganese-induced Parkinsonism and Parkinson’s disease: shared and distinguishable features," International Journal Of Environmental Research And Public Health, vol. 12, pp. 7519-7540, 2015.
[5] A. Gaeta and R. C. Hider, "The crucial role of metal ions in neurodegeneration: the basis for a promising therapeutic strategy," British Journal Of Pharmacology, vol. 146, pp. 1041-1059, 2005.
[6] R. A. Yokel, J. S. Crossgrove, and B. L. Bukaveckas, "Manganese distribution across the blood–brain barrier: II. Manganese efflux from the brain does not appear to be carrier mediated," Neurotoxicology, vol. 24, pp. 15-22, 2003.
[7] K. J. Barnham, C. L. Masters, and A. I. Bush, "Neurodegenerative diseases and oxidative stress," Nature Reviews Drug Discovery, vol. 3, pp. 205-214, 2004.
[8] I. Dalle-Donne, R. Rossi, F. Ceciliani, D. Giustarini, R. Colombo, and A. Milzani, "Proteins as sensitive biomarkers of human conditions associated with oxidative stress," Redox Proteomics : From Protein Modifications to Cellular Dysfunction and Diseases, vol. 21, pp. 485-525, 2006.
[9] H. Braak and E. Braak, "Pathoanatomy of Parkinson’s disease," Journal Of Neurology, vol. 247, pp. II3-II10, 2000.
[10] K. Del Tredici, U. Rüb, R. A. De Vos, J. R. Bohl, and H. Braak, "Where does Parkinson disease pathology begin in the brain?," Journal Of Neuropathology & Experimental Neurology, vol. 61, pp. 413-426, 2002.
[11] P. M. Kidd, "Parkinson's disease as multifactorial oxidative neurodegeneration: implications for integrative management," Alternative Medicine Review, vol. 5, pp. 502-529, 2000.
[12] L. Waite, G. Broe, H. Creasey, D. Grayson, D. Edelbrock, and B. O'toole, "Neurological signs, aging, and the neurodegenerative syndromes," Archives Of Neurology, vol. 53, pp. 498-502, 1996.
123
[13] L. E. Hebert, P. A. Scherr, J. L. Bienias, D. A. Bennett, and D. A. Evans, "Alzheimer disease in the US population: prevalence estimates using the 2000 census," Archives Of Neurology, vol. 60, pp. 1119-1122, 2003.
[14] J. W. Williams, B. L. Plassman, J. Burke, T. Holsinger, and S. Benjamin, "Preventing Alzheimer's disease and cognitive decline," Annals Of Internal Medicine, vol. 154, pp. 211-215, 2010.
[15] G. Liu, W. Huang, R. D. Moir, C. R. Vanderburg, B. Lai, Z. Peng, R. E. Tanzi, J. T. Rogers, and X. Huang, "Metal exposure and Alzheimer’s pathogenesis," Journal Of Structural Biology, vol. 155, pp. 45-51, 2006.
[16] C. Exley, "A molecular mechanism of aluminium-induced Alzheimer's disease?," Journal Of Inorganic Biochemistry, vol. 76, pp. 133-140, 1999.
[17] T. A. Clark, H. P. Lee, R. K. Rolston, X. Zhu, M. W. Marlatt, R. J. Castellani, A. Nunomura, G. Casadesus, M. A. Smith, and H.-g. Lee, "Oxidative stress and its implications for future treatments and management of Alzheimer disease," International Journal Of Biomedical Science, vol. 6, pp. 225, 2010.
[18] J. L. Cummings, H. V. Vinters, G. M. Cole, and Z. S. Khachaturian, "Alzheimer's disease Etiologies, pathophysiology, cognitive reserve, and treatment opportunities," Neurology, vol. 51, pp. S2-S17, 1998.
[19] B. D. Trapp and K.-A. Nave, "Multiple sclerosis: an immune or neurodegenerative disorder?," Annual Review of Neuroscience, vol. 31, pp. 247-269, 2008.
[20] E. Elemek and K. Almas, "Multiple sclerosis and oral health: an update," The New York State Dental Journal, vol. 79, pp. 16-21, 2013.
[21] B. Kornek, M. K. Storch, R. Weissert, E. Wallstroem, A. Stefferl, T. Olsson, C. Linington, M. Schmidbauer, and H. Lassmann, "Multiple sclerosis and chronic autoimmune encephalomyelitis: a comparative quantitative study of axonal injury in active, inactive, and remyelinated lesions," The American Journal Of Pathology, vol. 157, pp. 267-276, 2000.
[22] E. Fisher, A. Chang, R. J. Fox, J. A. Tkach, T. Svarovsky, K. Nakamura, R. A. Rudick, and B. D. Trapp, "Imaging correlates of axonal swelling in chronic multiple sclerosis brains," Annals Of Neurology, vol. 62, pp. 219-228, 2007.
[23] S. Turkson and V. Asamoah, "Body dysmorphic disorder in a Ghanaian male: case report," East African Medical Journal, vol. 76, pp. 111-114, 1999.
[24] E. Bramon and P. C. Sham, "The common genetic liability between schizophrenia and bipolar disorder: a review," Current Psychiatry Reports, vol. 3, pp. 332-337, 2001.
[25] S. L. Buka and A. P. Fan, "Association of prenatal and perinatal complications with subsequent bipolar disorder and schizophrenia," Schizophrenia Research, vol. 39, pp. 113-119, 1999.
[26] M. Cannon, A. Caspi, T. E. Moffitt, H. Harrington, A. Taylor, R. M. Murray, and R. Poulton, "Evidence for early-childhood, pan-developmental impairment specific to schizophreniform disorder: results from a longitudinal birth cohort," Archives Of General Psychiatry, vol. 59, pp. 449-456, 2002.
[27] A. G. Cardno, F. V. Rijsdijk, P. C. Sham, R. M. Murray, and P. McGuffin, "A twin study of genetic relationships between psychotic symptoms," American Journal Of Psychiatry, vol. 159, pp. 539-545, 2002.
[28] S. Shergill, J. van Os, and R. Murray, "Schizophrenia and depression: what is their relationship," Managing The Depressive Symptoms Of Schizophrenia, pp. 12-29, 1999.
124
[29] P. Jones and D. J. Done, "From birth to onset: a developmental," Neurodevelopment and adult psychopathology, pp. 119-136, 1997.
[30] P. Jones, R. Murray, B. Rodgers, and M. Marmot, "Child developmental risk factors for adult schizophrenia in the British 1946 birth cohort," The Lancet, vol. 344, pp. 1398-1402, 1994.
[31] S. Frangou, M. Hadjulis, X. Chitnis, D. Baxter, S. Donaldson, and V. Raymont, "The Maudsley Bipolar Disorder Project: brain structural changes in bipolar 1 disorder," Bipolar Disorder, vol. 4, pp. 123-124, 2002.
[32] R. M. Murray, P. Sham, J. Van Os, J. Zanelli, M. Cannon, and C. McDonald, "A developmental model for similarities and dissimilarities between schizophrenia and bipolar disorder," Schizophrenia Research, vol. 71, pp. 405-416, 2004.
[33] C. G. Fraga, "Relevance, essentiality and toxicity of trace elements in human health," Molecular Aspects Of Medicine, vol. 26, pp. 235-244, 2005.
[34] D. Auld, "Handbook on Metalloproteins, edited by I. Bertini, A. Sigel & H. Sigel," ed: New york: Marcel Dekker, inc, 200, pp. 881-941.
[35] H. Kozlowski, A. Janicka-Klos, J. Brasun, E. Gaggelli, D. Valensin, and G. Valensin, "Copper, iron, and zinc ions homeostasis and their role in neurodegenerative disorders (metal uptake, transport, distribution and regulation)," Coordination Chemistry Reviews, vol. 253, pp. 2665-2685, 2009.
[36] M. L. Hegde, P. Shanmugavelu, B. Vengamma, T. S. Rao, R. B. Menon, R. V. Rao, and K. J. Rao, "Serum trace element levels and the complexity of inter-element relations in patients with Parkinson's disease," Journal Of Trace Elements In Medicine And Biology, vol. 18, pp. 163-171, 2004.
[37] F. Jimenez-Jimenez and M. Luquin, "Mecanismos de muerte neuronal y neuroprotección en la enfermedad de Parkinson," Mecanismos de muerte neuronal y neuro proteccion en la enfermendad neurologicas. Neurologia, vol. 11, pp. 93-106, 1996.
[38] K. J. Barnham and A. I. Bush, "Metals in Alzheimer's and Parkinson's diseases," Current Opinion In Chemical Biology, vol. 12, pp. 222-228, 2008.
[39] A. I. Bush, "Metals and neuroscience," Current Opinion In Chemical Biology, vol. 4, pp. 184-191, 2000.
[40] G. Perry, L. M. Sayre, C. S. Atwood, R. J. Castellani, A. D. Cash, C. A. Rottkamp, and M. A. Smith, "The role of iron and copper in the aetiology of neurodegenerative disorders," CNS Drugs, vol. 16, pp. 339-352, 2002.
[41] S. C. Bondy, "The neurotoxicity of environmental aluminum is still an issue," Neurotoxicology, vol. 31, pp. 575-581, 2010.
[42] M. Fasano, B. Bergamasco, and L. Lopiano, "Is neuromelanin changed in Parkinson’s disease? Investigations by magnetic spectroscopies," Journal Of Neural Transmission, vol. 113, pp. 769-774, 2006.
[43] S. Yumoto, H. Nagai, H. Matsuzaki, H. Matsumura, W. Tada, E. Nagatsuma, and K. Kobayashi, "Aluminium incorporation into the brain of rat fetuses and sucklings," Brain Research Bulletin, vol. 55, pp. 229-234, 2001.
[44] R. A. Yokel, "The toxicology of aluminum," Neurotoxicology, vol. 21, pp. 813-828, 2000.
125
[45] R. Anane, M. Bonini, and E. E. Creppy, "Transplacental passage of aluminium from pregnant mice to fetus organs after maternal transcutaneous exposure," Human & Experimental Toxicology, vol. 16, pp. 501-504, 1997.
[46] C. Rivera, J. Voipio, J. A. Payne, and E. Ruusuvuori, "The K+/Cl-co-transporter KCC2 renders GABA hyperpolarizing during neuronal maturation," Nature, vol. 397, pp. 251-255, 1999.
[47] IPCS.No. 194 Environmental health criteria for aluminium. Effects on humans.Gene‘ve: WHO, " pp. 138-156, 1997.
[48] P. N. Alexandrov, Y. Zhao, A. I. Pogue, M. A. Tarr, T. Kruck, M. E. Percy, J.-G. Cui, and W. J. Lukiw, "Synergistic effects of iron and aluminum on stress-related gene expression in primary human neural cells," Journal Of Alzheimer's Disease, vol. 8, pp. 117-127, 2005.
[49] J. A. Duce and A. I. Bush, "Biological metals and Alzheimer's disease: implications for therapeutics and diagnostics," Progress In Neurobiology, vol. 92, pp. 1-18, 2010.
[50] T. P. Flaten, "Aluminium as a risk factor in Alzheimer’s disease, with emphasis on drinking water," Brain Research Bulletin, vol. 55, pp. 187-196, 2001.
[51] G. L. Klein, "Aluminum: new recognition of an old problem," Current Opinion In Pharmacology, vol. 5, pp. 637-640, 2005.
[52] W. A. Banks, M. L. Niehoff, D. Drago, and P. Zatta, "Aluminum complexing enhances amyloid β protein penetration of blood–brain barrier," Brain Research, vol. 1116, pp. 215-221, 2006.
[53] K. M. Erikson, T. Syversen, J. L. Aschner, and M. Aschner, "Interactions between excessive manganese exposures and dietary iron-deficiency in neurodegeneration," Environmental Toxicology And Pharmacology, vol. 19, pp. 415-421, 2005.
[54] C. Keen, J. Ensunsa, and M. Clegg, "Manganese metabolism in animals and humans including the toxicity of manganese," Metal Ions In Biological Systems, vol. 37, pp. 89-121, 2000.
[55] D. Boggio Bertinet, M. Tinivella, F. Alessandro Balzola, A. de Francesco, O. Davini, L. Rizzo, P. Massarenti, M. Antonietta Leonardi, and F. Balzola, "Brain manganese deposition and blood levels in patients undergoing home parenteral nutrition," Journal Of Parenteral And Enteral Nutrition, vol. 24, pp. 223-227, 2000.
[56] D. Krieger, S. Krieger, L. Theilmann, O. Jansen, P. Gass, and H. Lichtnecker, "Manganese and chronic hepatic encephalopathy," The Lancet, vol. 346, pp. 270-274, 1995.
[57] G. A. Wasserman, X. Liu, F. Parvez, H. Ahsan, D. Levy, P. Factor-Litvak, J. Kline, A. van Geen, V. Slavkovich, and N. J. Lolacono, "Water manganese exposure and children's intellectual function in Araihazar, Bangladesh," Environmental Health Perspectives, vol. 114, pp. 124-129, 2006.
[58] M. Krachler and E. Rossipal, "Concentrations of trace elements in extensively hydrolysed infant formulae and their estimated daily intakes," Annals Of Nutrition And Metabolism, vol. 44, pp. 68-74, 2000.
[59] B. Lönnerdal, "Nutritional aspects of soy formula," Acta Paediatrica, vol. 83, pp. 105-108, 1994.
[60] M. M. Finkelstein and M. Jerrett, "A study of the relationships between Parkinson's disease and markers of traffic-derived and environmental manganese air pollution in two Canadian cities," Environmental Research, vol. 104, pp. 420-432, 2007.
126
[61] M. P. Walsh, "The global experience with lead in gasoline and the lessons we should apply to the use of MMT," American Journal Of Industrial Medicine, vol. 50, pp. 853-860, 2007.
[62] M. Aschner, K. M. Erikson, and D. C. Dorman, "Manganese dosimetry: species differences and implications for neurotoxicity," Critical Reviews In Toxicology, vol. 35, pp. 1-32, 2005.
[63] A. H. Sadak and P. E. Schulz, "Welding-related parkinsonism: clinical features, treatment, and pathophysiology," Neurology, vol. 57, pp. 1738-1739, 2001.
[64] K. A. Josephs, J. Ahlskog, K. Klos, N. Kumar, R. Fealey, M. R. Trenerry, and C. T. Cowl, "Neurologic manifestations in welders with pallidal MRI T1 hyperintensity," Neurology, vol. 64, pp. 2033-2039, 2005.
[65] J. S. Schneider, E. Decamp, A. J. Koser, S. Fritz, H. Gonczi, T. Syversen, and T. R. Guilarte, "Effects of chronic manganese exposure on cognitive and motor functioning in non-human primates," Brain Research, vol. 1118, pp. 222-231, 2006.
[66] A. B. Bowman, G. F. Kwakye, E. H. Hernández, and M. Aschner, "Role of manganese in neurodegenerative diseases," Journal Of Trace Elements In Medicine And Biology, vol. 25, pp. 191-203, 2011.
[67] M. F. Struve, B. E. McManus, B. A. Wong, and D. C. Dorman, "Basal ganglia neurotransmitter concentrations in rhesus monkeys following subchronic manganese sulfate inhalation," American Journal Of Industrial Medicine, vol. 50, pp. 772-778, 2007.
[68] C. W. Olanow, "Manganese‐induced parkinsonism and Parkinson's disease," Annals Of The New York Academy Of Sciences, vol. 1012, pp. 209-223, 2004.
