Cogswell Review Paper on Chromatography in Disease Detection

8/10/2019 Cogswell Review Paper on Chromatography in Disease Detection

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An Overview and Critique of Chromatographic Methods being Used for Disease Detection

By

Kyle Cogswell


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Chromatography is an analytical technique that traces its origin back to the beginning of

the 20th

century. Invented by Mikhail Tswett, chromatographys first application was in Tswetts

separation of plant pigments, namely the chlorophyll and xanthophyll pigments. His apparatus

was a hollow tube packed full of chalk particles; simple in design but the principle of separation

due to differences in affinity between a mobile and a stationary phase would completely change

the field of separations chemistry. Before chromatography was invented, separations and

purifications were carried out through crystallization. Inherently this method has its advantages

due to its simplicity but the quality of the purification can sometimes be lacking and the amount

of material required to do this is quite large when compared to chromatography. Despite the

advanced potential of chromatography and the disadvantages associated with crystallization,

chromatography did not catch on when Tswett originally made use of it in 1903. More so, the

methods validity was criticized and was not widely adopted for some time. After a period of

roughly 20 years without usage, chromatography was reborn, thanks to Martin and Synge who

went on to win a Nobel Prize for their use of chromatography. The initial packed column used to

separate plant pigments gave way to more advanced columns both packed and wall coated, with

a large variety of stationary phases to be used and just as large a variety of mobile phases.

As it is commonly known chromatographic techniques are named after the mobile phase

that is used. Stationary phases used in these types of chromatography are dependent on the kind

of data, which one is trying to obtain The two main types of chromatography that have become

very useful in the medical world are gas chromatography (GC) and the high performance and

ultra high performance variants of liquid chromatography (HPLC and UHPLC). The higher

pressures lead to higher efficiencies. Efficiency of GC and HPLC methods vary, but on average a


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GC has an efficiency of around 100,000 theoretical plates, whereas HPLC usually has efficiency

in excess of 10,000 theoretical plates. Though GC is more efficient its applications are somewhat

limited when compared with the kinds of samples that are usually analyzed due to the sensitivity

of many compounds to heat.

Chromatography has grown so large and diverse due to the simplicity of the principle that

it is based on. However, even though the diversity of the methods is large, there are a few

methods that tend to stand out due to their larger range of applications or the repeatability of data

acquisition. The methods applied to disease detection have also fallen into a few distinct

categories, however The implementation of LC-MS/MS will increase in the next few years as

this technology continues to improve and the advantages become more well known (Wu 4)

Other methods that are often lumped with chromatography, but are not actual chromatographic

techniques can also be used to aid the traditional chromatographic techniques. Electrophoresis

can be used to overcome dynamic range problems associated with chromatography paired with

mass spectrometry. (Nagore 386)

As mentioned above GC and HPLC have applications in the separation of certain

compounds from a complex matrix. Human saliva, as an example of a complex matrix,

contains a number of biochemical components which may be useful for diagnosis/monitoring

of metabolic disorders, and as markers of cancer or heart disease. (Al-Shehri 140) Subsequent

detection can provide insight as to what types of analytes are present, and depending on the

detector, the amount of the aforementioned analyte. Detection of a disease can come in many

forms. A disease may arise when levels of one analyte are too high, such as increased levels of

keto acids which are thought to be linked to many degenerative diseases. (Olsen 116) High levels

of organic analytes, such as polyphenols, can indicate good health since many studies point to


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these compounds as lowering the risk of pathological conditions. (Nagy 162) However, other

organic compounds can be linked to disease. Volatile organic compounds (VOCs) are present in

human breath but relative levels can indicate the level of health (Boots) More so, understanding

the healthy and unhealthy VOCs can lead to a fast, inexpensive, and non-invasive early detection

technique. (Badjagbo 1893) What has remained unclear, however, is if the diseased cells are

directly responsible for these analytes or if the disease alters the hostsmetabolism and this

indirectly produces these analytes. In the realm of cancer, researchers are beginning to test

isolated samples and find that each tumor type has a distinct profile much like a fingerprint.

(Bartolazzi 483) Fingerprinting of a disease can also be done through blood samples, where

certain compounds may indicated oxidative stress or disease risk. (Moore 51) Any type of

sample that may be taken from a person may serve as a sample matrix, and there are a huge

number of components, which can be used to indicate the health of a person. Sometimes, the

components that signify health and sickness could be very similar and require complex analysis

with powerful separation techniques to find their relationship to health or disease.

