PRESENTATIONS Training Workshop on Laboratory Methods …inweh.unu.edu/.../05/Training_Workshop_Laboratory_Methods_Proced… · PRESENTATIONS Training Workshop on Laboratory Methods

PRESENTATIONS

Training Workshop on Laboratory Methods and Procedures

Caribbean Coastal Pollution Project (CCPP)

Assessment, Monitoring and Management of Persistent Organic Pollutants (POP) and Persistent Toxic Substances (PTS) in the Coastal Ecosystems of the Wider Caribbean Region

19‐20 January 2009 Reef Yucatan Hotel, Merida, Mexico

Day 1: Monday, 19 Jan Ken Drouillard, GLIER, University of Windsor, Canada

Quality Systems Documentation Overview of Analytical Methods for Stockholm Convention Persistent Organic Pollutants (POPs)

Chris Metcalfe, Trent University and Watershed Sciences Centre, Canada Chromatographic Techniques, Limits of Detection and Calibration Limits of Detection

Nargis Ismail, GLIER, University of Windsor, Canada How to Validate Your Method

Ken Drouillard, GLIER, University of Windsor, Canada Quality Control Charts and Evaluation of Data Integrity

Gerardo Gold Bouchot, CINVESTAV, Mexico & Nargis Ismail, GLIER, University of Windsor, Canada Micro‐extraction Technique

Ken Drouillard, GLIER, University of Windsor & Chris Metcalfe, Trent University and Watershed Sciences Centre, Canada

Clean‐up of POPs from Biological Samples

Day 2: Tuesday, 20 Jan Nargis Ismail, GLIER, University of Windsor, Canada

GC‐Methods and instrument maintenance

Ken Drouillard, GLIER, University of Windsor, Canada Data Reporting & Data Management

TABLE: Determination of PCBs and Pesticides in Tuna Homogenate (Iaea‐435) ‐ Dec 2008

TABLE: List of CBs, OCs and PCB’s, providing Instrument Detection Limit (IDL), Calibration (Working) Linear Range and Coefficient of Determination (R2).

Quality Systems DocumentationDocumentation

•What is its function and why is it needed?

•Elements of a Quality System Manual and Accessory Documents

•The GLIER QA Manual Example

(Provided as supplementary materials)

Quality Systems Documentation• Combined these Documents Demonstrate:

– how to produce valid resultshow to produce valid results– show others that the lab is capable of doing so– act as the ‘corporate memory’ for all details and procedures related to the laboratory

– used as a training manual to new staff– provide baseline on which to seek and pdemonstrate improvements in policies through time

– necessary component towards seeking laboratory accreditation

• Laboratory Competence– Personnel in a laboratory have specific knowledge and skills related to the science underlying their testing procedures

– Staff can demonstrated this knowledge

– Procedures conform to the requirements of the science

• A competent laboratory has:– People with the skills and knowledgePeople with the skills and knowledge

– Environment with the facilities and equipment

– Quality control and procedures to produce valid results

• Quality – Degree to which a set of procedures fulfills objective measurement requirementsmeasurement requirements

– For analytical chemistry • ability of methods to produce precise and accurate results

• ability to demonstrate that the method was implemented under controlled conditions during a set of the analyses for unknown samplesunknown samples

The Quality System Documentation Should Adhere To the Following Principles:

• Capacity – Resources (Personnel with skills), facilities & equipment,Resources (Personnel with skills), facilities & equipment, quality control, procedures to produce quality results

• Exercise of Responsibility– Persons in organization have authority to make decisions and perform functions within the scope of the work

S i ifi h d• Scientific Method – accepted scientific approaches utilized

Principles (Cont.): • Objectivity of Results

– Test results are mainly based on measurable quantities with repeatable measurement characteristicsrepeatable measurement characteristics

• Impartiality of Conduct– Pursuit of competent results through scientific approaches are the overriding influence on the work of persons performing the tests

T bilit f M t• Traceability of Measurement– Results are based on a recognized system of measurements

– Instrumentation used in such measurements are calibrated and functioning correctly

Principles (Cont.):

• Repeatability of Test– A test that produces objective results will produce the same results during subsequent re‐testing

• Transparency of the Process– Processes used to produce objective results are open to internal and external scrutiny