[69] A. B. Tabrizi, "Development of a dispersive liquid–liquid microextraction method for iron speciation and determination in different water samples," Journal Of Hazardous Materials, vol. 183, pp. 688-693, 2010.
[70] H. L. Bonkovsky, "Iron as a comorbid factor in chronic viral hepatitis," The American Journal Of Gastroenterology, vol. 97, pp. 1-4, 2002.
[71] C. Chacarolli, J. Andrade, O. Guimaraes, V. Balbo, C. Venezuela, and F. Teruel, "Spectrophotometric study of iron oxidation in the iron (II)/azide/tetrahydrofuran system and some analytical applications," Analytica Chimica Acta, vol. 411, pp. 217-222, 2000.
[72] R. R. Crichton, D. T. Dexter, and R. J. Ward, "Brain iron metabolism and its perturbation in neurological diseases," Monatshefte Für Chemie-Chemical Monthly, vol. 142, pp. 341-355, 2011.
[73] D. B. Kell, "Towards a unifying, systems biology understanding of large-scale cellular death and destruction caused by poorly liganded iron: Parkinson’s, Huntington’s, Alzheimer’s, prions, bactericides, chemical toxicology and others as examples, " Archives Of Toxicology, vol. 84, pp. 825-889, 2010.
[74] C. Morris, J. Candy, A. Oakley, C. Bloxham, and J. Edwardson, "Histochemical distribution of non-haem iron in the human brain," Cells Tissues Organs, vol. 144, pp. 235-257, 1992.
[75] M. Gerlach, D. Ben‐Shachar, P. Riederer, and M. Youdim, "Altered brain metabolism of iron as a cause of neurodegenerative diseases?," Journal Of Neurochemistry, vol. 63, pp. 793-807, 1994.
127
[76] E. Kienzl, K. Jellinger, H. Stachelberger, and W. Linert, "Iron as catalyst for oxidative stress in the pathogenesis of Parkinson's disease?," Life Sciences, vol. 65, pp. 1973-1976, 1999.
[77] P. Moreira, S. Siedlak, G. Aliev, X. Zhu, A. Cash, M. Smith, and G. Perry, "Oxidative stress mechanisms and potential therapeutics in Alzheimer disease," Journal Of Neural Transmission, vol. 112, pp. 921-932, 2005.
[78] T. A. Rouault and S. Cooperman, "Brain iron metabolism," in Seminars in pediatric neurology, 2006, pp. 142-148.
[79] D. Berg and M. B. Youdim, "Role of iron in neurodegenerative disorders," Topics In Magnetic Resonance Imaging, vol. 17, pp. 5-17, 2006.
[80] L. Zecca, M. B. Youdim, P. Riederer, J. R. Connor, and R. R. Crichton, "Iron, brain ageing and neurodegenerative disorders," Nature Reviews. Neuroscience, vol. 5, pp. 863-873, 2004.
[81] E. Gaggelli, H. Kozlowski, D. Valensin, and G. Valensin, "Copper homeostasis and neurodegenerative disorders (Alzheimer's, prion, and Parkinson's diseases and amyotrophic lateral sclerosis)," Chemical Reviews, vol. 106, pp. 1995-2044, 2006.
[82] L. Rossi, M. Arciello, C. Capo, and G. Rotilio, "Copper imbalance and oxidative stress in neurodegeneration," The Italian Journal Of Biochemistry, vol. 55, pp. 212-221, 2006.
[83] İ. Durukan, Ç. A. Şahin, N. Şatıroğlu, and S. Bektaş, "Determination of iron and copper in food samples by flow injection cloud point extraction flame atomic absorption spectrometry," Microchemical Journal, vol. 99, pp. 159-163, 2011.
[84] J. W. Crayton and W. J. Walsh, "Elevated serum copper levels in women with a history of post-partum depression," Journal Of Trace Elements In Medicine And Biology, vol. 21, pp. 17-21, 2007.
[85] V. Desai and S. G. Kaler, "Role of copper in human neurological disorders," The American Journal Of Clinical Nutrition, vol. 88, pp. 855S-858S, 2008.
[86] A.-E. A. Ghanem, E. M. Ali, A. A. El-Bakary, D. A. El-Morsy, S. M. Elkanishi, E.-S. Saleh, and H. El-Said, "Copper and Zinc levels in hair of both schizophrenic and depressed patients," Journal Of Forensic Medicine And Clinical Toxicology, vol. 17, pp. 89-102, 2009.
[87] P. M. Roos, O. Vesterberg, and M. Nordberg, "Metals in motor neuron diseases," Experimental Biology And Medicine, vol. 231, pp. 1481-1487, 2006.
[88] D. J. Waggoner, T. B. Bartnikas, and J. D. Gitlin, "The role of copper in neurodegenerative disease," Neurobiology Of Disease, vol. 6, pp. 221-230, 1999.
[89] S. Watanabe, S. Nagano, J. Duce, M. Kiaei, Q.-X. Li, S. M. Tucker, A. Tiwari, R. H. Brown, M. F. Beal, and L. J. Hayward, "Increased affinity for copper mediated by cysteine 111 in forms of mutant superoxide dismutase 1 linked to amyotrophic lateral sclerosis," Free Radical Biology And Medicine, vol. 42, pp. 1534-1542, 2007.
[90] R. S. MacDonald, "The role of zinc in growth and cell proliferation," The Journal Of Nutrition, vol. 130, pp. 1500S-1508S, 2000.
[91] A. Takeda, "Movement of zinc and its functional significance in the brain," Brain Research Reviews, vol. 34, pp. 137-148, 2000.
[92] R. K. Blanchard and R. J. Cousins, "Differential display of intestinal mRNAs regulated by dietary zinc," Proceedings Of The National Academy Of Sciences, vol. 93, pp. 6863-6868, 1996.
128
[93] M. A. Rahman, M. A. K. Azad, M. I. Hossain, M. S. Qusar, W. Bari, F. Begum, S. I. Huq, and A. Hasnat, "Zinc, manganese, calcium, copper, and cadmium level in scalp hair samples of schizophrenic patients," Biological Trace Element Research, vol. 127, pp. 102-108, 2009.
[94] C. J. Frederickson, K. Jae-Young, and A. I. Bush, "The neurobiology of zinc in health and disease," Nature Reviews. Neuroscience, vol. 6, pp. 449-462, 2005.
[95] C. J. Frederickson, W. Maret, and M. P. Cuajungco, "Zinc and excitotoxic brain injury: a new model," The Neuroscientist, vol. 10, pp. 18-25, 2004.
[96] M. Yanik, A. Kocyigit, H. Tutkun, H. Vural, and H. Herken, "Plasma manganese, selenium, zinc, copper, and iron concentrations in patients with schizophrenia," Biological Trace Element Research, vol. 98, pp. 109-117, 2004.
[97] M. L. Ackland and A. Michalczyk, "Zinc deficiency and its inherited disorders-a review," Genes & Nutrition, vol. 1, pp. 41-49, 2006.
[98] T. G. Kazi, N. F. Kolachi, H. I. Afridi, N. G. Kazi, and S. S. Arain, "Effects of mineral supplementation on liver cirrhotic/cancer male patients," Biological Trace Element Research, vol. 150, pp. 81-90, 2012.
[99] A. Taylor, "Measurement of zinc in clinical samples," Annals Of Clinical Biochemistry, vol. 34, pp. 142-150, 1997.
[100] P. Zatta, R. Lucchini, S. J. van Rensburg, and A. Taylor, "The role of metals in neurodegenerative processes: aluminum, manganese, and zinc," Brain Research Bulletin, vol. 62, pp. 15-28, 2003.
[101] A. Cote, M. Chiasson, M. R. Peralta, K. Lafortune, L. Pellegrini, and K. Toth, "Cell type‐specific action of seizure‐induced intracellular zinc accumulation in the rat hippocampus," The Journal Of Physiology, vol. 566, pp. 821-837, 2005.
[102] B. Szewczyk, M. Kubera, and G. Nowak, "The role of zinc in neurodegenerative inflammatory pathways in depression," Progress In Neuro-Psychopharmacology And Biological Psychiatry, vol. 35, pp. 693-701, 2011.
[103] B. K. Bitanihirwe and M. G. Cunningham, "Zinc: the brain's dark horse," Synapse, vol. 63, pp. 1029-1049, 2009.
[104] E. Mocchegiani, C. Bertoni-Freddari, F. Marcellini, and M. Malavolta, "Brain, aging and neurodegeneration: role of zinc ion availability," Progress In Neurobiology, vol. 75, pp. 367-390, 2005.
[105] S. W. Suh, S. J. Won, A. M. Hamby, B. H. Yoo, Y. Fan, C. T. Sheline, H. Tamano, A. Takeda, and J. Liu, "Decreased brain zinc availability reduces hippocampal neurogenesis in mice and rats," Journal Of Cerebral Blood Flow & Metabolism, vol. 29, pp. 1579-1588, 2009.
[106] A. Takeda and H. Tamano, "Insight into zinc signaling from dietary zinc deficiency," Brain Research Reviews, vol. 62, pp. 33-44, 2009.
[107] J. Dombovári and L. Papp, "Comparison of sample preparation methods for elemental analysis of human hair," Microchemical Journal, vol. 59, pp. 187-193, 1998.
[108] E. Barany, I. A. Bergdahl, A. SCHÜTZ, S. Skerfving, and A. Oskarsson, "Inductively coupled plasma mass spectrometry for direct multi-element analysis of diluted human blood and serum," Journal Of Analytical Atomic Spectrometry, vol. 12, pp. 1005-1009, 1997.
129
[109] I. Rodushkin, F. Ödman, R. Olofsson, and M. D. Axelsson, "Determination of 60 elements in whole blood by sector field inductively coupled plasma mass spectrometry," Journal Of Analytical Atomic Spectrometry, vol. 15, pp. 937-944, 2000.
[110] I. Rodushkin, T. Ruth, and Å. Huhtasaari, "Comparison of two digestion methods for elemental determinations in plant material by ICP techniques," Analytica Chimica Acta, vol. 378, pp. 191-200, 1999.
[111] A. Alimonti, F. Petrucci, F. Laurenti, P. Papoff, and S. Caroli, "Reference values for selected trace elements in serum of term newborns from the urban area of Rome," Clinica Chimica Acta, vol. 292, pp. 163-173, 2000.
[112] J. Begerow, M. Turfeld, and L. Dunemann, "New horizons in human biomonitoring of environmentally and occupationally relevant metals—sector-field ICP-MS versus electrothermal AAS," Journal Of Analytical Atomic Spectrometry, vol. 15, pp. 347-352, 2000.
[113] B. Bocca, A. Alimonti, F. Petrucci, N. Violante, G. Sancesario, G. Forte, and O. Senofonte, "Quantification of trace elements by sector field inductively coupled plasma mass spectrometry in urine, serum, blood and cerebrospinal fluid of patients with Parkinson's disease," Spectrochimica Acta Part B: Atomic Spectroscopy, vol. 59, pp. 559-566, 2004.
[114] C. Huang and D. Beauchemin, "Direct multielemental analysis of human serum by ICP-MS with on-line standard addition using flow injection," Journal Of Analytical Atomic Spectrometry, vol. 18, pp. 951-952, 2003.
[115] I. Rodushkin and M. D. Axelsson, "Application of double focusing sector field ICP-MS for multielemental characterization of human hair and nails. Part I. Analytical methodology," Science Of The Total Environment, vol. 250, pp. 83-100, 2000.
[116] P. C. D’haese, M.-M. Couttenye, L. V. Lamberts, M. M. Elseviers, W. G. Goodman, I. Schrooten, W. E. Cabrera, and M. E. De Broe, "Aluminum, iron, lead, cadmium, copper, zinc, chromium, magnesium, strontium, and calcium content in bone of end-stage renal failure patients," Clinical Chemistry, vol. 45, pp. 1548-1556, 1999.
[117] R. Sharma and S. Pervez, "Toxic metals status in human blood and breast milk samples in an integrated steel plant environment in Central India," Environmental Geochemistry And Health, vol. 27, pp. 39-45, 2005.
[118] H. I. Afridi, T. G. Kazi, N. Kazi, G. A. Kandhro, J. A. Baig, A. Q. Shah, S. Khan, N. F. Kolachi, S. K. Wadhwa, and F. Shah, "Evaluation of zinc, copper and iron in biological samples (scalp hair, blood and urine) of tuberculosis and diarrhea male human immunodeficiency virus patients," Clinical Laboratory, vol. 57, pp. 677-688, 2010.
[119] Q. Pasha, S. A. Malik, and M. H. Shah, "Statistical analysis of trace metals in the plasma of cancer patients versus controls," Journal Of Hazardous Materials, vol. 153, pp. 1215-1221, 2008.
[120] D. Smith, M. Hernandez-Avila, M. M. Téllez-Rojo, A. Mercado, and H. Hu, "The relationship between lead in plasma and whole blood in women," Environmental Health Perspectives, vol. 110, pp. 263-268, 2002.
[121] Ż. Polkowska, K. Kozłowska, J. Namieśnik, and A. Przyjazny, "Biological fluids as a source of information on the exposure of man to environmental chemical agents," Critical Reviews In Analytical Chemistry, vol. 34, pp. 105-119, 2004.
[122] M. Tüzen, "Determination of some trace elements in whole blood and serum by GFAAS," Trace Elements And Electrolytes, vol. 19, pp. 202-204, 2002.
130
[123] K. Srogi, "Heavy metals in human hair samples from Silesia province: the influence of sex, age and smoking habit," Changes, vol. 11, pp. 7-27, 2004.
[124] K. Srogi, "Hair analysis-a tool in biomedical, environmental and forensic sciences: a review of literature published after 1989," Chemia Analityczna, vol. 51, pp. 3-34, 2006.
[125] M. S. Arain, H. I. Afridi, T. G. Kazi, F. N. Talpur, M. B. Arain, A. Kazi, S. A. Arain, and J. Ali, "Correlation of aluminum and manganese concentration in scalp hair samples of patients having neurological disorders," Environmental Monitoring And Assessment, vol. 187, pp. 2-10, 2015.
[126] V. Vickackaite, S. Tautkus, and R. Kazlauskas, "Determination of heavy metals in natural waters by flame atomic absorption spectrometry," Chemia Analityczna, vol. 41, pp. 483-488, 1996.
[127] M. S. Arain, T. G. Kazi, H. I. Afridi, J. Ali, and N. Ullah, "An innovative modified dispersive liquid-phase microextraction method for trace level of iron in serum samples of neurogical disorders patients prior to determine by flame atomic absorption spectrometry," International Journal Of Scientific & Engineering Research, vol. 8, pp. 171-190, 2017.
[128] S. L. Ferreira, W. N. dos Santos, and V. A. Lemos, "On-line preconcentration system for nickel determination in food samples by flame atomic absorption spectrometry," Analytica chimica acta, vol. 445, pp. 145-151, 2001.
[129] F. S. Rojas, C. B. Ojeda, and J. C. Pavón, "Determination of Iron by Dispersive Liquid-Liquid Microextraction Procedure in Environmental Samples," American Journal Of Chemistry, vol. 2, pp. 28-32, 2012.
[130] S. Dadfarnia and A. M. H. Shabani, "Recent development in liquid phase microextraction for determination of trace level concentration of metals—A review," Analytica Chimica Acta, vol. 658, pp. 107-119, 2010.
[131] C. Nerín, "Focus on sample handling," Analytical And Bioanalytical Chemistry, vol. 388, pp. 1001-2, 2007.