The three types of diseases that are of interest for this paper are those that affect the mind

like Parkinsons and Alzheimers, those that affect the immune system like HIV-AIDS, and

those that can be associated with the growth of cancer. Separation of matrices, sampled from

people are the first and likely most important step of the process, the next is using a adequate

detection method to detect analytes and amounts of them present, finally, making use of

statistical software to link the analytes to the diseases is necessary.

Given the high accuracy and low detectability limits imposed by chromatographic

techniques, their usage is bound to find a place in disease detection. This is of such great

importance, because often many diseases have progressed quite far by the time they are detected


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with traditional means. If detected earlier, a person can begin the necessary regimens to rid

himself or herself of the disease or at least slow the spread. Cancer, for example, can require

heavy doses of radiation and/or chemotherapy, but with earlier detection the amount of treatment

can be minimized and the danger posed by the treatments can too be minimized. Research on

medicines used to treat or eliminate diseases like HIV-AIDS, Parkinsons, and Alzheimers

would also benefit from this early detection. In the case of HIV-AIDS it is a numbers game in

treatment. If caught early a person can be cured, a child who was functionally cured of AIDS is

the very example of this. Early detection in general can lead to new and rewarding forms of

treatment, which may not have been initially explored.

One disease that can benefit greatly from chromatographic separation is HIV, the

precursor to AIDS. According to the center for disease control HIV antibody is detectable in at

least 95% of patients within 3 months after infection. Although a negative antibody test result

usually indicates that a person is not infected, antibody tests cannot exclude recent infection.

(CDC). This implies that with current methods there is concentration dependence; that below a

certain concentration the antibodies are undetectable. These antibodies have to accumulate for

three months to have a high enough concentration in the blood to be detectable by current means.

While science moves closer and closer to a cure with a small number of individuals worldwide

being cured through one method or another; detection becomes the limiting factor in the process.

The authors of Rapid detection of HIV-1 p24 antigen using magnetic immuno-chromatography

(MICT)believe that detection of HIV can be improved from current methods; these researchers

recognize that the p24 antigen has aided in detection with traditional immunoassays, but has not

yet been applied to rapid lateral-flow HIV antibody detection assays. (Workman 14).


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HIV core antigen (p24) is usually present in the serum or plasma of HIV infected

individuals 7-10 days earlier than HIV antibody thereby reducing the window period of detection

by a similar time. (Workman 14) This antigen is a marker for the HIV before it fully develops

and is usually present in very small amounts early on. Workman and his cohorts use the

remainder of their paper to describe their methods for taking the HIV samples containing the p24

antigen, and the development of an apparatus for magnetic immuno-chromatographic testing

(MICT). This method of chromatography uses magnetic materials to improve the sensitivity.

Detection was carried out with a magnetic assay reader system (MAR), and the readouts

were normalized in order to minimize the instrument variability. (Workman 15) The paper made

use of many different sources of the HIV and associated p24 antigen; ranging from virus spiked

plasmas, in cultures containing HIV-1 subtypes A-G and O, and commercially available panels.

The testing over this wide range of panels allowed the researchers to reach the conclusion that

their minimum detection limit was in the range of 15-30 pg/mL. However, there are still ultra

sensitive methods that can detect the p24 on the order of ~1 pg/ml. This method is much more

time consuming and complicated. The authors state that The MICT assay presented here could

provide a simple, rapid and low cost alternative to current EIA protocols and continue later with

The MICT assay not only provides a sensitivity equivalent to most of the EIAs, the test is

simple, requiring the addition of only two reagents and can be completed in 40 min. (Workman

16) The author posits that this method could be used for infants who often have plasma

concentrations of the virus conducive for this type of testing. Early detection in infants allows for

more effective treatment, or possibly functional cures like the one witnessed in Mississippi

where an infant, upon birth, was given a very effective retroviral treatment to eliminate the HIV.


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The application of chromatography in HIV/AIDS detection is not as wide of a field as

one would think; the traditional detection enzyme-linked immunosorbent assay ELISA is a

technique that gives comparable results. Chromatography finds more of a niche in the separation

of a matrix containing the drugs used in treatment of HIV. (Faux) Given that todays retroviral

treatment contains a plethora of different analytes, it becomes very necessary to see how these

analytes are distributed throughout a human host. (Rezk) Knowing the concentrations of the

HIV-protease inhibitors allows for would logically allow doctors to prescribe more effective

treatments and scientists in their understanding of the HIV-protease inhibitors. (Bertucci)

Another disease that already has methods for detection but could benefit from a more

sensitive method is that of cancer. Current cancer treatments are varied for each type of cancer,

but quite often people have no clue about their cancer until a tumor is noticed or the person

begins to get ill. Current methods of cancer screening can reduce the deaths caused by cancer,

but screening is not always effective. (cancer.gov)

Types of cancer, such as breast and lung, have received a great deal of attention in the

media and as such they have also seen a great deal of effort put into the detection process.