– Improvement of documentation, methods, equipment can be identified and strived for

Quality Systems Documents• Quality System Manual (QSM)

– First level document that describes the policy implementation of the Lab Quality Management SystemSystem

• Management Requirements• Technical Requirements• Revision History

• Quality System Procedures (QSP)– Second level document detailing instructions & procedures in QSMprocedures in QSM

• Standard Operating Procedures (SOPs)– Laboratory testing procedures

• Related Procedures– Additional procedures common across different SOPs

1) Quality System Manual (QSM)A) Management Requirements– Organization and Management

• Legal identification of laboratory and organization• Legal identification of laboratory and organization• Scope of laboratory activities governed by QSM• Authorization of QSM by Laboratory Heads

– Quality Policy• Policy statements:

– Top management commitment to implementing a quality management system and continual improvement

– Roles and responsibilities of laboratory personnel» Technical management and quality management

• Document and Data control– Statement indicating that all QSM documents are current and have been reviewed by top management and technical management

– Identification and location of supporting procedures

1) Quality System Manual (QSM)A) Management Requirements– Control of Non‐conforming testing work

• Statement of policy on what happens when any non‐conformance is found.

– Corrective and Preventative Action System– Statement of policy to implement continual improvement in

laboratory procedures– Control of Quality Records

– All records, technical and non‐technical generated within the scope of the laboratory operations

– Records provide information sufficient for traceability:– all actions from sample receipt to report– responsibility for all involved in the process

– Audits ‐ internal and external of QSM– Management Review

‐statement of frequency of management review of quality systems

1) Quality System Manual (QSM)B) Technical Requirements

– Personnel– Controlled Environment and Facilities– Method Validation and Calibration– Equipment– Measurement Traceability– Handling of Test Items– Quality Assurance of Test Results– Results Reporting, Data Storage and Security

C) QSM Revision History

2) Quality System Procedures (QSP)(Focus on Technical Requirements)

– Technical Requirements

l

‐Each sub‐section reflects a stand‐alone document subject to revision‐Describes Purpose, Scope and Authorities and Responsibilities for tasks

– Personnel

– Controlled Environment and Facilities

– Method Validation and Calibration

– Equipmentq p

– Measurement Traceability

– Handling of Test Items

– Quality Assurance of Test Results

– Results Reporting


Personnel• Authorities and Responsibilities:

– Laboratory Head– Laboratory Head• Responsible for Management of Lab Operations

– Quality Manager• Ensures the Implementation of QSM and QSP• Reviews documentation, data quality, internal audits, proficiency testing, implementation of continual improvement policies

– Laboratory Supervisor• laboratory productivity, competency of technical personnel/training, safety, laboratory productivity, competency of technical personnel/training, safety,method validation, equipment use and maintenance, data reporting, data security and storage

– Laboratory Technician• Implementation of Standard Operating Procedures• Instrument calibration and log book recording, sample processing, instrument analysis, implementation of related procedures

• Personnel (Cont.)


• Technician Competence, Qualification & Training– Describe education and experience requirements for all personnel descriptions

– In‐house training program for technicians

– Analyst Proficiency testing• Analyst must demonstrate proficiency prior to implementing assigned tasks by satisfactorily analyzing quality control sample

• Analyst testing results are documented

• Environmental ConditionsF iliti d t t t t ti


– Facilities are adequate to carry out testing– Descriptions of

• Laboratory space – Criteria for lighting, temperature, humidity, air quality

• Safety considerations – Use/availability of fumehoods, venting, solvent cabinets,

h i l t h / h t ti fi tchemical storage, emergency showers/eyewash stations, first aid kits, emergency exits, other institutional safety procedures

• Security Considerations– Documentation storage areas, sample storage areas, preparation laboratory, instrument access

• Test Method and Method Calibration


– Method Selection• Statement of persons responsible for selecting methods adopted within SOP. That such methods are based on published international, national and/or regional standards or deemed fit for testing criteria

– Analytical MethodsAnalytical Methods• Index of all SOPs and Related Procedures Implemented by Lab

• Test Method and Method CalibrationV lid ti


– Validation • SOPs & related procedures are validated for control samples

• Documentation of performance criteria for accuracy, recovery, precision, detection limits, calibration and linearity