[132] H. I. Afridi, T. G. Kazi, N. Kazi, M. K. Jamali, M. B. Arain, N. Jalbani, J. A. Baig, and R. A. Sarfraz, "Evaluation of status of toxic metals in biological samples of diabetes mellitus patients," Diabetes Research And Clinical Practice, vol. 80, pp. 280-288, 2008.
[133] D. Citak, M. Tuzen, and M. Soylak, "Speciation of Mn (II), Mn (VII) and total manganese in water and food samples by coprecipitation–atomic absorption spectrometry combination," Journal Of Hazardous Materials, vol. 173, pp. 773-777, 2010.
[134] T. G. Kazi, G. A. Kandhro, H. I. Afridi, J. A. Baig, A. Q. Shah, S. K. Wadhwa, S. Khan, and N. F. Kolachi, "Evaluation of iodine, iron, and selenium in biological samples of thyroid mother and their newly born babies," Early Human Development, vol. 86, pp. 649-655, 2010.
[135] H. I. Afridi, T. G. Kazi, M. K. Jamali, G. H. Kazi, M. B. Arain, N. Jalbani, and G. Q. Shar, "Analysis of heavy metals in scalp hair samples of hypertensive patients by conventional and microwave digestion methods," Spectroscopy Letters, vol. 39, pp. 203-214, 2006.
[136] H. I. Afridi, T. G. Kazi, N. Kazi, M. K. Jamali, M. B. Arain, N. Jalbani, R. A. Sarfaraz, A. Shah, G. A. Kandhro, and A. Q. Shah, "Potassium, calcium, magnesium, and sodium levels in biological samples of hypertensive and nonhypertensive diabetes mellitus patients," Biological Trace Element Research, vol. 124, p. 206-224, 2008.
131
[137] M. d. A. Bezerra, M. A. Z. Arruda, and S. L. C. Ferreira, "Cloud point extraction as a procedure of separation and pre‐concentration for metal determination using spectroanalytical techniques: a review," Applied Spectroscopy Reviews, vol. 40, pp. 269-299, 2005.
[138] F. Shah, M. Soylak, T. G. Kazi, and H. I. Afridi, "Preconcentration of lead from aqueous solution with activated carbon cloth prior to analysis by flame atomic absorption spectrometry: a multivariate study," Journal Of Analytical Atomic Spectrometry, vol. 28, pp. 601-605, 2013.
[139] L. Zhao, S. Zhong, K. Fang, Z. Qian, and J. Chen, "Determination of cadmium (II), cobalt (II), nickel (II), lead (II), zinc (II), and copper (II) in water samples using dual-cloud point extraction and inductively coupled plasma emission spectrometry," Journal Of Hazardous Materials, vol. 239, pp. 206-212, 2012.
[140] M. Ghaedi, A. Shokrollahi, K. Niknam, E. Niknam, and M. Soylak, "Development of efficient method for preconcentration and determination of copper, nickel, zinc and iron ions in environmental samples by combination of cloud point extraction and flame atomic absorption spectrometry," Open Chemistry, vol. 7, pp. 148-154, 2009.
[141] T. G. Kazi, N. Jalbani, J. A. Baig, H. I. Afridi, G. A. Kandhro, M. B. Arain, M. K. Jamali, and A. Q. Shah, "Determination of toxic elements in infant formulae by using electrothermal atomic absorption spectrometer," Food And Chemical Toxicology, vol. 47, pp. 1425-1429, 2009.
[142] Y. Yamini, M. Faraji, S. Shariati, R. Hassani, and M. Ghambarian, "On-line metals preconcentration and simultaneous determination using cloud point extraction and inductively coupled plasma optical emission spectrometry in water samples," Analytica Chimica Acta, vol. 612, pp. 144-151, 2008.
[143] Ç. A. Şahin, M. Efeçınar, and N. Şatıroğlu, "Combination of cloud point extraction and flame atomic absorption spectrometry for preconcentration and determination of nickel and manganese ions in water and food samples," Journal Of Hazardous Materials, vol. 176, pp. 672-677, 2010.
[144] F. H. Quina and W. L. Hinze, "Surfactant-mediated cloud point extractions: an environmentally benign alternative separation approach," Industrial & Engineering Chemistry Research, vol. 38, pp. 4150-4168, 1999.
[145] C. D. Stalikas, "Micelle-mediated extraction as a tool for separation and preconcentration in metal analysis," Trends In Analytical Chemistry, vol. 21, pp. 343-355, 2002.
[146] V. Doroschuk, S. Lelyushok, V. Ishchenko, and S. Kulichenko, "Flame atomic absorption determination of manganese (II) in natural water after cloud point extraction," Talanta, vol. 64, pp. 853-856, 2004.
[147] S. A. Arain, T. G. Kazi, H. I. Afridi, A. R. Abbasi, A. H. Panhwar, B. Shanker, and M. B. Arain, "Application of dual-cloud point extraction for the trace levels of copper in serum of different viral hepatitis patients by flame atomic absorption spectrometry: a multivariate study," Spectrochimica Acta Part A: Molecular And Biomolecular Spectroscopy, vol. 133, pp. 651-656, 2014.
[148] N. Kolachi, T. Kazi, S. Khan, S. Wadhwa, J. Baig, H. Afridi, A. Shah, and F. Shah, "Multivariate optimization of cloud point extraction procedure for zinc determination in aqueous extracts of medicinal plants by flame atomic absorption spectrometry," Food And Chemical Toxicology, vol. 49, pp. 2548-2556, 2011.
132
[149] A. R. Rod, S. Borhani, and F. Shemirani, "Cloud point preconcentration and flame atomic absorption spectrometry: application to the determination of manganese in milk and water samples," European Food Research And Technology, vol. 223, p. 649-653, 2006.
[150] S. S. Arain, T. G. Kazi, J. B. Arain, H. I. Afridi, and K. D. Brahman, "Preconcentration of toxic elements in artificial saliva extract of different smokeless tobacco products by dual-cloud point extraction," Microchemical Journal, vol. 112, pp. 42-49, 2014.
[151] C. B. Ojeda and F. S. Rojas, "Separation and preconcentration by a cloud point extraction procedure for determination of metals: an overview," Analytical And Bioanalytical Chemistry, vol. 394, pp. 759-782, 2009.
[152] M. S. Arain, T. G. Kazi, H. I. Afridi, S. A. Arain, J. Ali, S. S. Arain, A. H. Panhwar, and B. Shanker, "Preconcentration and determination of manganese in biological samples by dual-cloud point extraction coupled with flame atomic absorption spectrometry," Journal Of Analytical Atomic Spectrometry, vol. 29, pp. 2349-2355, 2014.
[153] M. S. Arain, S. A. Arain, T. G. Kazi, H. I. Afridi, J. Ali, S. S. Arain, K. D. Brahman, and M. A. Mughal, "Temperature controlled ionic liquid-based dispersive micro-extraction using two ligands, for determination of aluminium in scalp hair samples of Alzheimer’s patients: A multivariate study," Spectrochimica Acta Part A: Molecular And Biomolecular Spectroscopy, vol. 137, pp. 877-885, 2015.
[154] M. Rezaee, Y. Yamini, A. Khanchi, M. Faraji, and A. Saleh, "A simple and rapid new dispersive liquid–liquid microextraction based on solidification of floating organic drop combined with inductively coupled plasma-optical emission spectrometry for preconcentration and determination of aluminium in water samples," Journal Of Hazardous Materials, vol. 178, pp. 766-770, 2010.
[155] S. B. Erdemoglu, K. Pyrzyniska, and S. Gucer, "Speciation of aluminum in tea infusion by ion-exchange resins and flame AAS detection," Analytica Chimica Acta, vol. 411, pp. 81-89, 2000.
[156] S. Knezevic, R. Milacic, and M. Veber, "ETAAS determination of aluminium and copper in dialysis concentrates after microcolumn chelating ion-exchange preconcentration," Fresenius' Journal Of Analytical Chemistry, vol. 362, pp. 162-166, 1998.
[157] M. Luo and S. Bi, "Solid phase extraction–spectrophotometric determination of dissolved aluminum in soil extracts and ground waters," Journal Of Inorganic Biochemistry, vol. 97, pp. 173-178, 2003.
[158] L. Xia, B. Hu, Z. Jiang, Y. Wu, L. Li, and R. Chen, "8-Hydroxyquinoline–chloroform single drop microextraction and electrothermal vaporization ICP-MS for the fractionation of aluminium in natural waters and drinks," Journal Of Analytical Atomic Spectrometry, vol. 20, pp. 441-446, 2005.
[159] M. Shokri, A. Beiraghi, and S. Seidi, "In situ emulsification microextraction using a dicationic ionic liquid followed by magnetic assisted physisorption for determination of lead prior to micro-sampling flame atomic absorption spectrometry," Analytica Chimica Acta, vol. 889, pp. 123-129, 2015.
[160] L. Zaijun, C. Jie, S. Haixia, and P. Jiaomai, "Advance of room temperature ionic liquid as solvent for extraction and separation," Reviews In Analytical Chemistry, vol. 26, pp. 109-153, 2007.
[161] F. Shah, T. G. Kazi, H. I. Afridi, and M. Soylak, "Temperature controlled ionic liquid-dispersive liquid phase microextraction for determination of trace lead level in blood
133
samples prior to analysis by flame atomic absorption spectrometry with multivariate optimization," Microchemical Journal, vol. 101, pp. 5-10, 2012.
[162] P. Sun and D. W. Armstrong, "Ionic liquids in analytical chemistry," Analytica Chimica Acta, vol. 661, pp. 1-16, 2010.
[163] A. Berthod, M. Ruiz-Angel, and S. Carda-Broch, "Ionic liquids in separation techniques," Journal Of Chromatography A, vol. 1184, pp. 6-18, 2008.
[164] A. H. Panhwar, T. G. Kazi, H. I. Afridi, A. R. Abbasi, M. B. Arain, S. A. Arain, S. S. Arain, and J. Ali, "Ultrasonic-assisted ionic liquid-based microextraction for preconcentration and determination of aluminum in drinking water, blood and urine samples of kidney failure patients: a multivariate study," Analytical Methods, vol. 6, pp. 8277-8283, 2014.
[165] G.-T. Wei, Z. Yang, and C.-J. Chen, "Room temperature ionic liquid as a novel medium for liquid/liquid extraction of metal ions," Analytica Chimica Acta, vol. 488, pp. 183-192, 2003.
[166] C. E. Song and E. J. Roh, "Practical method to recycle a chiral (salen) Mn epoxidation catalyst by using an ionic liquid," Chemical Communications,vol.10, pp. 837-838, 2000.
[167] D. R. MacFarlane, M. Forsyth, P. C. Howlett, J. M. Pringle, J. Sun, G. Annat, W. Neil, and E. I. Izgorodina, "Ionic liquids in electrochemical devices and processes: managing interfacial electrochemistry," Accounts Of Chemical Research, vol. 40, pp. 1165-1173, 2007.
[168] M. Grotti, F. Soggia, F. Ardini, and R. Frache, "Determination of sub-nanomolar levels of iron in sea-water using reaction cell inductively coupled plasma mass spectrometry after Mg (OH)2 coprecipitation," Journal Of Analytical Atomic Spectrometry, vol. 24, pp. 522-527, 2009.
[169] S. Sacmaci and S. Kartal, "Selective extraction, separation and speciation of iron in different samples using 4-acetyl-5-methyl-1-phenyl-1H-pyrazole-3-carboxylic acid," Analytica Chimica Acta, vol. 623, pp. 46-52, 2008.
[170] G. Khayatian, S. Hassanpoor, F. Nasiri, and A. Zolali, "Preconcentration, determination and speciation of iron by solid-phase extraction using dimethyl (E)-2-[(Z)-1-acetyl)-2-hydroxy-1-propenyl]-2-butenedioate," Química Nova, vol. 35, pp. 535-540, 2012.
[171] E. Pehlivan and D. Kara, "Iron speciation by solid phase extraction and flame atomic absorption spectrometry using N, N′-bis-(2-hydroxy-5-bromobenzyl)-2-hydroxy-1, 3-diiminopropane," Microchimica Acta, vol. 158, pp. 137-144, 2007.
[172] M. Ghaedi, A. Shokrollahi, R. Mehrnoosh, O. Hossaini, and M. Soylak, "Combination of cloud point extraction and flame atomic absorption spectrometry for preconcentration and determination of trace iron in environmental and biological samples," Central European Journal Of Chemistry, vol. 6, pp. 488-496, 2008.
[173] S. Nitiyanontakit, P. Varanusupakul, and P. Ngamukot, "Single strand hollow fiber membrane (SSHFM): An on-line sample preparation for the flow based colorimetric determination of free iron in fruit juices," Talanta, vol. 84, pp. 1304-1308, 2011.
[174] S. A. Arain, T. G. Kazi, H. I. Afridi, A. R. Abbasi, N. Ullah, A. H. Panhwar, and S. Siraj, "Determination of trace levels of iron in serum samples of hepatitis B and C patients using dispersive liquid–liquid microextraction," Analytical Methods, vol. 7, pp. 9211-9217, 2015.
134
[175] J. Chen and K. C. Teo, "Determination of cadmium, copper, lead and zinc in water samples by flame atomic absorption spectrometry after cloud point extraction," Analytica Chimica Acta, vol. 450, pp. 215-222, 2001.
[176] M. Mirzaei and M. Behzadi, "A simple and rapid dispersive liquid–liquid microextraction based on solidification of floating organic drop method combined with flame atomic absorption spectrometry for preconcentration and determination of copper," Journal Of AOAC International, vol. 96, pp. 441-446, 2013.
[177] S. Shariati and M. Golshekan, "Dispersive liquid–liquid microextraction of copper ions as neocuproine complex in environmental aqueous samples," Acta Chim. Slov, vol. 58, pp. 311-317, 2011.
[178] M. S. Arain, T. G. Kazi, H. I. Afridi, J. Ali, and A. Akhtar, "Ultrasonic energy enhanced the efficiency of advance extraction methodology for enrichment of trace level of copper in serum samples of patients having neurological disorders," Ultrasonics Sonochemistry, vol. 37, pp. 23-28, 2017.
[179] N. Manutsewee, W. Aeungmaitrepirom, P. Varanusupakul, and A. Imyim, "Determination of Cd, Cu, and Zn in fish and mussel by AAS after ultrasound-assisted acid leaching extraction," Food Chemistry, vol. 101, pp. 817-824, 2007.
[180] A. Moreno-Cid and M. Yebra, "Flow injection determination of copper in mussels by flame atomic absorption spectrometry after on-line continuous ultrasound-assisted extraction," Spectrochimica Acta Part B: Atomic Spectroscopy, vol. 57, pp. 967-974, 2002.
[181] F. P. Capote and M. L. de Castro, "Ultrasound in analytical chemistry," Analytical And Bioanalytical Chemistry, vol. 387, pp. 249-257, 2007.
[182] T. G. Kazi, H. I. Afridi, G. H. Kazi, M. K. Jamali, M. B. Arain, and N. Jalbani, "Evaluation of essential and toxic metals by ultrasound-assisted acid leaching from scalp hair samples of children with macular degeneration patients," Clinica Chimica Acta, vol. 369, pp. 52-60, 2006.
[183] J. B. Ghasemi and E. Zolfonoun, "Ultrasound-assisted ionic liquid-based microextraction combined with least squares support vector machines regression for the simultaneous determination of aluminum, gallium, and indium in water and coal samples," Environmental Monitoring And Assessment, vol. 184, pp. 3971-3981, 2012.