Whereas cancers that researchers are just beginning to understand or cancers that are of a more

rare type have not received the same amount of attention and as such are not as advanced when it

comes to the detection process. Detection of metabolites is useful for all types of cancer and all

types of disease in general. In Metalabolomic Signatures of Aggressive Prostate Cancer

general markers for many types of cancer are given, 2-hydroxy-glutarate in the brain, blood

born and colorectal tumors, quinolinate in renal cell carcinoma, and sarcosine in prostate and

colorectal cancer.(McDunn 1547)


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It is less common and aggressive forms of cancer that can benefit most from a

chromatographic separation technique paired with an adequate system for detection, since their

methods of detection are currently more limited than the more common cancers. Thus the

importance lies in the novelty of finding an appropriate way to detect these cancers; researchers

are looking for the metabolites associated with these types of cancer. The more well known

cancers can too reap the benefits of the chromatographic applications. Much more is known

about these cancers, but earlier detection is always desirable. Arguably, the metabolic signatures

of these more common cancers are better understood than the less common forms given the

amount of research that has gone into them. Due to the above classifications, it is logical to start

with the cancers that have well understood causal factors; which will benefit from the strength of

chromatographic separations. Afterwards one can move on with the types of cancer that are still

relatively new in their treatments. In general, understanding of what is known will be conducted

first followed by the development of new methods and detection of new causes.

Lung cancer is one of the more common types of cancer weighing in with more than

240,000 new cases each year and contributing to over 160,000 deaths a year. (Sigel) According

to the national cancer institute, researchers are trying to come up with more effective methods for

screening. The traditional screening procedures of chest x-rays and mucus testing have not

proven to be effective for screening. (cancer.gov) The previous statement is logical in that these

methods fail at screening or early detection; by the time they are actually able to detect the

presence of lung cancer the tumor has to have grown large enough to be seen on an x-ray.

Given the extensive level of research put into lung cancer over the years, there is a lot of

data to work with, thus the biomarker understanding could be considered better defined. This is

only partly true; researchers are still trying to find the most efficient biomarker to screen lung


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cancer. The authors of Analysis of Volatile Organic Compounds Released from Human Lung

Cancer Cells and from the Urine of Tumor-Bearing Mice,(Hanai)A Sensor Array and GC

Study About VOCs and Cancer Cells (Bartolazzi) and A Study of the Volatile Organic

Compounds Exhaled by Lung Cancer Cells In Vitro for Breath Diagnosis (Chen)believe

volatile organic compounds are the biomarkers of choice.

In Analysis of Volatile Organic Compounds Released from Human Lung CancerCells

and from the Urine of Tumor-Bearing Mice, the author, Hanai, implants human cancer cells in

mice. He then takes samples of the Urine and uses head-space solid phase micro extraction HS-

SPME for the sample preparation before injection into the GC with time of flight mass

spectrometry. The mobile and stationary phases of the GC were not well documented. Certain

ketones and alcohols were found to be in greater abundance dimethyl succinate, diethyl ether,

ethanol, 2,2,4-trimethyl-1,3-pentanediol diisobutyrate, isobutyric acid 2-ethyl-3-hydroxyhexyl

ester, 2-butanone, 1-dodecanol, 3-butene-2-one, orthoformic acid tri-sec-butyl ester and 2,5-

hexanedione. (Hanai)

In another paper utilizing VOCs A Study of the Volatile Organic Compounds Exhaled

by Lung Cancer Cells In Vitro for Breath Diagnosis Instead of urine, Chen and his team used

breath samples and cell cultures as the sample matrix. However, they were concentrated using

SPME like the last paper and tested with a GC-MS system. The GC used here was of the

common dimensions, 250-micron inner diameter with 30 meters of length. Like the last article

there was no description of the stationary and mobile phases used, however, it was mentioned

that the GC was temperature programmed between 40 and 250 degrees Celsius with a ramp of 1

degree per minute. The biomarkers found here styrene, decane, isoprene, benzene, undecane, 1-

hexene, hexanal, propyl benzene, 1,2,3-trimethyl benzene, heptanal and methyl cyclopentane.