• Frequency of method validation (re validation• Frequency of method validation (re‐validation exercises) for control samples

• Review of method performance– Periodic examination of internal quality control, method calibration, method quality control data, performance history of validation data

• EquipmentEquipment inventory


– Equipment inventory– Records and Procedures

• Logs of: instrument, date of commission, history of modifications/repairs, calibration history, performance history

– Out of Service/Return to Service– Calibration Status

• SOPs provide specific details and include:• Calibration blank• Adequate number of standards used to define calibration, curve fitting procedures and statistical measurements of curve fit

• Low standard detection limit• Recovery standards, Certified Reference Tissues, Sample Duplicates

• Measurement and Traceability– Origins and certificates of analytical standards, certified


reference materials– Instrument calibration

• Periodic scheduled calibration against known standards (Cross check standards)

• Calibration conducted routinely prior to each use (i.e. Working standards)

– Frequency of calibration for:• Balances, volumetric glassware, temperature of storage ( l / d d) h h(sample/standard) storage areas, gas chromatographs

– Quality Control Samples:• Reference control standard (calibration accuracy); reference material (method accuracy); sample duplicate (method precision); standard recovery (method recovery); method blank (biological contamination)

– Level of Control Effort:• Frequency of analysis of QC samples relative to unknown samples

• Handling of test items– Sample reception procedures and forms


– Sample storage and Disposal– Chain of Custody

• Quality Assurance– QA/QC Sample Preparation & Effort of Control Sample Analyses– Evaluation of Interferences– Statistical Control

• Control Charting and evaluation of control chart dataControl Charting and evaluation of control chart data• Non‐conformances and trending monitored for analytical standards, reference materials, blanks, and standard recoveries

– Proficiency testing• Record of participation in interlaboratory comparisons and testing results

• Handling of test items– Sample reception procedures and forms


– Sample storage and Disposal– Chain of Custody

• Quality Assurance– QA/QC Sample Preparation & Effort of Control Sample Analyses– Evaluation of Interferences– Statistical Control

• Control Charting and evaluation of control chart dataControl Charting and evaluation of control chart data• Non‐conformances and trending monitored for analytical standards, reference materials, blanks, and standard recoveries

– Proficiency testing• Record of participation in interlaboratory comparisons and testing results

• Test Reports


Results Reporting

• Test Reports– QA signoff

– Flagged results

– ND, <DL

– Sig. digits

Selected Procedures

Selected Procedures

Selected Procedures

Selected Procedures

Selected Procedures

Selected Procedures

More Information:

• Canadian Association for Laboratory A dit ti (f l CAEAL)Accreditation (formerly CAEAL)– http://www.cala.ca/T27‐17025_Handbook.pdf

• GLIER – Quality System Manual Example– Electronic files supplied

Overview of Analytical Methods for Stockholm Convention PersistentStockholm Convention Persistent

Organic Pollutants (POPs)

Ken DrouillardGLIER, University of Windsor

Main Source for Presentation Materials:

Muir, D.C.G., E.D. Sverko. 2006. Analytical u , .C.G., . . S e o. 006. a yt camethods for PCBs and organochlorine pesticides in enviroinmental monitoring ans surveillance: a critical appraisal. Anal. Bioanal. Chem. 386: 769‐789

File available in Electronic PDF format.

Stockholm Convention “POPs”(The Dirty Dozen)

• Organochlorine Pesticides– Aldrin– ChlordaneChlordane– DDT– Dieldrin– Endrin– Heptachlor– Mirex– Toxaphene

• Industrial Chemicals• Industrial Chemicals– Polychlorinated Biphenyls (PCBs)– Hexachlorobenzene

• Industrial By‐Products– Dioxins– Furans

Challenges and Issues• Environmental analytical chemistry methods developed over past 30‐40 years

• Large numbers of methodsLarge numbers of methods – different selectivity and sensitivities

• GC‐MSD and GC‐ECD common

– different environmental matrices• All POPs relatively hydrophobic, most likely detected in organic phases: soils sediments tissue samples relative to air or waterphases: soils, sediments, tissue samples relative to air or water