[184] M. Roosta, M. Ghaedi, N. Shokri, A. Daneshfar, R. Sahraei, and A. Asghari, "Optimization of the combined ultrasonic assisted/adsorption method for the removal of malachite green by gold nanoparticles loaded on activated carbon: experimental design," Spectrochimica Acta Part A: Molecular And Biomolecular Spectroscopy, vol. 118, pp. 55-65, 2014.
[185] A. Väisänen and R. Suontamo, "Comparison of ultrasound-assisted extraction, microwave-assisted acid leaching and reflux for the determination of arsenic, cadmium and copper in contaminated soil samples by electrothermal atomic absorption spectrometry," Journal Of Analytical Atomic Spectrometry, vol. 17, pp. 739-742, 2002.
[186] S. Armenta, S. Garrigues, and M. De la Guardia, "Green analytical chemistry," Trends In Analytical Chemistry, vol. 27, pp. 497-511, 2008.
[187] P. G. Jessop, D. J. Heldebrant, X. Li, C. A. Eckert, and C. L. Liotta, "Green chemistry: Reversible nonpolar-to-polar solvent," Nature, vol. 436, pp. 1102-1102, 2005.
[188] P. G. Jessop, L. Phan, A. Carrier, S. Robinson, C. J. Dürr, and J. R. Harjani, "A solvent having switchable hydrophilicity," Green Chemistry, vol. 12, pp. 809-814, 2010.
135
[189] J. Alauzun, E. Besson, A. Mehdi, C. Reyé, and R. J. Corriu, "Reversible covalent chemistry of CO2: an opportunity for nano-structured hybrid organic–inorganic materials," Chemistry Of Materials, vol. 20, pp. 503-513, 2007.
[190] E. D. Bates, R. D. Mayton, I. Ntai, and J. H. Davis, "CO 2 capture by a task-specific ionic liquid," Journal Of The American Chemical Society, vol. 124, pp. 926-927, 2002.
[191] K. E. Gutowski and E. J. Maginn, "Amine-functionalized task-specific ionic liquids: a mechanistic explanation for the dramatic increase in viscosity upon complexation with CO2 from molecular simulation," Journal Of The American Chemical Society, vol. 130, pp. 14690-14704, 2008.
[192] T. Yamada, P. J. Lukac, M. George, and R. G. Weiss, "Reversible, room-temperature ionic liquids. Amidinium carbamates derived from amidines and aliphatic primary amines with carbon dioxide," Chemistry Of Materials, vol. 19, pp. 967-969, 2007.
[193] D. J. Heldebrant, C. R. Yonker, P. G. Jessop, and L. Phan, "Organic liquid CO 2 capture agents with high gravimetric CO2 capacity," Energy & Environmental Science, vol. 1, pp. 487-493, 2008.
[194] P. K. Kilaru, R. A. Condemarin, and P. Scovazzo, "Correlations of low-pressure carbon dioxide and hydrocarbon solubilities in imidazolium-, phosphonium-, and ammonium-based room-temperature ionic liquids. Part 1. Using surface tension," Industrial & Engineering Chemistry Research, vol. 47, pp. 900-909, 2008.
[195] P. Singh, J. P. Niederer, and G. F. Versteeg, "Structure and activity relationships for amine based CO2 absorbents—I," International Journal Of Greenhouse Gas Control, vol. 1, pp. 5-10, 2007.
[196] T. Yu, T. Yamada, G. C. Gaviola, and R. G. Weiss, "Carbon dioxide and molecular nitrogen as switches between ionic and uncharged room-temperature liquids comprised of amidines and chiral amino alcohols," Chemistry Of Materials, vol. 20, pp. 5337-5344, 2008.
[197] A. P. Abbott, J. C. Barron, K. S. Ryder, and D. Wilson, "Eutectic ‐Based Ionic Liquids with Metal‐Containing Anions and Cations," Chemistry–A European Journal, vol. 13, pp. 6495-6501, 2007.
[198] A. P. Abbott, G. Capper, D. L. Davies, H. L. Munro, R. K. Rasheed, and V. Tambyrajah, "Preparation of novel, moisture-stable, Lewis-acidic ionic liquids containing quaternary ammonium salts with functional side chainsElectronic supplementary information (ESI) available: plot of conductivity vs. temperature for the ionic liquid formed from zinc chloride and choline chloride (2 ∶ 1)." Chemical Communications, pp. 2010-2011, 2001.
[199] A. P. Abbott, G. Capper, D. L. Davies, and R. Rasheed, "Ionic liquids based upon metal halide/substituted quaternary ammonium salt mixtures," Inorganic Chemistry, vol. 43, pp. 3447-3452, 2004.
[200] A. P. Abbott, G. Capper, D. L. Davies, K. J. McKenzie, and S. U. Obi, "Solubility of metal oxides in deep eutectic solvents based on choline chloride," Journal Of Chemical & Engineering Data, vol. 51, pp. 1280-1282, 2006.
[201] M. J. Earle and K. R. Seddon, "Ionic liquids. Green solvents for the future," Pure And Applied Chemistry, vol. 72, pp. 1391-1398, 2000.
[202] M. A. Kareem, F. S. Mjalli, M. A. Hashim, and I. M. AlNashef, "Phosphonium-based ionic liquids analogues and their physical properties," Journal Of Chemical & Engineering Data, vol. 55, pp. 4632-4637, 2010.
136
[203] E. L. Smith, A. P. Abbott, and K. S. Ryder, "Deep eutectic solvents (DESs) and their applications," Chemical Reviews, vol. 114, pp. 11060-11082, 2014.
[204] Q. Zhang, K. D. O. Vigier, S. Royer, and F. Jérôme, "Deep eutectic solvents: syntheses, properties and applications," Chemical Society Reviews, vol. 41, pp. 7108-7146, 2012.
[205] W. Bi, M. Tian, and K. H. Row, "Evaluation of alcohol-based deep eutectic solvent in extraction and determination of flavonoids with response surface methodology optimization," Journal Of Chromatography A, vol. 1285, pp. 22-30, 2013.
[206] E. Habibi, K. Ghanemi, M. Fallah-Mehrjardi, and A. Dadolahi-Sohrab, "A novel digestion method based on a choline chloride–oxalic acid deep eutectic solvent for determining Cu, Fe, and Zn in fish samples," Analytica Chimica Acta, vol. 762, pp. 61-67, 2013.
[207] J. D. Mota‐Morales, M. C. Gutiérrez, M. L. Ferrer, I. C. Sanchez, E. A. Elizalde ‐Peña, J. A. Pojman, F. D. Monte, and G. Luna‐Bárcenas, "Deep eutectic solvents as both active fillers and monomers for frontal polymerization," Journal Of Polymer Science Part A: Polymer Chemistry, vol. 51, pp. 1767-1773, 2013.
[208] Q. Zeng, Y. Wang, Y. Huang, X. Ding, J. Chen, and K. Xu, "Deep eutectic solvents as novel extraction media for protein partitioning ," Analyst, vol. 139, pp. 2565-2573, 2014.
[209] M. G. Pereira and M. A. Z. Arruda, "Trends in preconcentration procedures for metal determination using atomic spectrometry techniques," Microchimica Acta, vol. 141, pp. 115-131, 2003.
[210] E. K. Paleologos, C. D. Stalikas, and M. I. Karayannis, "An optimised single-reagent method for the speciation of chromium by flame atomic absorption spectrometry based on surfactant micelle-mediated methodology," Analyst, vol. 126, pp. 389-393, 2001.
[211] R. M. Martin, Electronic structure: basic theory and practical methods: Cambridge university press, 2004.
[212] A. Safavi, M. Mirzaee, and H. Abdollahi, "Simultaneous spectrophotometric determination of iron, titanium, and aluminum by partial least-squares calibration method in micellar medium," Analytical Letters, vol. 36, pp. 699-712, 2003.
[213] S. Khan, T. G. Kazi, N. F. Kolachi, J. A. Baig, H. I. Afridi, and F. Shah, "A simple separation/preconcentration method for the determination of aluminum in drinking water and biological sample," Desalination, vol. 281, pp. 215-220, 2011.
[214] F. R. de Amorim, C. Bof, M. B. Franco, J. B. B. da Silva, and C. C. Nascentes, "Comparative study of conventional and multivariate methods for aluminum determination in soft drinks by graphite furnace atomic absorption spectrometry," Microchemical Journal, vol. 82, pp. 168-173, 2006.
[215] S. L. Ferreira, W. N. Dos Santos, C. M. Quintella, B. c. B. Neto, and J. M. Bosque-Sendra, "Doehlert matrix: a chemometric tool for analytical chemistry—review," Talanta, vol. 63, pp. 1061-1067, 2004.
[216] I. Lavilla, J. Capelo, and C. Bendicho, "Determination of cadmium and lead in mussels by electrothermal atomic absorption spectrometry using an ultrasound-assisted extraction method optimized by factorial design," Fresenius' Journal Of Analytical Chemistry, vol. 363, pp. 283-288, 1999.
[217] T. Kazi, M. Arain, M. K. Jamali, N. Jalbani, H. Afridi, R. Sarfraz, J. Baig, and A. Q. Shah, "Assessment of water quality of polluted lake using multivariate statistical
137
techniques: A case study," Ecotoxicology And Environmental Safety, vol. 72, pp. 301-309, 2009.
[218] M. Zougagh, A. G. de Torres, and J. M. C. Pavón, "Application of Doehlert matrix and factorial designs in the optimization of experimental variables associated with the on-line preconcentration and determination of zinc by flow injection inductively coupled plasma atomic emission spectrometry," Journal Of Analytical Atomic Spectrometry, vol. 15, pp. 1589-1594, 2000.
[219] S. Khodadoust and M. Ghaedi, "Optimization of dispersive liquid–liquid microextraction with central composite design for preconcentration of chlordiazepoxide drug and its determination by HPLC‐UV," Journal Of Separation Science, vol. 36, pp. 1734-1742, 2013.
[220] R. H. Myers, D. C. Montgomery, G. G. Vining, C. M. Borror, and S. M. Kowalski, "Response surface methodology: a retrospective and literature survey," Journal Of Quality Technology, vol. 36, pp. 53-77, 2004.
[221] J. Emerit, M. Edeas, and F. Bricaire, "Neurodegenerative diseases and oxidative stress," Biomedicine & Pharmacotherapy, vol. 58, pp. 39-46, 2004.
[222] J. K. Andersen, "Oxidative stress in neurodegeneration: cause or consequence?," pp. 18-25, 2004.
[223] J. S. Bains and C. A. Shaw, "Neurodegenerative disorders in humans: the role of glutathione in oxidative stress-mediated neuronal death," Brain Research Reviews, vol. 25, pp. 335-358, 1997.
[224] M. F. Beal, "Aging, energy, and oxidative stress in neurodegenerative diseases," Annals Of Neurology, vol. 38, pp. 357-366, 1995.
[225] G. Benzi and A. Moretti, "Age-and peroxidative stress-related modifications of the cerebral enzymatic activities linked to mitochondria and the glutathione system," Free Radical Biology And Medicine, vol. 19, pp. 77-101, 1995.
[226] B. Halliwell, A. Zentella, E. O. Gomez, and D. Kershenobich, "Antioxidants and human disease: a general introduction," Nutrition Reviews, vol. 55, p. S44-S52, 1997.
[227] P. Evans, "Free radicals in brain metabolism and pathology," British Medical Bulletin, vol. 49, pp. 577-587, 1993.
[228] B. Halliwell, "Cellular stress and protection mechanisms," Biochem Soc Trans, vol. 24, pp. 1023-1027, 1996.
[229] R. J. Reiter, "Oxidative processes and antioxidative defense mechanisms in the aging brain," The FASEB Journal, vol. 9, pp. 526-533, 1995.
[230] N. Simonian and J. Coyle, "Oxidative stress in neurodegenerative diseases," Annual Review Of Pharmacology And Toxicology, vol. 36, pp. 83-106, 1996.
[231] J.-Y. Li, E. Englund, J. L. Holton, D. Soulet, P. Hagell, A. J. Lees, T. Lashley, N. P. Quinn, S. Rehncrona, and A. Björklund, "Lewy bodies in grafted neurons in subjects with Parkinson's disease suggest host-to-graft disease propagation," Nature Medicine, vol. 14, pp. 501-503, 2008.
[232] S. Fahn and G. Cohen, "The oxidant stress hypothesis in Parkinson's disease: evidence supporting it," Annals Of Neurology, vol. 32, pp. 804-812, 1992.
[233] K. Jellinger, "Cell death mechanisms in Parkinson's disease," Journal Of Neural Transmission, vol. 107, pp. 1-29, 2000.
138
[234] P. Foley and P. Riederer, "Influence of neurotoxins and oxidative stress on the onset and progression of Parkinson’s disease," Journal Of Neurology, vol. 247, pp. II82-II94, 2000.
[235] C. D. Saunders and K. C. Allison, Parkinson's disease: a new hope: Harvard Health Publications, 2000.
[236] P. Jenner, "Oxidative mechanisms in nigral cell death in Parkinson's disease," Movement disorders: Official journal of the Movement Disorder Society, vol. 13, pp. 24-34, 1997.
[237] A. Tessitore, A. R. Hariri, F. Fera, W. G. Smith, T. N. Chase, T. M. Hyde, D. R. Weinberger, and V. S. Mattay, "Dopamine modulates the response of the human amygdala: a study in Parkinson's disease," Journal Of Neuroscience, vol. 22, pp. 9099-9103, 2002.
[238] J. Xu, S.-Y. Kao, F. J. Lee, W. Song, L.-W. Jin, and B. A. Yankner, "Dopamine-dependent neurotoxicity of α-synuclein: a mechanism for selective neurodegeneration in Parkinson disease," Nature Medicine, vol. 8, pp. 600-606, 2002.
[239] H. Braak, E. Ghebremedhin, U. Rüb, H. Bratzke, and K. Del Tredici, "Stages in the development of Parkinson’s disease-related pathology," Cell And Tissue Research, vol. 318, pp. 121-134, 2004.
[240] R. Abbott, G. Ross, L. White, C. Tanner, K. Masaki, J. Nelson, J. Curb, and H. Petrovitch, "Excessive daytime sleepiness and subsequent development of Parkinson disease," Neurology, vol. 65, pp. 1442-1446, 2005.
[241] R. D. Abbott, G. W. Ross, H. Petrovitch, C. M. Tanner, D. G. Davis, K. H. Masaki, L. J. Launer, J. D. Curb, and L. R. White, "Bowel movement frequency in late ‐life and incidental Lewy bodies," Movement Disorders, vol. 22, pp. 1581-1586, 2007.
[242] A. Haehner, T. Hummel, C. Hummel, U. Sommer, S. Junghanns, and H. Reichmann, "Olfactory loss may be a first sign of idiopathic Parkinson's disease," Movement Disorders, vol. 22, pp. 839-842, 2007.
[243] S. K. Van Den Eeden, C. M. Tanner, A. L. Bernstein, R. D. Fross, A. Leimpeter, D. A. Bloch, and L. M. Nelson, "Incidence of Parkinson’s disease: variation by age, gender, and race/ethnicity," American Journal Of Epidemiology, vol. 157, pp. 1015-1022, 2003.
[244] F. M. LaFerla, K. N. Green, and S. Oddo, "Intracellular amyloid-β in Alzheimer's disease," Nature Reviews Neuroscience, vol. 8, pp. 499-509, 2007.
[245] P. Zatta, D. Drago, S. Bolognin, and S. L. Sensi, "Alzheimer's disease, metal ions and metal homeostatic therapy," Trends In Pharmacological Sciences, vol. 30, pp. 346-355, 2009.