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(Chen) These do not match any of the biomarkers from the first paper. This is likely due to the

difference of the sample medium, urine vs. breath samples as well as the use of a FID detector as

opposed to a MS detector. It is likely that the compounds in the urine have gone through many

more of the bodys mechanisms for detoxification whereas the breath cultures are straight from

the lung.

Another possible way to detect lung cancer is to pick up on the genetic mutation as soon

as possible. Mutations of the epidermal growth factor receptor gene are thought to play a role in

the formation of lung cancers. These mutations are commonly identified using DNA sequencing

methods. Although considered the gold standard, this approach is time consuming. (Cohen2858-

2865) Denaturing high-performance liquid chromatography (dHPLC) can be utilized in detecting

some of the mutations in a much shorter time frame than the traditional methods. This type of

column is similar to a standard HPLC column except denaturing of an analyte takes place during

elution. The mobile phase mix of triethylamonium acetate and acetonitrile going from 0.05%

acetonitrile to 25% acetonitrile was partially responsible for the denaturing. Temperature was

also used to denature. Exon 19 deletions and exon 21-point mutations are very strong indicators

of cancer presence. Some of the authors from this paper went on to publish a follow up paper in

which they continue the research of the first. It is found that these two defects indicated above

are the two most common accounting for 90+% of the defects associated with lung

adenocarcinomas. (Cohen 4309-4317) These papers are written primarily by M.D.s and are not

quite as focused as one might want.

Peptides and proteins are another category of compounds that researchers are hoping to

use for early detection. In one research article dansyl depeptides are the biomarker of interest.

Liquid chromatography is used as the separation technique where MS is used to generate the


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library of 361 dansyl dipeptides and check to see if any of those are detected in the samples. The

researcher found that 279 of these dipeptides were found out of the 361 and that 90 of these were

associated with increased cancer risk. A validation experiment showed that 12 dipeptides were

selectively increased. (Wu 2091-2098) The value of statistics is demonstrated here as a large

sample matrix is brought down into a handful of analytes useful for cancer detection. This is

further backed up by a robust chromatographic regimen, where both HPLC and UPLC are used

to great effect. A larger 5mm by 150 mm column was used for one method as well as a 2.1mm

by 100 mm column. The first column was coated with C18 stationary phase and the second was

packed with 2.6 micron particles with C18stationary phase. A gradient elution was used, with

mobile phase varying from 95%/5% water acetonitrile to 5%/95% water acetonitrile, both 0.1%

formic acid. It is hinted that certain types of dipeptides may be in play when it comes to cancer

and the researchers test this assumption with a full panel of tests followed by a computerized

data-mining problem.

Glycoproteins can also prove to be a useful biomarker. A paper by Swiss researchers,

Soltermann et al., N-Glycoprotein Profiling of Lung Adenocarcinoma Pleural Effusions by

Shotgun Proteomics uses the pleural fluid as a sample matrix for the detection of glycoproteins,

which may be linked to cancer. The study was conducted using the effusions of 5 healthy

patients and 5 with malignant effusions. Capillary HPLC-MS was chosen, as it is commonly

chosen for biomolecules due to heat sensitivity. The 75-micron by 10 cm column is packed with

3 micron C18coated columns. A gradient elution is also chosen for the mobile phase using

varying amounts of water and acetonitrile with small amounts of formic acid. The issue of small

sample size id demonstrated here; further validation may be needed. However, the author

minimized conclusions likely due to sample size concerns; instead choosing to present this a


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preliminary study in the formation of a catalogue for adenocarcinoma. Their method showed the

detection of more than 100 N-GPs in a sample analysis(Soltermann 124-133)

In Glycoproteomic Analysis of Bronchoalveoar Lavage (BAL) Fluid Identities Tumor-

Associated Glycoproteins from Lung Adenocarcinoma, researchers from Johns Hopkins

medical institutions chose a wider approach. The sample size was still small but they tested

different types of cancer. These researchers observed about 80 glycoproteins in their samples;

roughly the same order of glycoproteins as the previous paper. Solid phase micro extraction was

used to concentrate the samples before analysis in the LC with tandem MS. The LC was done

with a 75 micron by 10 cm column packed with 5-micron particles with a C18stationary phase.

Gradient elution of the mobile phase was conducted using a mobile phase mixture of water and

acetonitrile with 0.2% formic acid. Acetonitrile began at 5% and was raised to 40% by the end.