• Instrument availability & limitations– E.g. High Resolution GC‐MSD for dioxins & furans, toxaphene

• High costs of ‘best practice’ techniques– Availability of analytical standards

Challenges and Issues

– Use of 13C‐labelled recovery standards– Certified reference materials– Costs of implementing QA/QC programs (personnel, time and consumables)

M t t d l b t dit ti• Movement towards laboratory accreditation– Standardized SOPs– Participation in Interlaboratory comparison exercises

Analytes of Concern• PCBs

– Total PCBs, sum PCBs (which congeners?)• IADN (Integrated Atmospheric Deposition Network – GreatIADN (Integrated Atmospheric Deposition Network Great Lakes) includes 100 congeners

• UNEP/World Bank/GEF Projects: 15‐30 congeners• Environment Canada – 42 or 71 congeners• Key Issue – guidelines based on sum or total PCBs, while analytical methods vary in # congeners analyzed

– Dioxin‐like PCBs (NO‐PCBs and MO‐PCBs)• NO‐PCBs: IUPAC #’s: 81, 77, 126, 169• MO‐PCBs: IUPAC #’s: 105, 114, 118, 123, 156, 157, 167, 189• Key issue – dioxin‐like activity often mostly related to NO‐and MO‐PCBs. Analytical methods require specialized separation procedures for testing (Particularly NO‐PCBs)

Analytes of Concern• UNEP Global POPs Monitoring workshop categorized essential PCBs to be monitored in human tissues and food products:human tissues and food products:– IUPAC #: 28/31, 52, 101/90, 118, 138, 153, 180– Most robust interlab comparisons

• UNEP Recommended PCBs for Sum PCB estimation in environmental matrices

IUPAC # 8/5 18 28/31 44 49 52 95/66 87 99– IUPAC #: 8/5, 18, 28/31, 44, 49, 52, 95/66, 87, 99, 101, 105/132, 110, 118, 128, 146, 149, 151, 153, 138/163, 156, 183, 187, 201/157, 170, 180, 194, 195, 206, 209

– Congeners in bold present in Quebec Ministry Analytical Standard

Analytes of Concern• OC Pesticides

– Chlordane• Essential: cis‐ and trans‐chlordane, cis‐ and trans‐nonachlor, oxychlordane• Recommended: MC4, MC5, MC6

– Heptachlor• Essential: Heptachlor, heptachlorepoxide

– DDT• Essential: 4,4’‐DDE, 4,4’‐DDD, 4,4’‐DDT• Recommended: 2,4’‐DDE, 2,4’‐DDD, 2,4’‐DDT

– Mirex• Essential: Mirex• Recommended: photomirexi ld i– Dieldrin

– Endrin– Aldrin– Toxaphene – few analytical standards available: Parlar (P) 26, 50, 62 most

common congeners analyzed for– Dioxins and Furans – several congeners – requires HR‐GC/HR‐MSD

No Consensus SOPs in Place for PCB/OCs

• UNEP POPs workshop on Global monitoring:– ‘Given broad range of technical expertise for analysis of PCBs and OCPs, as evident from the extensive international participation in interlaboratory calibration projects for these compounds, no single detailed, step‐by‐step analytical method can be recommended Insteadanalytical method can be recommended. Instead laboratories would use methods best‐suited to their situation and take part in international interlaboraotry comparisons to verify their work’

Internet Accessible PCB/OC SOPs• U.S. EPA & U.S. Geological Survey

– www.nemi.gov• Japan Environment AgencyJapan Environment Agency

– www.env.go.jp/en/index.html• Oslo‐Paris Commission

– www.ospar.org• Helsinki Commission

– www.helcom.fi• International Organization for Standardization

– www.iso.org• Association of Official Analytical Chemists International

– www.aoac.org• Several Others

Sample storage and handling• Norstrom and Won (1985); Wells et al (1997)

• Losses of more volatile OC‐pesticides (tetrachlorobenzenes and HCB) and reaction of DDT during freeze‐drying

• Recommend avoiding freeze drying• Recommend avoiding freeze drying

• Kiriluk et al (1996)– No effect of PCBs in fish when stored at ‐20 or ‐80oC

• Boer and Smedes (1997)– Reductions y1pin extractable lipids of samples stored at ‐20oC after 2 years, affects expression of lipid normalized residuesnormalized residues