[246] C. C. Curtain, F. Ali, I. Volitakis, R. A. Cherny, R. S. Norton, K. Beyreuther, C. J. Barrow, C. L. Masters, A. I. Bush, and K. J. Barnham, "Alzheimer's disease amyloid-β binds copper and zinc to generate an allosterically ordered membrane-penetrating structure containing superoxide dismutase-like subunits," Journal Of Biological Chemistry, vol. 276, pp. 20466-20473, 2001.
[247] A. Dedeoglu, K. Cormier, S. Payton, K. A. Tseitlin, J. N. Kremsky, L. Lai, X. Li, R. D. Moir, R. E. Tanzi, and A. I. Bush, "Preliminary studies of a novel bifunctional metal chelator targeting Alzheimer's amyloidogenesis," Experimental Gerontology, vol. 39, pp. 1641-1649, 2004.
[248] X. Huang, C. S. Atwood, M. A. Hartshorn, G. Multhaup, L. E. Goldstein, R. C. Scarpa, M. P. Cuajungco, D. N. Gray, J. Lim, and R. D. Moir, "The Aβ peptide of Alzheimer's
139
disease directly produces hydrogen peroxide through metal ion reduction," Biochemistry, vol. 38, pp. 7609-7616, 1999.
[249] L. E. Hebert, P. A. Scherr, L. A. Beckett, M. S. Albert, D. M. Pilgrim, M. J. Chown, H. H. Funkenstein, and D. A. Evans, "Age-specific incidence of Alzheimer's disease in a community population," Jama, vol. 273, pp. 1354-1359, 1995.
[250] C. R. Jack, M. S. Albert, D. S. Knopman, G. M. McKhann, R. A. Sperling, M. C. Carrillo, B. Thies, and C. H. Phelps, "Introduction to the recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease," Alzheimer's & Dementia, vol. 7, pp. 257-262, 2011.
[251] M. J. West, P. D. Coleman, D. G. Flood, and J. C. Troncoso, "Differences in the pattern of hippocampal neuronal loss in normal ageing and Alzheimer's disease," The Lancet, vol. 344, pp. 769-772, 1994.
[252] J. Walton, "Aluminum in hippocampal neurons from humans with Alzheimer's disease," Neurotoxicology, vol. 27, pp. 385-394, 2006.
[253] J. Busser, D. S. Geldmacher, and K. Herrup, "Ectopic cell cycle proteins predict the sites of neuronal cell death in Alzheimer’s disease brain," Journal Of Neuroscience, vol. 18, pp. 2801-2807, 1998.
[254] W. R. Markesbery, "Oxidative stress hypothesis in Alzheimer's disease," Free Radical Biology And Medicine, vol. 23, pp. 134-147, 1997.
[255] D. Praticò, "Evidence of oxidative stress in Alzheimer's disease brain and antioxidant therapy," Annals Of The New York Academy Of Sciences, vol. 1147, pp. 70-78, 2008.
[256] H. McFarland and D. McFarlin, "Immunologically mediated demyelinating diseases of the central and peripheral nervous system," Samter's Immunologic Diseases. Philadelphia, PA: lippincott williams & wilkins, pp. 1081-101, 2001.
[257] L. Brundin, E. Morcos, T. Olsson, N. P. Wiklund, and M. Andersson, "Increased intrathecal nitric oxide formation in multiple sclerosis; cerebrospinal fluid nitrite as activity marker," European Journal Of Neurology, vol. 6, pp. 585-590, 1999.
[258] E. Santiago, L. Perez-Mediavilla, and N. Lopez-Moratalla, "The role of nitric oxide in the pathogenesis of multiple sclerosis," Journal Of Physiology And Biochemistry, vol. 54, pp. 229-237, 1998.
[259] S. J. Heales, J. P. Bolaños, V. C. Stewart, P. S. Brookes, J. M. Land, and J. B. Clark, "Nitric oxide, mitochondria and neurological disease," Biochimica Et Biophysica Acta (BBA)-Bioenergetics, vol. 1410, pp. 215-228, 1999.
[260] J. E. Merrill and E. N. Benveniste, "Cytokines in inflammatory brain lesions: helpful and harmful," Trends In Neurosciences, vol. 19, pp. 331-338, 1996.
[261] K. J. Smith, R. Kapoor, and P. A. Felts, "Demyelination: the role of reactive oxygen and nitrogen species," Brain Pathology, vol. 9, pp. 69-92, 1999.
[262] R. Dutta and B. D. Trapp, "Mechanisms of neuronal dysfunction and degeneration in multiple sclerosis," Progress In Neurobiology, vol. 93, pp. 1-12, 2011.
[263] J. M. Frischer, S. Bramow, A. Dal-Bianco, C. F. Lucchinetti, H. Rauschka, M. Schmidbauer, H. Laursen, P. S. Sorensen, and H. Lassmann, "The relation between inflammation and neurodegeneration in multiple sclerosis brains," Brain, vol. 132, pp. 1175-1189, 2009.
[264] C. Bjartmar, R. P. Kinkel, G. Kidd, R. A. Rudick, and B. D. Trapp, "Axonal loss in normal-appearing white matter in a patient with acute MS," Neurology, vol. 57, pp. 1248-1252, 2001.
140
[265] B. Ferguson, M. K. Matyszak, M. M. Esiri, and V. H. Perry, "Axonal damage in acute multiple sclerosis lesions," Brain, vol. 120, pp. 393-399, 1997.
[266] P. Ganter, C. Prince, and M. M. Esiri, "Spinal cord axonal loss in multiple sclerosis: a post-mortem study," Neuropathology And Applied Neurobiology, vol. 25, pp. 459-467, 1999.
[267] G. Lovas, N. Szilágyi, K. Majtényi, M. Palkovits, and S. Komoly, "Axonal changes in chronic demyelinated cervical spinal cord plaques," Brain, vol. 123, pp. 308-317, 2000.
[268] B. D. Trapp, J. Peterson, R. M. Ransohoff, R. Rudick, S. Mörk, and L. Bö, "Axonal transection in the lesions of multiple sclerosis," New England Journal Of Medicine, vol. 338, pp. 278-285, 1998.
[269] D. H. Miller, F. Barkhof, J. A. Frank, G. J. Parker, and A. J. Thompson, "Measurement of atrophy in multiple sclerosis: pathological basis, methodological aspects and clinical relevance," Brain, vol. 125, pp. 1676-1695, 2002.
[270] R. Rudick, E. Fisher, J.-C. Lee, J. Simon, L. Jacobs, and M. S. C. R. Group, "Use of the brain parenchymal fraction to measure whole brain atrophy in relapsing-remitting MS," Neurology, vol. 53, pp. 1698-1698, 1999.
[271] J. Simon, L. Jacobs, M. Campion, R. Rudick, D. Cookfair, R. Herndon, J. Richert, A. Salazar, J. Fischer, and D. Goodkin, "A longitudinal study of brain atrophy in relapsing multiple sclerosis," Neurology, vol. 53, pp. 139-139, 1999.
[272] D. L. Arnold, G. T. Riess, P. M. Matthews, G. S. Francis, D. L. Collins, C. Wolfson, and J. P. Antel, "Use of proton magnetic resonance spectroscopy for monitoring disease progression in multiple sclerosis," Annals Of Neurology, vol. 36, pp. 76-82, 1994.
[273] N. De Stefano, P. M. Matthews, L. Fu, S. Narayanan, J. Stanley, G. S. Francis, J. P. Antel, and D. L. Arnold, "Axonal damage correlates with disability in patients with relapsing-remitting multiple sclerosis. Results of a longitudinal magnetic resonance spectroscopy study," Brain, vol. 121, pp. 1469-1477, 1998.
[274] P. Matthews, E. Pioro, S. Narayanan, N. De Stefano, L. Fu, G. Francis, J. Antel, C. Wolfson, and D. Arnold, "Assessment of lesion pathology in multiple sclerosis using quantitative MRI morphometry and magnetic resonance spectroscopy," Brain, vol. 119, pp. 715-722, 1996.
[275] S. L. Hauser and J. R. Oksenberg, "The neurobiology of multiple sclerosis: genes, inflammation, and neurodegeneration," Neuron, vol. 52, pp. 61-76, 2006.
[276] J. H. Noseworthy, C. Lucchinetti, M. M Rodriguez, and B. G. Weinshenker, "Medical progress," multiple sclerosis. New England Journal Of Medicine, vol. 343, pp. 938-952, 2000.
[277] B. G. Weinshenker, "Epidemiology of multiple sclerosis," Neurologic Clinics, vol. 14, pp. 291-308, 1996.
[278] T. Ho, B. Mishu, C. Li, C. Gao, D. Cornblath, J. Griffin, A. Asbury, M. Blaser, and G. McKhann, "Guillain-Barre syndrome in northern China Relationship to Campylobacter jejuni infection and anti-glycolipid antibodies," Brain, vol. 118, pp. 597-605, 1995.
[279] M. S. Hansen, P. Fink, L. Søndergaard, and M. Frydenberg, "Mental illness and health care use: a study among new neurological patients," General Hospital psychiatry, vol. 27, pp. 119-124, 2005.
[280] W. Owiredu, O. Osei, N. Amidu, J. Appiah-Poku, and Y. Osei, "Prevalence of metabolic syndrome among Psychiatric Patients in the Kumasi Metropolis, Ghana," Journal of Medical And Biomedical Sciences, vol. 1, pp. 38-49, 2012.
141
[281] E. Fuller, M. Torrey, and M. B. Knable, "Are Schizophrenia and Bipolar Disorder One Disease or Two?: Introduction to the Symposium," Schizophrenia Research, vol. 39, pp. 93-94, 1999.
[282] R. G. Steen, C. Mull, R. Mcclure, R. M. Hamer, and J. A. Lieberman, "Brain volume in first-episode schizophrenia," The British Journal Of Psychiatry, vol. 188, pp. 510-518, 2006.
[283] R. M. Bilder, H. Wu, B. Bogerts, M. Ashtari, D. Robinson, M. Woerner, J. A. Lieberman, and G. Degreef, "Cerebral volume asymmetries in schizophrenia and mood disorders: a quantitative magnetic resonance imaging study," International Journal Of Psychophysiology, vol. 34, pp. 197-205, 1999.
[284] E. A. Hoge, L. Friedman, and S. C. Schulz, "Meta-analysis of brain size in bipolar disorder," Schizophrenia Research, vol. 37, pp. 177-181, 1999.
[285] I. C. Wright, S. Rabe-Hesketh, P. W. Woodruff, A. S. David, R. M. Murray, and E. T. Bullmore, "Meta-analysis of regional brain volumes in schizophrenia," American Journal Of Psychiatry, vol. 157, pp. 16-25, 2000.
[286] H. Elkis, L. Friedman, A. Wise, and H. Y. Meltzer, "Meta-analyses of studies of ventricular enlargement and cortical sulcal prominence in mood disorders: comparisons with controls or patients with schizophrenia," Archives Of General Psychiatry, vol. 52, pp. 735-746, 1995.
[287] C. E. Bearden, K. M. Hoffman, and T. D. Cannon, "The neuropsychology and neuroanatomy of bipolar affective disorder: a critical review," Bipolar Disorders, vol. 3, pp. 106-150, 2001.
[288] P. Brambilla, K. Harenski, M. Nicoletti, A. G. Mallinger, E. Frank, D. J. Kupfer, M. S. Keshavan, and J. C. Soares, "MRI study of posterior fossa structures and brain ventricles in bipolar patients," Journal Of Psychiatric Research, vol. 35, pp. 313-322, 2001.
[289] S. M. Strakowski, M. P. DelBello, M. E. Zimmerman, G. E. Getz, N. P. Mills, J. Ret, P. Shear, and C. M. Adler, "Ventricular and periventricular structural volumes in first-versus multiple-episode bipolar disorder," American Journal Of Psychiatry, vol. 159, pp. 1841-1847, 2002.
[290] H. Hafner, W. Löffler, K. Maurer, and M. Hambrecht, "Depression, negative symptoms, social stagnation and social decline in the early course of schizophrenia," Acta Psychiatrica Scandinavica, vol. 100, pp. 105-118, 1999.
[291] A. R. Koreen, S. G. Siris, M. Chakos, and J. Alvir, "Depression in first-episode schizophrenia," The American Journal Of Psychiatry, vol. 150, pp. 1643-1648, 1993.
[292] F. Murphy, B. Sahakian, J. Rubinsztein, A. Michael, R. Rogers, T. Robbins, and E. Paykel, "Emotional bias and inhibitory control processes in mania and depression," Psychological Medicine, vol. 29, pp. 1307-1321, 1999.
[293] J. Adair, "Trace element and selenium speciation analysis of human body fluids by ICP MS," University of surrey, 2002.
[294] R. Hänsch and R. R. Mendel, "Physiological functions of mineral micronutrients (Cu, Zn, Mn, Fe, Ni, Mo, B, Cl)," Current Opinion In Plant Biology, vol. 12, pp. 259-266, 2009.
[295] S. B. Goldhaber, "Trace element risk assessment: essentiality vs. toxicity," Regulatory Toxicology And Pharmacology, vol. 38, pp. 232-242, 2003.
142
[296] A. I. Bush, "The metallobiology of Alzheimer's disease," Trends In Neurosciences, vol. 26, pp. 207-214, 2003.
[297] M. Lovell, J. Robertson, W. Teesdale, J. Campbell, and W. Markesbery, "Copper, iron and zinc in Alzheimer's disease senile plaques," Journal Of The Neurological Sciences, vol. 158, pp. 47-52, 1998.
[298] J. A. Wright and D. R. Brown, "Alpha‐synuclein and its role in metal binding: Relevance to Parkinson's disease," Journal Of Neuroscience Research, vol. 86, pp. 496-503, 2008.
[299] P. Lima, M. Vasconcellos, R. Montenegro, and R. Burbano, Alzheimer's Disease and Metal Contamination: Aspects on genotoxicity: INTECH open access publisher, 2011.
[300] L. Kochian and D. Jones, "Aluminum toxicity and resistance in plants," Research Issues in Aluminum Toxicity. Eds. RA Yokel and MS Golub. Taylor and francis publishers, bristol, PA, 1997.
[301] A. Shokrollahi, M. Ghaedi, M. Niband, and H. Rajabi, "Selective and sensitive spectrophotometric method for determination of sub-micro-molar amounts of aluminium ion," Journal Of Hazardous Materials, vol. 151, pp. 642-648, 2008.
[302] W. H. Organization, "Environmental health criteria for aluminium," Environmental Health Criteria, 1997.
[303] R. F. Itzhaki, "Possible factors in the etiology of Alzheimer's disease," Molecular neurobiology, vol. 9, pp. 1-13, 1994.
[304] N. Roberts, A. Clough, J. Bellia, and J. Kim, "Increased absorption of aluminium from a normal dietary intake in dementia," Journal Of Inorganic Biochemistry, vol. 69, pp. 171-176, 1998.
[305] C. Exley, "Human exposure to aluminium," Environmental Science: Processes & Impacts, vol. 15, pp. 1807-1816, 2013.
[306] R. J. Mailloux, R. Hamel, and V. D. Appanna, "Aluminum toxicity elicits a dysfunctional TCA cycle and succinate accumulation in hepatocytes," Journal Of Biochemical And Molecular Toxicology, vol. 20, pp. 198-208, 2006.
[307] S. Candura, L. Manzo, and L. Costa, "Role of occupational neurotoxicants in psychiatric and neurodegenerative disorders," by Costa LG, Manzo L, pp. 131-67, 1998.