Using this method the team was able to find noticeably elevated levels of several glycoproteins

that multiple types of lung cancer had in common. Of 80 glycoproteins found in BAL

specimens, 32 were identified in both cancer BAL and cancer tissues, with levels of 25

glycoproteins showing at least a 2-fold difference between cancer and benign BAL. (Li 3689-

3696)

Prostate cancer is the most common male malignancy.(McDunn 1547) McDunn

continues on to state that though detection is improving it is definitely not perfect. This type of

cancer has been receiving more attention in the past few years as it is often a silent killer or men

and is often found far too late for effective treatment. Though this cancer could be considered a

more common form as it affects a great number of men, 240,000 new cases and 28,000 deaths in

the United States according to McDunn via Siegel (Siegel), it can in fact be one of the more

aggressive forms of cancer.


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McDunn and his colleagues ran their metabolomics studies using multiple prostate

samples 331 with prostate cancer and 178 which were cancer free. Analysis was carried out with

ultra high performance liquid chromatography with mass spectrometry as well as gas

chromatography with mass spectrometry. Though the gas chromatographic set up is not

described in detail, the UHPLC apparatus is. The UHPLC column used was a 2.1 mm by 100

mm. It was packed with 1.7 micron particles coated in C18stationary phase. The mobile phase

was composed of solvent A (water with 0.01% formic acid) and solvent B ((3:1) methanol:

acetonitrile) operated in gradient elution mode. They are able to classify over 300 compounds

and were then able to show the differentiation of these compounds in healthy and cancerous

prostate tissues. By the end they are ale to propose their research as a possible diagnostic to

compare patient tissues against.

In another article Application of Holistic Liquid Chromatography-High Resolution Mass

Spectrometry Based Urinary Metabolomics for Prostate Cancer Detection and Biomarker

Discovery by Zhang et. Al, the authors state Disappointingly, sarcosine as a (urinary or

plasma) biomarker in prostate cancer has not been supported by several independent

studies.(Zhang).Zhang settled on 4 metabolites, ureido isobutyric acid, indolylacryoloyglycine,

acetylvanilalinine, and 2-oxoglutarate. The author claims that these metabolites have not been

explored before but are also related to the occurrence of other diseases as well. Zhang like

previous researchers also does his work with the use of LC but uses a high-resolution mass

spectrometer and uses urinary samples. The LC methods used are reversed phase HPLC as well

as the newer HILIC. The reversed phase HPLC uses a traditional 4.6 mm by 150 mm column

with 5-micron particles coated in C18stationary phase. The HILIC column is the same

dimensions but uses special HILIC particles. The mobile phases used in HILIC conditions were


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about some of the first biomarker work done with the liver. It was found that workers who were

near vinyl chloride were very apt to develop liver sarcoma. Using chromatography this paper

found that glycosaminoglycan levels are an indicator of liver cancer. Urinary heparin sulfate

increases and chondroitin sulfates decrease in patients with liver cancer. (Curran 3050-3053).

Two more recent papers move in another direction when it comes to the detection of

metabolites involved in liver cancer. One paper by Mochalski et. Al details the uptake and the

emission of volatile organic components by hepatic carcinoma cells (Mochalski) and the other

paper by Liu et. al attempts to link the concentrations of certain amines to the incidence of

hepatic cancer. (Liu 36-45) Mochalski chooses to focus on VOC metabolites in the diseased

cells, but did not focus much on samples from healthy individuals. It is important to have

controls and the addition of samples from healthy individuals provides that. The VOCs were pre-

concentrated manually using needle trap devices. The needles were used in the sealed cultures

that contained the cells. Afterwards they were injected into the GC-MS and analyzed using a

gradient temperature program. The GC column used was from Agilent technologies; it was a

column with split splitless injection 1:20 ratio, with 320-micron diameter and 25 meters of

length. The stationary phase was 5 microns thick but was listed by the brand name PoraBond Q.

Helium was used as the mobile phase, as is common in high quality chromatography carried out

with GC.

The other paper, which focused on the amines present in hepatic cancer cells, observed

amines in multiple matrices both plasma and urine. The researchers also took time to match

hepatic cancer patients and healthy age-matched volunteers (Liu 36 -45) Both sample

matrices under went similar pretreatments using a solution of methanol with trace amounts of


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acetic acid to deproteinize the solution. Proteins are removed as they may diminish the

concentration of the target analytes and generally make the chromatographic steps more difficult.