– Recommended sample storage at ‐70oC when samples will be stored for > 2 year time period

•Short term storage of biological samples (< 2 years) for PCBs/OCs should be frozen at ‐20oC

Sample storage and handling• Storage containers:

– Preferred: Soap‐washed, Solvent (acetone, followed by hexane)‐rinsed glass jars fitted withfollowed by hexane) rinsed glass jars fitted with Teflon lined caps or hexane‐rinsed foil liner under lid

– Wrap excised sample in hexane‐rinsed foil and place inside a ziplock bag or whirlpack bag

Clear polyethylene bags or polypropylene jars for– Clear polyethylene bags or polypropylene jars for temporary storage i.e. sample transfer from field to laboratory

• Sample dissection – use of solvent‐cleaned stainless steel tools

Sample Preparation

• Sample homogenization:– Large Samples:

• Care in cleaning between samples

– Small samples & homogenate subsamples:• Mortar and pestle with Na2SO42 4

• Na2SO4 – ACS‐grade, muffled at 450oC overnight and maintained at 110oC until use

• Other desiccants: Celite; Hydromatrix must be solvent extracted prior to use

• Samples are with a non‐interfering chemical with similar elution and chromatographic properties of analytes. Unversally adopted for PCBs/OCs

• GC‐MSD

Sample Recovery Spikes

GC MSD– Isotopically labelled PCBs or OC‐pesticides– Identical physical properties to native analytes, similar

adsorption/partitioning properties– Requires MSD to distinguish native and labelled analyte

• GC‐ECD– Environmentally rare chemicals that respond well to ECD and

have similar physical properties of PCB/Ocshave similar physical properties of PCB/Ocs– PCBs #30, 204– Pentachloronitrobenzene– endrin ketone– 1,3,5‐tribromobenzene

• When to spike?

Analyte Extraction• Considerations

– Degree of adsorption/partitioning to sample matrix components (e.g. soils, blood proteins, tissue lipids)Extraction solvents (Pesticide analysis grade)– Extraction solvents (Pesticide analysis grade)

• Polar:apolar binary mixture more efficient for recovery of OCs from tissue samples of low lipid content and from sediments

– Acetone/hexane– Hexane/dichloromethane– Acetone/dichloromethane

– Solvent/matrix contact time and processing timeAmount of solvents utilized per sample– Amount of solvents utilized per sample

• Tissues: cold column, sonication, soxhlet, microwave‐assisted, supercritical fluid, pressurized liquid extraction

• Sediments: soxhlet, microwave‐assisted, pressurized liquid

Extraction

Extraction – blood and milk• Blood

– plasma preferred, then serum, then whole blood– Liquid‐liquid (shake flask), vortex unit, sonication + centrifugation &

dryingdrying• ethanol/hexane• methanol/hexane• Alcohol shown to be important in disrupting lipoproteins increasing extraction

efficiency– Solid phase extraction (C18‐cartridges) and elution with hexane,

hexane/DCM

• MilkLi id Li id i ( h k fl k)– Liquid‐Liquid extraction (shake flask) common

• Acetone/hexane• Dichloromethane/hexane

– Waring blender with acetonitrile and potassium oxalate + extraction by SPE (C18 cartridge)

Tissue Lipid Content• >95% of PCB/OCs in biological samples contained within neutral lipids (triglycerides) of samples with > 1% total lipid content

i f li id li d O id f l• Expression of lipid normalized POP residues useful to understand environmental fate and food web biomagnifications, not relevant for fish advice information

• Aliquot of sample extracts (usually 10%) removed following extraction to determine lipids by weighing

• Total lipids: chloroform:methanol:water (Bligh and Dyer 1959)– chloroform:methanol:water (Bligh and Dyer 1959)

– Propanol:cyclohexane:water (Smedes 1999)• Neutral lipids:

• Hexane:DCM or Extraction solvent combination

• Blood plasma lipids (neutural lipids):– Colorimetric approaches (phospho‐vanillin reaction)

Lipid Content

Li id t t i diff t ti f k ll

•Total lipids sometimes determined for other purposes, i.e. to establish health and energy density of animal•For tissues with low lipid content (<1%); total lipids ≠ neutral lipids!