[308] A. Becaria, A. Campbell, and S. Bondy, "Aluminum as a toxicant," Toxicology And Industrial Health, vol. 18, pp. 309-320, 2002.
[309] R. A. Yokel, "Brain uptake, retention, and efflux of aluminum and manganese," Environmental Health Perspectives, vol. 110, p. 699-704, 2002.
[310] C. Exley, "Elucidating aluminium's exposome," Current Inorganic Chemistry, vol. 2, pp. 3-7, 2012.
[311] C. Exley, "A biogeochemical cycle for aluminium?," Journal Of Inorganic Biochemistry, vol. 97, pp. 1-7, 2003.
[312] C. Exley and E. R. House, Aluminium in the human brain: Monatshefte Für Chemie-Chemical Monthly, vol. 142, pp. 357-363, 2011.
[313] O. Egbuna and A. Bose, "Acute aluminum neurotoxicity secondary to treatment of severe hyperphosphatemia of acute renal failure and the K/DOQI guidelines: a case report and review of the literature," Journal Nephrol, vol. 2, pp. 1-8, 2005.
[314] I. Rodushkin and M. D. Axelsson, "Application of double focusing sector field ICP-MS for multielemental characterization of human hair and nails. Part II. A study of the
143
inhabitants of northern Sweden," Science Of The Total Environment, vol. 262, pp. 21-36, 2000.
[315] R. M. Bowler, S. Gysens, E. Diamond, S. Nakagawa, M. Drezgic, and H. A. Roels, "Manganese exposure: neuropsychological and neurological symptoms and effects in welders," Neurotoxicology, vol. 27, pp. 315-326, 2006.
[316] P. K. Pal, A. Samii, and D. Calne, "Manganese neurotoxicity: a review of clinical features, imaging and pathology," Neurotoxicology, vol. 20, pp. 227-238, 1998.
[317] B. Sjögren, A. Iregren, W. Frech, M. Hagman, L. Johansson, M. Tesarz, and A. Wennberg, "Effects on the nervous system among welders exposed to aluminium and manganese," Occupational And Environmental Medicine, vol. 53, pp. 32-40, 1996.
[318] B. S. Levy and W. J. Nassetta, "Neurologic effects of manganese in humans: a review," International Journal Of Occupational And Environmental Health, vol. 9, pp. 153-163, 2003.
[319] M. S. Arain, H. I. Afridi, T. G. Kazi, A. Kazi, J. Ali, S. A. Arain, and A. H. Panhwar, "Variation in the Levels of Aluminum and Manganese in Scalp Hair Samples of the Patients Having Different Psychiatric Disorders with Related to Healthy Subjects," Biological Trace Element Research, vol. 168, pp. 67-73, 2015.
[320] J. Crossgrove and W. Zheng, "Manganese toxicity upon overexposure," Nuclear Magnetic Resonance In Biomedicine, vol. 17, pp. 544-553, 2004.
[321] L. Soko, L. Chimuka, E. Cukrowska, and S. Pole, "Extraction and preconcentration of manganese (II) from biological fluids (water, milk and blood serum) using supported liquid membrane and membrane probe methods," Analytica Chimica Acta, vol. 485, pp. 25-35, 2003.
[322] R. A. Yokel, "Blood-brain barrier flux of aluminum, manganese, iron and other metals suspected to contribute to metal-induced neurodegeneration," Journal Of Alzheimer's Disease, vol. 10, pp. 223-253, 2006.
[323] N. Inoue, "Occupational neurotoxicology due to heavy metals-especially manganese poisoning," Brain And Nerve, vol. 59, pp. 581-589, 2007.
[324] D. Mergler, G. Huel, R. Bowler, A. Iregren, S. Belanger, M. Baldwin, R. Tardif, A. Smargiassi, and L. Martin, "Nervous system dysfunction among workers with long-term exposure to manganese," Environmental Research, vol. 64, pp. 151-180, 1994.
[325] J. Fell, N. Meadows, K. Khan, S. Long, P. Milla, A. Reynolds, G. Quaghebeur, and W. Taylor, "Manganese toxicity in children receiving long-term parenteral nutrition," The Lancet, vol. 347, pp. 1218-1221, 1996.
[326] A. Woolf, R. Wright, C. Amarasiriwardena, and D. Bellinger, "A child with chronic manganese exposure from drinking water," Environmental Health Perspectives, vol. 110, p. 613-616, 2002.
[327] L. Zecca, M. Gallorini, V. Schünemann, A. X. Trautwein, M. Gerlach, P. Riederer, P. Vezzoni, and D. Tampellini, "Iron, neuromelanin and ferritin content in the substantia nigra of normal subjects at different ages: consequences for iron storage and neurodegenerative processes," Journal Of Neurochemistry, vol. 76, pp. 1766-1773, 2001.
[328] P. Ponka, "Cellular iron metabolism," Kidney International, vol. 55, pp. S2-S11, 1999.
[329] N. C. Andrews and P. J. Schmidt, "Iron homeostasis," Annual Review of Physiology, vol. 69, pp. 69-85, 2007.
144
[330] P. Ponka, "Hereditary causes of disturbed iron homeostasis in the central nervous system," Annals Of The New York Academy Of Sciences, vol. 1012, pp. 267-281, 2004.
[331] M. Thomas and J. Jankovic, "Neurodegenerative disease and iron storage in the brain," Current Opinion In Neurology, vol. 17, pp. 437-442, 2004.
[332] K. L. Double, M. Gerlach, V. Schünemann, A. X. Trautwein, L. Zecca, M. Gallorini, M. B. Youdim, P. Riederer, and D. Ben-Shachar, "Iron-binding characteristics of neuromelanin of the human substantia nigra," Biochemical Pharmacology, vol. 66, pp. 489-494, 2003.
[333] G. A. Salvador, R. M. Uranga, and N. M. Giusto, "Iron and mechanisms of neurotoxicity," International Journal Of Alzheimer’s Disease, vol. 2011,pp. 1-9, 2010.
[334] M. A. Smith, K. Wehr, P. L. Harris, S. L. Siedlak, J. R. Connor, and G. Perry, "Abnormal localization of iron regulatory protein in Alzheimer's disease," Brain Research, vol. 788, pp. 232-236, 1998.
[335] R. J. Castellani, P. L. Harris, A. Lecroisey, N. Izadi-Pruneyre, C. Wandersman, G. Perry, and M. A. Smith, "Evidence for a novel heme-binding protein, HasAh, in Alzheimer disease," Antioxidants & Redox Signaling, vol. 2, pp. 137-142, 2000.
[336] P. Odetti, S. Garibaldi, R. Norese, G. Angelini, L. Marinelli, S. Valentini, S. Menini, N. Traverso, D. Zaccheo, and S. Siedlak, "Lipoperoxidation is selectively involved in progressive supranuclear palsy," Journal Of Neuropathology & Experimental Neurology, vol. 59, pp. 393-397, 2000.
[337] T. Kawamata, I. Tooyama, T. Yamada, D. G. Walker, and P. L. McGeer, "Lactotransferrin immunocytochemistry in Alzheimer and normal human brain," The American Journal Of Pathology, vol. 142, pp. 1574-1585, 1993.
[338] R. J. Castellani, S. L. Siedlak, G. Perry, and M. A. Smith, "Sequestration of iron by Lewy bodies in Parkinson’s disease," Acta Neuropathologica, vol. 100, pp. 111-114, 2000.
[339] Z. M. Qian and Q. Wang, "Expression of iron transport proteins and excessive iron accumulation in the brain in neurodegenerative disorders," Brain Research Reviews, vol. 27, pp. 257-267, 1998.
[340] A. H. Koeppen, "A brief history of brain iron research," Journal Of The Neurological Sciences, vol. 207, pp. 95-97, 2003.
[341] P. T. Lieu, M. Heiskala, P. A. Peterson, and Y. Yang, "The roles of iron in health and disease," Molecular Aspects Of Medicine, vol. 22, pp. 1-87, 2001.
[342] J. Connor, B. Snyder, J. Beard, R. Fine, and E. Mufson, "Regional distribution of iron and iron‐regulatory proteins in the brain in aging and Alzheimer's disease," Journal Of Neuroscience Research, vol. 31, pp. 327-335, 1992.
[343] E. D. Gaier, B. A. Eipper, and R. E. Mains, "Copper signaling in the mammalian nervous system: synaptic effects," Journal Of Neuroscience Research, vol. 91, pp. 2-19, 2013.
[344] G. G. Zucconi, S. Cipriani, R. Scattoni, I. Balgkouranidou, D. Hawkins, and K. Ragnarsdottir, "Copper deficiency elicits glial and neuronal response typical of neurodegenerative disorders," Neuropathology And Applied Neurobiology, vol. 33, pp. 212-225, 2007.
[345] M. Mazloum-Ardakani, Z. Akrami, H. Kazemian, and H. R. Zare, "Preconcentration and electroanalysis of copper at zeolite modified carbon paste electrode," International Journal Of Electrochemical Science, vol. 4, pp. 308-319, 2009.
145
[346] C. S. Atwood, R. C. Scarpa, X. Huang, R. D. Moir, W. D. Jones, D. P. Fairlie, R. E. Tanzi, and A. I. Bush, "Characterization of copper interactions with Alzheimer amyloid β peptides," Journal Of Neurochemistry, vol. 75, pp. 1219-1233, 2000.
[347] J. R. Turnlund, R. A. Jacob, C. L. Keen, J. Strain, D. S. Kelley, J. M. Domek, W. R. Keyes, J. L. Ensunsa, J. Lykkesfeldt, and J. Coulter, "Long-term high copper intake: effects on indexes of copper status, antioxidant status, and immune function in young men," The American Journal Of Clinical Nutrition, vol. 79, pp. 1037-1044, 2004.
[348] E. Madsen and J. D. Gitlin, "Copper and iron disorders of the brain," Annual Review of Neuroscience, vol. 30, pp. 317-337, 2007.
[349] J. Tolls, "Book Review: Environmental Chemistry: A Global Perspective," Human & Experimental Toxicology, vol. 19, pp. 540-540, 2000.
[350] L. J. Harvey and H. J. McArdle, "Biomarkers of copper status: a brief update," British Journal Of Nutrition, vol. 99, pp. S10-S13, 2008.
[351] S. Lutsenko, "Human copper homeostasis: a network of interconnected pathways," Current Opinion In Chemical Biology, vol. 14, pp. 211-217, 2010.
[352] C. Frederickson and D. Moncrieff, "Zinc-containing neurons," Neurosignals, vol. 3, pp. 127-139, 1994.
[353] H. H. Sandstead, C. J. Frederickson, and J. G. Penland, "History of zinc as related to brain function," The Journal Of Nutrition, vol. 130, pp. 496S-502S, 2000.
[354] M. Maes, E. Vandoolaeghe, H. Neels, P. Demedts, A. Wauters, H. Y. Meltzer, C. Altamura, and R. Desnyder, "Lower serum zinc in major depression is a sensitive marker of treatment resistance and of the immune/inflammatory response in that illness," Biological Psychiatry, vol. 42, pp. 349-358, 1997.
[355] T. Pepersack, P. Rotsaert, F. Benoit, D. Willems, M. Fuss, P. Bourdoux, and J. Duchateau, "Prevalence of zinc deficiency and its clinical relevance among hospitalised elderly," Archives Of Gerontology And Geriatrics, vol. 33, pp. 243-253, 2001.
[356] P. Mecocci, A. Cherubini, E. Mariani, C. Ruggiero, and U. Senin, "Depression in the elderly: new concepts and therapeutic approaches," Aging Clinical And Experimental Research, vol. 16, pp. 176-189, 2004.
[357] M. a. J. Salgueiro, M. B. Zubillaga, A. E. Lysionek, R. A. Caro, R. Weill, and J. R. Boccio, "The role of zinc in the growth and development of children," Nutrition, vol. 18, pp. 510-519, 2002.
[358] B. L. Vallee, J. E. Coleman, and D. S. Auld, "Zinc fingers, zinc clusters, and zinc twists in DNA-binding protein domains," Proceedings Of The National Academy Of Sciences, vol. 88, pp. 999-1003, 1991.
[359] B. L. Vallee and K. H. Falchuk, "The biochemical basis of zinc physiology," Physiological reviews, vol. 73, pp. 79-118, 1993.
[360] R. Tudor, P. Zalewski, and R. Ratnaike, "Zinc in health and chronic disease," The Journal Of Nutrition, Health & Aging, vol. 9, pp. 45-51, 2004.
[361] Z. Wang, J.-Y. Li, A. Dahlstrom, and G. Danscher, "Zinc-enriched GABAergic terminals in mouse spinal cord," Brain Research, vol. 921, pp. 165-172, 2001.
[362] A. Takeda, M. Nakamura, H. Fujii, and H. Tamano, "Synaptic Zn 2+ homeostasis and its significance," Metallomics, vol. 5, pp. 417-423, 2013.
146
[363] V. Bancila, I. Nikonenko, Y. Dunant, and A. Bloc, "Zinc inhibits glutamate release via activation of pre‐synaptic KATP channels and reduces ischaemic damage in rat hippocampus," Journal Of Neurochemistry, vol. 90, pp. 1243-1250, 2004.
[364] S. L. Sensi, P. Paoletti, A. I. Bush, and I. Sekler, "Zinc in the physiology and pathology of the CNS," Nature Reviews Neuroscience, vol. 10, pp. 780-791, 2009.
[365] A. Kocyigit, F. Armutcu, A. Gurel, and B. Ermis, "Alterations in plasma essential trace elements selenium, manganese, zinc, copper, and iron concentrations and the possible role of these elements on oxidative status in patients with childhood asthma," Biological Trace Element Research, vol. 97, pp. 31-41, 2004.
[366] S.-J. Lee and J.-Y. Koh, "Roles of zinc and metallothionein-3 in oxidative stress-induced lysosomal dysfunction, cell death, and autophagy in neurons and astrocytes," Molecular Brain, vol. 3, pp. 2-9, 2010.
[367] C.-S. Hsiung, J. D. Andrade, R. Costa, and K. O. Ash, "Minimizing interferences in the quantitative multielement analysis of trace elements in biological fluids by inductively coupled plasma mass spectrometry," Clinical Chemistry, vol. 43, pp. 2303-2311, 1997.
[368] V. Spevackova, K. Kratzer, M. Cejchanova, and B. Benes, "Determination of some metals in biological samples for monitoring purposes," Central European Journal Of Public Health, vol. 5, pp. 177-179, 1997.
[369] H. I. Afridi, T. G. Kazi, N. G. Kazi, M. K. Jamali, R. A. Sarfaraz, M. B. Arain, G. A. Kandhro, A. Q. Shah, J. A. Baig, and N. Jalbani, "Determination of copper and iron in biological samples of viral hepatitis (A–E) female patients," Biological Trace Element Research, vol. 129, pp. 78-87, 2009.
[370] S. Skerfving, V. Bencko, M. Vahter, A. Schütz, and L. Gerhardsson, "Environmental health in the Baltic region—toxic metals," Scandinavian Journal Of Work, Environment & Health, pp. 40-64, 1999.
[371] J. Moon, S. Kang, Y. Chung, and O. Lee, "Elemental analysis of Korean women’s blood serums using instrumental neutron activation analysis," Journal Of Radioanalytical And Nuclear Chemistry, vol. 271, pp. 155-158, 2006.
[372] H. Cunzhi, J. Jiexian, Z. Xianwen, G. Jingang, Z. Shumin, and D. Lili, "Serum and tissue levels of six trace elements and copper/zinc ratio in patients with cervical cancer and uterine myoma," Biological Trace Element Research, vol. 94, p. 113, 2003.