The separation in this article was different from the last one in that UPLC was used

instead of GC. The column was a reversed phase column given the polar nature of the mobile

phase used. The column dimensions were 75 mm in length 3.0 mm in diameter packed with 2.2-

micron particles. The mobile phase was composed of 0.05% heptafluorobutyric acid (HFBA) in

water (solvent A) and 0.05% HFBA in methanol (solvent B). Gradient elution was achieved by

increasing the amount of solution B from 20% to 50% by ramping up to the higher concentration

over time.

The study found that in both urine and plasma spermidine was a marker. Plasma also had

putrescine as a significant marker and the urine had a spermidine like compound as well as

spermine. Inspection of the structures of these components in an earlier section results in the

realization that they are all very similar in structure but different from the other amines in that

they are more nitrogen rich.

Another possible place to look at biomarkers is in the DNA itself, how it changes and

mutates in the presence of cancer. Researchers from Wuhan University were able to find with

hydrophilic-interaction liquid chromatography with in-source fragmentation and tandem mas

spectrometry that drops in 5-hydroxymethycytosine (5-hmC) effects epigenetic regulation in

hepatocellular carcinoma. This falls into the category of a biomarker that is missing as opposed

to the cancer generating the biomarker (Chen).

The final disease type of interest is the class of diseases that affect the mind. These

diseases attack a certain function in the brain, whether it is memory in the case of Alzheimers or

motor function in the case of Parkinsons. Unlike the other diseases such as HIV-AIDS or


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cancers, Alzheimers and Parkinsons do not kill on their own, but make life very difficult for

those who have theses diseases.

Alzheimers and Parkinsons are both getting a great deal ofattention in the scientific

world, with researchers searching for appropriate bio-markers to diagnose each case. As such,

separation is needed for both and will fall to chromatographic methods. The methods for the two

are similar in nature so they will be addressed one after the other starting with Alzheimers and

moving on to Parkinsons. The paper Addressing the Need for Biomarker Liquid

Chromatograph/Mass Spectrometry Assays: A Protocol for Effective Method Development for

the Bio analysis of Endogenous Compounds in Cerebrospinal fluid makes a very important

point which applies to both of these diseases; that analyzing the biomarkers effectively becomes

a challenge due to the relatively small samples of cerebrospinal fluid available from an

individual as well as the low concentration of the target analytes and interference from other

compounds. (Benitex 1882-1886) Another possible problem is biomarker overlap, where one of

these biomarkers can be common for many neurological disorders. Certain neurotransmitters are

responsible for many functions in the brain so it should stand to reason that altered levels of

these neurotransmitters may lead to many different diseases. Rapid Analysis of

Neurotransmitters in Rat Brain Using Ultra-Fast Liquid Chromatography and Tandem Mass

Spectrometry: Application to a Comparative Study in Normal and Insomniac Rats goes on to

posit that the things that are learned from the neurotransmitter levels in insomniac mice can be

applied to Alzheimers and Parkinsons research. (He 969-978) A reversed phase column is used

in this experiment with 5-micron packing particles and a 4.6 mm diameter by 150 mm length

column with a water/methanol mobile phase with small amounts of acetic acid. The use of


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reversed phase columns will be shown in multiple articles below using either a water/acetonitrile

or water/methanol mobile phase with a low percentage of acetic or formic acid.

Given that liquid chromatography methods are likely to be the most useful with

cerebrospinal fluid it is necessary to find operating conditions, which can give good separations.

Quantitative Profiling of Polar Cationic Metabolites in Human Cerebrospinal Fluid by

Reversed-Phase Nano liquid Chromatography/Mass Spectrometry Gives a plethora of

experimental conditions that can be used on cerebrospinal fluid. Perhaps the most useful thing

provided is some of the common stationary phases used and the types of mobile phase

combinations, which may be used in conjunction with these stationary phases to do separations.

(Myint 1121-1129) This article does not make conclusions as to what biomarker fluctuations are

present in mental diseases, but instead gives procedures for detecting anything that could

possibly be present in the cerebrospinal fluid. Many of these experimental conditions are used

for both Alzheimers and Parkinsons diseases. Addressing the Need for Biomarker Liquid

Chromatography/Mass Spectrometry Assays: A Protocol for Effective Method Development for

the Bio analysis of Endogenous Compounds in Cerebrospinal Fluid is another article that

focuses on the possible procedures. This article takes a more general stance by using a 2.1 mm

diameter by 50 mm length column with C18stationary phase to detect analytes by using a water

acetonitrile mobile phase mixture. This article takes time to go into the benefits of derivatization

and makes mention of some compounds of interest, those being histamines and taurine. The

detection of amines and amino acids has been shown to be a powerful marker for some of the

previous diseases discussed but will also be a marker for Alzheimers and Parkinsons.

There are two chromatographic routes to detect the biomarkers for Alzheimers disease:

gas and liquid chromatographic methods. Both methods are used over a variety of different


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sample matrices from the human body in hopes of finding an abnormality to qualify as a marker

for the disease. The vast majority of resources out there focus on using a liquid chromatographic

technique on cerebrospinal fluid, bodily plasma, with mass spectrometry detection for analysis.

Fewer resources were found on gas chromatography, but a few were found with GC on

cerebrospinal fluid and in breath. Detection of Alzheimers and Parkinsons disease from

Exhaled Breath Using Nanomaterial-based Sensors makesthe conclusion that both of these

diseases can be differentiated from a normal specimen as well as each other by a simple breath

sample. (Tisch 43-56) Logically, researches likely believe the cerebrospinal fluid to be the best

source of analytes related to brain related diseases and that many of the analytes may be too

sensitive for the temperatures involved in gas chromatography. (Bazenet 441-454)

As stated before most of the work done here is done with HPLC or UPLC. In A New

Metabolomic Workflow for Early Detection of Alzheimers Disease the researchers used

reversed phase UPLC-MS (RP/UHPLC-MS) as well as hydrophilic interaction liquid

chromatography (HILIC/UHPLC-MS). The sample was cerebrospinal fluid filtered and

centrifuged so that compounds less than 3 kDa could be analyzed. (Ibanez 65-71). The two

UHPLC methods differed in the stationary and mobile phase. The UHPLC uses a reversed phase

column with C18stationary phase, and the HILIC was done using a HILIC column. The standard

reverse phase UHPLC used a water/acetonitrile gradient elution with 0.1% amounts of formic

acid, whereas the HILIC used differing pH by use of a 10mM ammonium formate in the

water/pure acetonitrile mobile phase system. Many analytes were found from the separation and

subsequent MS testing. Some analytes such as methylsalsolinol and methylthiadenosine were

found to be increased for patients suffering for mild cognitive impairment (MCI) and


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Alzheimers (AD). Other analytes such as Creatinine, were decreased for (MCI) and (AD). There

were also some analytes that were increased for MCI but not for AD and others that showed no

distinct patterns. More time and research will help here and with a larger sample the results may

become clearer. It is important to note that many of these markers are amino acids or amine like

compounds. These compounds are polar and as such would have to be derivatized before

entering a GC-FID. The large structures of these materials lend to the likelihood of

decomposition at higher temperature.

Metabolite Profiling of Alzheimers Disease Cerebrospinal Fluid had a larger sample

size and made use of both HPLC-MS and GC-MS methods. This article also spent more time on

the important issue of age and gender and had an extensive statistical evaluation to help cope

with this. Solid phase extraction with LC-MS was used for steroid and catcholamine

determination. Further work with proteins was done by precipitating them out and separating

them based on polarity. Any compound entering the GC was derivatized so that amino acids

yielded the methyl esters. The actual description of the experimental apparatuses was missing for

both GC and LC. However, the statistical programming is robust and describes analytes, which

have been discussed in other articles. (Czech) It is interesting to note that one of the trends of

decreased uridine goes against the A New Metabolomic Workflow for Early Detection of

Alzheimers Disease conclusions. This could be due to sample size or methodology but is

something that should be evaluated.

Metabolic Profiling of Alzheimers Brains followed an UHPLC-MS procedure, but

instead used samples from brain tissues of people with Alzheimers. The shortcoming is that the

people were diagnosed with the disease, and that ideally one wants a range of samples from

control samples, to mild impairment, to full fledged Alzheimers. A UHPLC column was used


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with a diameter of 2.1mm and a length of 100mm. The stationary phase was C18 and as such is

considered reverse phased when the water acetonitrile mobile phase is used. The mobile phase

was used in gradient elution mode at 0.4mL per min. Again 0.1% formic acid was present in the

water and acetonitrile. Here amines were of concern especially spermine and its many

derivatives. (Inoue)

Parkinsons disease is just one disease of the mind that affects motor control in human

beings, another ailment that has similar effects on motor function in Huntingtons disease. Both

of these diseases suffer from poor diagnosis early on and can be treated more effectively if

detection is early. Development of an Enzyme-Linked Immunosorbent Assay (ELISA) to

Measure the Level of Tyrosine Hydroxylase Protein in Brain Tissue from Parkinsons disease

Models talks about the significance of tyrosine hydroxylase (TH), and that when it is not in

correct proportions it is related to all kinds of severe neurological conditions. Monitoring TH

expression would allow for effective treatment. Ideally, one wants to be able to follow the TH

concentration from the lowest concentrations and up. The TH is key in the later formation of

catecholamines such as dopamine, norepinephrine and epinephrine. (Fauss 245-257)

Measurement of these catcholamines is important and the use of HPLC can be used for

separation.