[373] G. A. Kandhro, T. G. Kazi, H. I. Afridi, N. Kazi, M. B. Arain, R. A. Sarfraz, N. Syed, J. A. Baig, and A. Q. Shah, "Evaluation of iron in serum and urine and their relation with thyroid function in female goitrous patients," Biological Trace Element Research, vol. 125, p. 203-212, 2008.
[374] V. Bencko, "Use of human hair as a biomarker in the assessment of exposure to pollutants in occupational and environmental settings," Toxicology, vol. 101, pp. 29-39, 1995.
[375] I. M. Kempson and W. M. Skinner, "ToF-SIMS analysis of elemental distributions in human hair," Science Of The Total Environment, vol. 338, pp. 213-227, 2005.
[376] S. A. Macko, M. H. Engel, V. Andrusevich, G. Lubec, T. C. O'Connell, and R. E. Hedges, "Documenting the diet in ancient human populations through stable isotope analysis of hair," Philosophical Transactions Of The Royal Society Of London B: Biological Sciences, vol. 354, pp. 65-76, 1999.
[377] H. Sang, P. Liang, and D. Du, "Determination of trace aluminum in biological and water samples by cloud point extraction preconcentration and graphite furnace atomic
147
absorption spectrometry detection," Journal Of Hazardous Materials, vol. 154, pp. 1127-1132, 2008.
[378] E. L. Silva, P. dos Santos Roldan, and M. F. Giné, "Simultaneous preconcentration of copper, zinc, cadmium, and nickel in water samples by cloud point extraction using 4-(2-pyridylazo)-resorcinol and their determination by inductively coupled plasma optic emission spectrometry," Journal Of Hazardous Materials, vol. 171, pp. 1133-1138, 2009.
[379] E. Z. Jahromi, A. Bidari, Y. Assadi, M. R. M. Hosseini, and M. R. Jamali, "Dispersive liquid–liquid microextraction combined with graphite furnace atomic absorption spectrometry: Ultra trace determination of cadmium in water samples," Analytica Chimica Acta, vol. 585, pp. 305-311, 2007.
[380] H. Liu and S.-J. Jiang, "Determination of vanadium in water samples by reaction cell inductively coupled plasma quadrupole mass spectrometry," Journal Of Analytical Atomic Spectrometry, vol. 17, pp. 556-559, 2002.
[381] K. Pyrzynska and T. Wierzbicki, "Solid-phase extraction for preconcentration and separation of vanadium species in natural waters," Microchimica Acta, vol. 147, pp. 59-64, 2004.
[382] L. Yang, R. E. Sturgeon, D. Prince, and S. Gabos, "Determination of vanadium in biological fluids using HR-ICP-MS," Journal Of Analytical Atomic Spectrometry, vol. 17, pp. 1300-1303, 2002.
[383] P. Liang, H. Sang, and Z. Sun, "Cloud point extraction and graphite furnace atomic absorption spectrometry determination of manganese (II) and iron (III) in water samples," Journal Of Colloid And Interface Science, vol. 304, pp. 486-490, 2006.
[384] A. N. Anthemidis, G. A. Zachariadis, C. G. Farastelis, and J. A. Stratis, "On-line liquid–liquid extraction system using a new phase separator for flame atomic absorption spectrometric determination of ultra-trace cadmium in natural waters ," Talanta, vol. 62, pp. 437-443, 2004.
[385] M. Ghaedi, A. Shokrollahi, K. Niknam, E. Niknam, S. Derki, and M. Soylak, "A cloud point extraction procedure for preconcentration/flame atomic absorption spectrometric determination of silver, zinc, and lead at subtrace levels in environmental samples," Journal Of AOAC International, vol. 92, pp. 907-913, 2009.
[386] M. Soylak, B. Kaya, and M. Tuzen, "Copper (II)-8-hydroxquinoline coprecipitation system for preconcentration and separation of cobalt (II) and manganese (II) in real samples," Journal Of Hazardous Materials, vol. 147, pp. 832-837, 2007.
[387] A. N. Anthemidis, "Automatic sequential injection liquid–liquid micro-extraction system for on-line flame atomic absorption spectrometric determination of trace metal in water samples," Talanta, vol. 77, pp. 541-545, 2008.
[388] M. A. Farajzadeh, M. Bahram, B. G. Mehr, and J. Å. Jönsson, "Optimization of dispersive liquid–liquid microextraction of copper (II) by atomic absorption spectrometry as its oxinate chelate: Application to determination of copper in different water samples," Talanta, vol. 75, pp. 832-840, 2008.
[389] M. Ghaedi, A. Shokrollahi, F. Ahmadi, H. Rajabi, and M. Soylak, "Cloud point extraction for the determination of copper, nickel and cobalt ions in environmental samples by flame atomic absorption spectrometry," Journal Of Hazardous Materials, vol. 150, pp. 533-540, 2008.
[390] N. F. Kolachi, T. G. Kazi, H. I. Afridi, N. Kazi, G. A. Kandhro, A. Q. Shah, J. A. Baig, S. K. Wadhwa, S. Khan, and F. Shah, "Distribution of copper, iron, and zinc in
148
biological samples (scalp hair, serum, blood, and urine) of Pakistani viral hepatitis (A–E) patients and controls," Biological Trace Element Research, vol. 143, pp. 116-130, 2011.
[391] L. Tavakoli, Y. Yamini, H. Ebrahimzadeh, and S. Shariati, "Homogeneous liquid–liquid extraction for preconcentration of polycyclic aromatic hydrocarbons using a water/methanol/chloroform ternary component system," Journal Of Chromatography A, vol. 1196, pp. 133-138, 2008.
[392] S. Yalcin, H. Filik, and R. Apak, "Speciation analysis of manganese in tea samples using flame atomic absorption spectrometry after cloud point extraction," Journal Of Analytical Chemistry, vol. 67, pp. 47-55, 2012.
[393] M. d. A. Bezerra, A. L. Conceição, and S. L. Ferreira, "A pre-concentration procedure using cloud point extraction for the determination of manganese in saline effluents of a petroleum refinery by flame atomic absorption spectrometry," Microchimica Acta, vol. 154, pp. 149-152, 2006.
[394] V. A. Lemos, P. X. Baliza, A. L. de Carvalho, R. V. Oliveira, L. S. G. Teixeira, and M. A. Bezerra, "Development of a new sequential injection in-line cloud point extraction system for flame atomic absorption spectrometric determination of manganese in food samples," Talanta, vol. 77, pp. 388-393, 2008.
[395] K. C. Teo and J. Chen, "Determination of manganese in water samples by flame atomic absorption spectrometry after cloud point extraction," Analyst, vol. 126, pp. 534-537, 2001.
[396] E. Paleologos, D. Giokas, S. Tzouwara-Karayanni, and M. Karayannis, "Micelle mediated methodology for the determination of free and bound iron in wines by flame atomic absorption spectrometry," Analytica Chimica Acta, vol. 458, pp. 241-248, 2002.
[397] X.-B. Yin, "Dual-cloud point extraction as a preconcentration and clean-up technique for capillary electrophoresis speciation analysis of mercury," Journal Of Chromatography A, vol. 1154, pp. 437-443, 2007.
[398] M. Bilal, T. G. Kazi, H. I. Afridi, M. B. Arain, J. A. Baig, M. Khan, and N. Khan, "Application of conventional and modified cloud point extraction for simultaneous enrichment of cadmium, lead and copper in lake water and fish muscles," Journal Of Industrial And Engineering Chemistry, vol. 40, pp. 137-144, 2016.
[399] S. S. Arain, T. G. Kazi, J. B. Arain, H. I. Afridi, K. D. Brahman, F. Shah, S. Arain, and A. H. Panhwar, "Simultaneous preconcentration of toxic elements in artificial saliva extract of smokeless tobacco product, mainpuri by cloud point extraction method," Ecotoxicology And Environmental Safety, vol. 92, pp. 289-296, 2013.
[400] M. Rezaee, Y. Assadi, M.-R. M. Hosseini, E. Aghaee, F. Ahmadi, and S. Berijani, "Determination of organic compounds in water using dispersive liquid–liquid microextraction," Journal Of Chromatography A, vol. 1116, pp. 1-9, 2006.
[401] E. Aguilera-Herrador, R. Lucena, S. Cárdenas, and M. Valcárcel, "Direct coupling of ionic liquid based single-drop microextraction and GC/MS," Analytical Chemistry, vol. 80, pp. 793-800, 2008.
[402] X. Han and D. W. Armstrong, "Ionic liquids in separations," Accounts Of Chemical Research, vol. 40, pp. 1079-1086, 2007.
[403] H. Weingartner, "Understanding ionic liquids at the molecular level: facts, problems, and controversies," Angewandte Chemie International Edition, vol. 47, pp. 654-670, 2008.
149
[404] C. A. Hawkins, S. L. Garvey, and M. L. Dietz, "Structural variations in room-temperature ionic liquids: Influence on metal ion partitioning modes and extraction selectivity," Separation And Purification Technology, vol. 89, pp. 31-38, 2012.
[405] J. Luczak, J. Hupka, J. Thoming, and C. Jungnickel, "Self-organization of imidazolium ionic liquids in aqueous solution," Colloids And Surfaces A: Physicochemical And Engineering Aspects, vol. 329, pp. 125-133, 2008.
[406] H. Abdolmohammad-Zadeh and G. Sadeghi, "Combination of ionic liquid-based dispersive liquid–liquid micro-extraction with stopped-flow spectrofluorometry for the pre-concentration and determination of aluminum in natural waters, fruit juice and food samples," Talanta, vol. 81, pp. 778-785, 2010.
[407] H. Zhao, S. Xia, and P. Ma, "Use of ionic liquids as ‘green’solvents for extractions," Journal Of Chemical Technology And Biotechnology, vol. 80, pp. 1089-1096, 2005.
[408] Naeemullah, M. Tuzen, T. G. Kazi, and D. Citak, "Dispersive ionic liquid microextraction of aluminium from environmental water samples by effervescent generation of carbon dioxide," International Journal Of Environmental Analytical Chemistry, vol. 96, pp. 729-738, 2016.
[409] F. Pena-Pereira, I. Lavilla, and C. Bendicho, "Miniaturized preconcentration methods based on liquid–liquid extraction and their application in inorganic ultratrace analysis and speciation: a review," Spectrochimica Acta Part B: Atomic Spectroscopy, vol. 64, pp. 1-15, 2009.
[410] X. Liao, B. Liang, Z. Li, and Y. Li, "A simple, rapid and sensitive ultraviolet-visible spectrophotometric technique for the determination of ultra-trace copper based on injection-ultrasound-assisted dispersive liquid–liquid microextraction," Analyst, vol. 136, pp. 4580-4586, 2011.
[411] É. C. Lima, F. Barbosa Jr, F. J. Krug, M. M. Silva, and M. G. Vale, "Comparison of ultrasound-assisted extraction, slurry sampling and microwave-assisted digestion for cadmium, copper and lead determination in biological and sediment samples by electrothermal atomic absorption spectrometry," Journal Of Analytical Atomic Spectrometry, vol. 15, pp. 995-1000, 2000.
[412] S. Ozcan, A. Tor, and M. E. Aydin, "Determination of selected polychlorinated biphenyls in water samples by ultrasound-assisted emulsification-microextraction and gas chromatography-mass-selective detection," Analytica Chimica Acta, vol. 647, pp. 182-188, 2009.
[413] Q. Chang, J. Zhang, X. Du, J. Ma, and J. Li, "Ultrasound-assisted emulsification solidified floating organic drop microextraction for the determination of trace amounts of copper in water samples," Frontiers Of Environmental Science & Engineering In China, vol. 4, pp. 187-195, 2010.
[414] M. Yebra-Biurrun and R. Cespón-Romero, "Fast ultrasound-assisted extraction of copper, iron, manganese and zinc from human hair samples prior to flow injection flame atomic absorption spectrometric detection," Analytical And Bioanalytical Chemistry, vol. 388, pp. 711-716, 2007.
[415] E. Privalova, M. Nurmi, M. Marañón, E. Murzina, P. Mäki-Arvela, K. Eränen, D. Y. Murzin, and J.-P. Mikkola, "CO2 removal with ‘switchable’versus ‘classical’ionic liquids," Separation And Purification Technology, vol. 97, pp. 42-50, 2012.
[416] F. Karadas, M. Atilhan, and S. Aparicio, "Review on the use of ionic liquids (ILs) as alternative fluids for CO2 capture and natural gas sweetening," Energy & Fuels, vol. 24, pp. 5817-5828, 2010.
150
[417] M. J. Muldoon, S. N. Aki, J. L. Anderson, J. K. Dixon, and J. F. Brennecke, "Improving carbon dioxide solubility in ionic liquids," The Journal Of Physical Chemistry B, vol. 111, pp. 9001-9009, 2007.
[418] R. J. Soukup-Hein, M. M. Warnke, and D. W. Armstrong, "Ionic liquids in analytical chemistry," Annual Review Of Analytical Chemistry, vol. 2, pp. 145-168, 2009.
[419] R. C. Harris, "Physical Properties of Alcohol Based Deep Eutectic Solvents, PhD Thesis," university of leicester, 2009.
[420] A. P. Abbott, D. Boothby, G. Capper, D. L. Davies, and R. K. Rasheed, "Deep eutectic solvents formed between choline chloride and carboxylic acids: versatile alternatives to ionic liquids," Journal Of The American Chemical Society, vol. 126, pp. 9142-9147, 2004.
[421] A. P. Abbott, G. Capper, D. L. Davies, R. K. Rasheed, and V. Tambyrajah, "Novel solvent properties of choline chloride/urea mixtures," Chemical Communications, pp. 70-71, 2003.
[422] J. M.S.S Esperancca, J. N. Canongia Lopes, M. Tariq, L. s. M. Santos, J. W. Magee, and L. s. P. N. Rebelo, "Volatility of Aprotic Ionic Liquids A Review," Journal Of Chemical & Engineering Data, vol. 55, pp. 3-12, 2009.
[423] C. Rub and B. Konig, "Low melting mixtures in organic synthesis–an alternative to ionic liquids?," Green Chemistry, vol. 14, pp. 2969-2982, 2012.
[424] Naeemullah, M. Tuzen, T. G. Kazi, and J. Ali, "Green and Deep Eutectic Solvent Microextraction Method for FAAS Determination of Trace Level Cadmium in Water Samples Using Multivariate Strategic Approach," Atomic Spectroscopy, vol. 37, pp. 244-251, 2016.
[425] R. G. Brereton, Applied chemometrics for scientists: John Wiley & Sons, 2007.
[426] S. L. C. Ferreira, M. d. G. A. Korn, H. S. Ferreira, E. G. P. da Silva, R. G. O. Araújo, A. S. Souza, S. M. Macedo, D. d. C. Lima, R. M. de Jesus, and F. A. C. Amorim, "Application of multivariate techniques in optimization of spectroanalytical methods," Applied Spectroscopy Reviews, vol. 42, pp. 475-491, 2007.
[427] S. Dinc, A. Bozdogan, G. Alpdogan, B. Asci, and S. Sungur, "Factorial Design in the Optimization of Preconcentration Procedure for Aluminum Determination by AAS," Innovations In Chemical Biology, pp. 313-317, 2009.
[428] N. Jalbani, T. G. Kazi, B. M. Arain, M. Jamali, H. I. Afridi, and R. Sarfraz, "Application of factorial design in optimization of ultrasonic-assisted extraction of aluminum in juices and soft drinks," Talanta, vol. 70, pp. 307-314, 2006.