Thalamic noradrenaline in Parkinson's disease: Deficits suggest role in motor and non-

motor symptoms agrees in principal with the previous article going into the details of

norepinephrine also known as noradrenaline. Parkinsons samples had greatly reduced amounts

of norepinephrine. (Pifl 1618-1624) This article used HPLC with electrochemical detection,

electron capture detection is somewhat poorly represented in the articles found due to the nuclear

nature of its apparatus but is very sensitive and great for charged analytes like norepinephrine. A


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HPLC column was used with a 5-micron C18 stationary phase. Given the detector type a

different kind of mobile phase had to be used containing 0.1 M sodium phosphate, pH 4.9, 1 mM

EDTA, 0.5 mM l-octane sulfonic acid and 9% methanol were used. This complex mobile phase

was likely developed over the course of many experiments and is compatible with the ECD.

Cerebrospinal fluid biomarkers of central catecholamine deficiency in Parkinsons

disease and other synucleinopathies followed the same analytes as the previous articles and

found that they were reduced in people with Parkinsons even at early stages. Liquid

chromatography with ECD was used on cerebrospinal fluid this time instead of thalamus tissue

like the previous article though similar results were arrived at. (Goldstein 1900-1913)

Another neurological condition, which has motor effects similar to Parkinsons disease, is

Huntingtons disease. It is thought that methylation of guanine is a normal process but when it

proceeds at an irregular pace it can cause neurological disorders. HPLC was used here with UV

and electrochemical detectors. Two columns of 4.6mm diameter were used in series one with

75mm length and the other with 250 mm length. Mobile phase was acetonitrile and methanol

both spiked with lithium phosphate. The mobile phase was operated in various gradients for

separation. Samples of many bodily types were used including, whole blood, buffy coat, and

brain samples. Changes in the methylated guanine were observed and this was to be expected

with the Huntingtons diseased mice. (Thomas 112-120)

In summary, it can be shown that chromatography is a powerful separation technique

useful for splitting a complex matrix into desirable target analytes. Whether it is gas

chromatography or liquid chromatography, depends on the sample matrix to be separated and

also the target analyte type to be analyzed. Gas chromatography excels for analytes that are not

too polar and are able to withstand high temperatures. Compounds like volatile organic


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compounds produced by human respiration are a prime matrix for separation through gas

chromatography. Respiration samples were found to be useful for any type of disease

determination. Perhaps the biggest problem was that many of the analytes associated with one

disease could be a potential analyte for another disease. Increasing sample size and diversity of

the sample population would allow for a more in depth statistical analysis of the analytes and the

diseases that they are associated with. It would be necessary to combine the data from many

different diseases as well as data from individuals suffering from multiple diseases, this may

allow for a more encompassing set of biomarkers for diseases.

Liquid chromatography, especially the high performance variety is used to a great effect

in determining the metabolites that result due to disease. The strength of liquid chromatography

methods here is that the sample can be obtained from a persons fluids, such as blood, urine, and

cerebrospinal fluid. Additionally, the risk of destroying analytes through high temperature is not

present like it is for gas chromatography. Damaging the analytes with the mobile phase is a

possibility but a large variety of mobile phases exist and additionally the sample may be

derivatized to prevent damage by the mobile phase.

No matter the method of chromatographic separation, sample contamination and damage

is a very real possibility so care must be taken when doing the sampling and conducting

extractions. Also, the sample cannot be allowed to sit too long and if it is to be stored it should be

stored in low temperature conditions in well-sealed packaging.

Both methods require post separation detection. Traditionally GC relies on FID and LC

relies on UV detection. This is not always the case with the research done for disease

biomarkers. Quite common to both methods of chromatographic separation are mass


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spectrometry based detectors. Electron capture detectors, electrochemical detectors, and others

being used to a lesser extent.

Given more time and research, using chromatography as a method for separation of

complex matrices followed by detection through MS or another method based on the target

analyte will likely develop into a very effective way of screening people for a variety of diseases.


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Documents

Cogswell Review Paper on Chromatography in Disease Detection