[429] A. A. Momen, G. A. Zachariadis, A. N. Anthemidis, and J. A. Stratis, "Use of fractional factorial design for optimization of digestion procedures followed by multi-element determination of essential and non-essential elements in nuts using ICP-OES technique," Talanta, vol. 71, pp. 443-451, 2007.
[430] M. T. Castro and N. Baccan, "Application of factorial design in optimization of preconcentration procedure for copper determination in soft drink by flame atomic absorption spectrometry," Talanta, vol. 65, pp. 1264-1269, 2005.
[431] L. M. Costa, S. L. Ferreira, A. R. A. Nogueira, and J. A. Nóbrega, "Use of factorial design for optimization of microwave-assisted digestion of lubricating oil," Journal Of The Brazilian Chemical Society, vol. 16, pp. 1269-1274, 2005.
151
[432] L. M. Costa, M. d. G. A. Korn, J. T. Castro, W. P. Santos, E. V. Carvalho, and A. R. A. Nogueira, "Factorial design employed for microwave-assisted digestion of bean samples," Química Nova, vol. 29, pp. 149-152, 2006.
[433] W. N. dos Santos, C. M. Santos, and S. L. Ferreira, "Application of three-variables Doehlert matrix for optimisation of an on-line pre-concentration system for zinc determination in natural water samples by flame atomic absorption spectrometry," Microchemical Journal, vol. 75, pp. 211-221, 2003.
[434] S. L. Ferreira, M. A. Bezerra, and W. N. dos Santos, "Application of Doehlert designs for optimisation of an on-line preconcentration system for copper determination by flame atomic absorption spectrometry," Talanta, vol. 61, pp. 295-303, 2003.
[435] A. Baranda, N. Etxebarria, R. Jimenez, and R. Alonso, "Development of a liquid–liquid extraction procedure for five 1, 4-dihydropyridines calcium channel antagonists from human plasma using experimental design," Talanta, vol. 67, pp. 933-941, 2005.
[436] I. Lavilla, B. Perez-Cid, and C. Bendicho, "Optimization of digestion methods for sewage sludge using the Plackett-Burman saturated design," Fresenius' Journal Of Analytical Chemistry, vol. 361, pp. 164-167, 1998.
[437] F. Shah, T. G. Kazi, H. I. Afridi, N. Kazi, J. A. Baig, A. Q. Shah, S. Khan, N. F. Kolachi, and S. K. Wadhwa, "Evaluation of status of trace and toxic metals in biological samples (scalp hair, blood, and urine) of normal and anemic children of two age groups," Biological Trace Element Research, vol. 141, pp. 131-149, 2011.
[438] T. G. Kazi, H. I. Afridi, M. K. Jamali, M. B. Arain, N. Jalbani, and N. Syed, "Evaluation of zinc status in whole blood and scalp hair of female cancer patients," Clinica Chimica Acta, vol. 379, pp. 66-70, 2007.
[439] W. Horwitz, Official methods of analysis of the AOAC International vol. 18: The association, 2000.
[440] F. Shah, E. Yilmaz, T. G. Kazi, H. I. Afridi, and M. Soylak, "Vortex-assisted liquid–liquid microextraction coupled to flame atomic absorption spectrometry for lead determination: ionic liquid based microextraction using Triton X-100 as dispersant," Analytical Methods, vol. 4, pp. 4091-4095, 2012.
[441] Z. Fang, Flow injection separation and preconcentration: VCH, 1993.
[442] A. Abbott, K. El Ttaib, K. Ryder, and E. Smith, "Electrodeposition of nickel using eutectic based ionic liquids," Transactions Of The IMF, vol. 86, pp. 234-240, 2008.
[443] K. Powers, T. Smith-Weller, G. Franklin, W. Longstreth, P. Swanson, and H. Checkoway, "Parkinson’s disease risks associated with dietary iron, manganese, and other nutrient intakes," Neurology, vol. 60, pp. 1761-1766, 2003.
[444] B. L. Batista, D. Grotto, J. L. Rodrigues, V. C. de Oliveira Souza, and F. Barbosa, "Determination of trace elements in biological samples by inductively coupled plasma mass spectrometry with tetramethylammonium hydroxide solubilization at room temperature," Analytica Chimica Acta, vol. 646, pp. 23-29, 2009.
[445] J. Borkowska-Burnecka, A. Szymczycha-Madeja, and W. Żyrnicki, "Determination of toxic and other trace elements in calcium-rich materials using cloud point extraction and inductively coupled plasma emission spectrometry," Journal Of Hazardous Materials, vol. 182, pp. 477-483, 2010.
[446] K. Soltyk, A. Lozak, P. Ostapczuk, and Z. Fijalek, "Determination of chromium and selected elements in multimineral and multivitamin preparations and in pharmaceutical raw material," Journal Of Pharmaceutical And Biomedical Analysis, vol. 32, pp. 425-432, 2003.
152
[447] D. Wang, X. Du, and W. Zheng, "Alteration of saliva and serum concentrations of manganese, copper, zinc, cadmium and lead among career welders," Toxicology Letters, vol. 176, pp. 40-47, 2008.
[448] C. J. Frederickson, S. W. Suh, D. Silva, C. J. Frederickson, and R. B. Thompson, "Zinc and health: current status and future directions,"Journal Of Nutrition vol. 130, pp. 1471S-1483S, 2000.
[449] M. M. Black, "The evidence linking zinc deficiency with children's cognitive and motor functioning," The Journal Of Nutrition, vol. 133, pp. 1473S-1476S, 2003.
[450] S. Bhatnagar and S. Taneja, "Zinc and cognitive development," British Journal Of Nutrition, vol. 85, pp. S139-S145, 2001.
[451] T. P. Flaten, "Geographical associations between aluminium in drinking water and death rates with dementia (including Alzheimer's disease), Parkinson's disease and amyotrophic lateral sclerosis in Norway," Environmental Geochemistry And Health, vol. 12, pp. 152-167, 1990.
[452] D. Forster, A. Newens, D. Kay, and J. Edwardson, "Risk factors in clinically diagnosed presenile dementia of the Alzheimer type: a case-control study in northern England," Journal Of Epidemiology And Community Health, vol. 49, pp. 253-258, 1995.
[453] C. N. Martyn, D. N. Coggon, H. Inskip, R. F. Lacey, and W. F. Young, "Aluminum concentrations in drinking water and risk of Alzheimer's disease," Epidemiology, vol. 8, pp. 281-286, 1997.
[454] S. Rifat, M. Eastwood, D. C. McLachlan, and P. Corey, "Effect of exposure of miners to aluminium powder," The Lancet, vol. 336, pp. 1162-1165, 1990.
[455] A. B. Graves, D. Rosner, D. Echeverria, J. A. Mortimer, and E. B. Larson, "Occupational exposures to solvents and aluminium and estimated risk of Alzheimer's disease," Occupational And Environmental Medicine, vol. 55, pp. 627-633, 1998.
[456] R. Williams, "Aluminium and biological systems: an introduction," Coordination Chemistry Reviews, vol. 149, pp. 1-9, 1996.
[457] S. Bondy, A. Becaria, and A. Campbell, "Aluminum As A Toxicant," vol. 18, pp. 309-320, 2004.
[458] M. Amjadi and A. Jouyban, "Development of a dispersive liquid-liquid micro-extraction (DLLME) method for preconcentration and determination of Al in biological fluids," Research In Pharmaceutical Sciences, vol. 7, p. 652, 2012.
[459] N. Satıroglu and İ. Tokgoz, "Cloud point extraction of aluminum (III) in water samples and determination by electrothermal atomic absorption spectrometry, flame atomic absorption spectrometry and UV-visible spectrophotometry," International Journal Of Environmental And Analytical Chemistry, vol. 90, pp. 560-572, 2010.
[460] M. Sun and Q. Wu, "Determination of ultra-trace aluminum in human albumin by cloud point extraction and graphite furnace atomic absorption spectrometry," Journal Of Hazardous Materials, vol. 176, pp. 901-905, 2010.
[461] H. İ. Ulusoy, R. Gurkan, U. Aksoy, and M. Akcay, "Development of a cloud point extraction and preconcentration method for determination of trace aluminum in mineral waters by FAAS," Microchemical Journal, vol. 99, pp. 76-81, 2011.
[462] S. A. Arain, T. G. Kazi, H. I. Afridi, A. R. Abbasi, J. A. Baig, A. H. Panhwar, and N. Ullah, "Solid phase microextraction of trace levels of copper in serum samples of hepatitis B patients, on activated carbon cloth modified with an ionic liquid by using a
153
syringe mountable filter technique," Journal Of Analytical Atomic Spectrometry, vol. 29, pp. 2362-2370, 2014.
[463] M. Ghaedi, K. Niknam, K. Taheri, H. Hossainian, and M. Soylak, "Flame atomic absorption spectrometric determination of copper, zinc and manganese after solid-phase extraction using 2, 6-dichlorophenyl-3, 3-bis (indolyl) methane loaded on Amberlite XAD-16," Food And Chemical Toxicology, vol. 48, pp. 891-897, 2010.
[464] N. Pourreza and R. Hoveizavi, "Simultaneous preconcentration of Cu, Fe and Pb as methylthymol blue complexes on naphthalene adsorbent and flame atomic absorption determination," Analytica Chimica Acta, vol. 549, pp. 124-128, 2005.
[465] K. Shrivas, "Monitoring of copper level in water and soil samples by using liquid–liquid extraction," Environmental Monitoring And Assessment, vol. 168, pp. 315-319, 2010.
[466] K. Shrivas and N. K. Jaiswal, "Dispersive liquid–liquid microextraction for the determination of copper in cereals and vegetable food samples using flame atomic absorption spectrometry," Food Chemistry, vol. 141, pp. 2263-2268, 2013.
[467] S. Tokalıoglu and S. Yıldız, "A comparative study on the preconcentration of some metal ions in water samples with Cu (II) and Ni (II) salicylaldoxime coprecipitants," Microchimica Acta, vol. 165, pp. 129-133, 2009.
[468] X. Wen, Q. Deng, and J. Guo, "Ionic liquid-based single drop microextraction of ultra-trace copper in food and water samples before spectrophotometric determination," Spectrochimica Acta Part A: Molecular And Biomolecular Spectroscopy, vol. 79, pp. 1941-1945, 2011.
[469] C. D. Vulpe and S. Packman, "Cellular copper transport," Annual Review Of Nutrition, vol. 15, pp. 293-322, 1995.
[470] R. Hatano, M. Ebara, H. Fukuda, M. Yoshikawa, N. Sugiura, F. Kondo, M. Yukawa, and H. Saisho, "Accumulation of copper in the liver and hepatic injury in chronic hepatitis C," Journal Of Gastroenterology And Hepatology, vol. 15, pp. 786-791, 2000.
[471] C. Tanasescu, R. Baldescu, and Z. Chirulescu, "Interdependence between Zn and Cu serum concentrations and serum immunoglobulins (IgA, IgM, IgG) in liver diseases," Romanian Journal Of Internal Medicine= Revue Roumaine De Medecine Interne, vol. 34, pp. 217-224, 1995.
[472] I. Hossain, S. N. Islam, M. N. I. Khan, S. Islam, and A. Hasnat, "Serum level of copper, zinc and manganese in somatization disorder patients," German Journal Of Psychiatry, vol. 10, pp. 41-45, 2007.
[473] X. Huang, C. S. Atwood, R. D. Moir, M. A. Hartshorn, R. E. Tanzi, and A. I. Bush, "Trace metal contamination initiates the apparent auto-aggregation, amyloidosis, and oligomerization of Alzheimer’s Aβ peptides,"Journal Of Biological Inorganic Chemistry, vol. 9, pp. 954-960, 2004.
[474] M. A. Smith, P. L. Harris, L. M. Sayre, and G. Perry, "Iron accumulation in Alzheimer disease is a source of redox-generated free radicals," Proceedings Of The National Academy Of Sciences, vol. 94, pp. 9866-9868, 1997.
[475] J. Hu and J. R. Connor, "Demonstration and characterization of the iron regulatory protein in human brain," Journal Of Neurochemistry, vol. 67, pp. 838-844, 1996.
[476] Z. Liu, P. Hu, X. Meng, R. Zhang, H. Yue, C. Xu, and Y. Hu, "Synthesis and properties of switchable polarity ionic liquids based on organic superbases and fluoroalcohols," Chemical Engineering Science, vol. 108, pp. 176-182, 2014.
154
[477] M. Kawahara and M. Kato-Negishi, "Link between aluminum and the pathogenesis of Alzheimer's disease: the integration of the aluminum and amyloid cascade hypotheses," International Journal Of Alzheimer’s Disease, vol. 2011, pp. 1-17, 2011.
[478] L. Neri and D. Hewitt, "Aluminium, Alzheimer's disease, and drinking water," The lancet, vol. 338, p. 390, 1991.
[479] P. Nayak, "Aluminum: impacts and disease," Environmental Research, vol. 89, pp. 101-115, 2002.
[480] E. Hosovski, Z. Mastelica, D. Sunderić, and D. Radulović, "Mental abilities of workers exposed to aluminium," La Medicina Del Lavoro, vol. 81, pp. 119-123, 1990.
[481] E. Salib and V. Hillier, "A case-control study of Alzheimer's disease and aluminium occupation," The British Journal Of Psychiatry, vol. 168, pp. 244-249, 1996.
[482] R. T. Gun, A. Korten, A. Jorm, A. Henderson, G. Broe, H. Creasey, E. McCusker, and A. Mylvaganam, "Occupational risk factors for Alzheimer disease: a case-control study," Alzheimer Disease & Associated Disorders, vol. 11, pp. 21-27, 1997.
[483] J. Naser, F. Mjalli, B. Jibril, S. Al-Hatmi, and Z. Gano, "Potassium Carbonate as a Salt for Deep Eutectic Solvents," International Journal Of Chemical Engineering And Applications, vol. 4, p. 114-118, 2013.
[484] M. Aschner, T. R. Guilarte, J. S. Schneider, and W. Zheng, "Manganese: recent advances in understanding its transport and neurotoxicity," Toxicology And Applied Pharmacology, vol. 221, pp. 131-147, 2007.
[485] C. J. Martin, "Manganese neurotoxicity: connecting the dots along the continuum of dysfunction," Neurotoxicology, vol. 27, pp. 347-349, 2006.
[486] J. E. Myers, M. L. Thompson, S. Ramushu, T. Young, M. F. Jeebhay, L. London, E. Esswein, K. Renton, A. Spies, and A. Boulle, "The nervous system effects of occupational exposure on workers in a South African manganese smelter," Neurotoxicology, vol. 24, pp. 885-894, 2003.
[487] M. Verity, "Manganese neurotoxicity: a mechanistic hypothesis," Neurotoxicology, vol. 20, pp. 489-497, 1998.
[488] J. Schneider, E. Decamp, K. Clark, C. Bouquio, T. Syversen, and T. Guilarte, "Effects of chronic manganese exposure on working memory in non-human primates," Brain Research, vol. 1258, pp. 86-95, 2009.
[489] B. Racette, S. Tabbal, D. Jennings, L. Good, J. Perlmutter, and B. Evanoff, "Prevalence of parkinsonism and relationship to exposure in a large sample of Alabama welders," Neurology, vol. 64, pp. 230-235, 2005.
[490] M. Aschner and M. Gannon, "Manganese (Mn) transport across the rat blood-brain barrier: saturable and transferrin-dependent transport mechanisms," Brain Research bulletin, vol. 33, pp. 345-349, 1994.
155
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