Lipid content in different tissues of muskellunge determined by chloroform/methanol extraction and by hexane/DCM extraction

Drouillard et al. 2004. Chemosphere 55:395

Clean‐up• Clean‐up involves separating analytes from coextractedmatrix components that can interfere with analysis– Lipids, pigments, hydrophobic proteins, sulfur, other contaminants

– Separation of co‐eluting analytes into separate fractions for examination by GC/ECD

• Common clean‐up methods– Gel permeation chromatography (GPC) – size exclusion– Silica gel (removal lipids and separation)– Alumina (separation)– Florisil (separation)– Activated copper (sulfur removal)

Method 1668

Method 1668

EPA1668

Env. Can.

Instrumental Analysis

• GC‐ECD, GC‐MSD most common for PCB/OCs• GC Injection Port

– Splitless most common, on‐column injection (less common)– pressure programming can improve chromatography

• GC Columns– Open tubular capillary columns– DB‐5 columns, 30 m or 60 m (0.25 mm i.d. x 0.1 µm film thickness) commonly used) y

• 30 m column shows co‐elution of several PCBs; 60 m can resolve greater numbers of PCBs

– DB‐XLB (J&W Scientific); HT‐8 (SGE Inc.) considered advanced in separation of PCB congeners but less commonly adopted

Co‐eluting PCB congeners on 30m DB‐column As determined by GC‐ECD

60 m column:28, 31 resolved33,20 resolved149, 118 resolved153 resolved from 132/105

71/ 41/ 64 – co‐elute101/90 – co‐elute151/82 – co‐elute77/110 – co‐elute (fractionated on florisil)101/132 – co‐elute135/144 – co‐elute156‐171 – co‐elute195/208 – co‐elute

‐Co‐eluting congeners, particularly for mixedCo eluting congeners, particularly for mixed homologue groups, can lead to error in quantitation when the proportion of congeners in the standard differs from the proportion of congeners in the sample!

‐Occasional confirmation by GC‐MSD is useful

Quebec Ministry of Environment StandardGC‐ECD with 60 m DB‐5 column

Co‐elution of OCs with PCB congeners(60 m DB‐5 column, GC‐ECD)

Without Fractionation• Heptachlor epoxide / oxychlordane/ PCB 70• p,p’‐DDE/dieldrin/PCB 87

– p,p’‐DDE/dieldrin readily resolved on new 60 m column• o’p‐DDE/PCB110• Endosulfan II/PCB118• p,p’‐DDE with cis‐nonachlor (Resolved on our system)• Trans‐nonachlor with PCB 99 (Resolved on our system)• PCB 180 and PBDE 47

• **Some issues with supplied standards: mixed heptachlor epoxide+ oxychlordane and p,p’‐DDE/dieldrin could result in quantitationerrors for these compounds unless very good chromatography is realized

GC‐ECD of supplied OC‐StandardMixture (60 m DB‐5 column)

Fractionation Alumina or Florisil

• Can further separate PCBs/OC pesticides for cleaner chromatography removing some (not all) co‐elution issues highlighted previously

• Additional processing time, i.e. solvent reduction and instrument analysis of multiple fractions per sample

• Florisil Cleanup (3 fractions)– Fraction 1:

• PCBs – all congeners (except NO‐PCBs), HCB (F1,F2), Heptachlor (F1, F2), Aldrin (F1, F2), Endosulfan II (f1, f2), DDE (F1, F2 – both isomers), DDD (F1, F2) ; mirex (F1)

– Fraction 2:• HCHs (100 F2), HCB (F1,F2), Heptachlor (F1,F2), heptachlor epoxide (Isomer a) Aldrin (F1,

F2), oxychlordane (F2, F3), trans‐chlordane (F2); Endosulfan I (F1+F2), cis‐chlordane (F2); DDE (F1, F2), DDD (F1, F2); DDT (F2)

– Fraction 3:• Endrin, heptachlor epoxide (F2, F3) heptachlor epoxide (isomer B), oxychlordante (F2, F3),

Dieldrin (F3), methoxychlor (F3)

Chromatographic Resolution• Good instrument maintenance

– Diligence in good sample clean‐up • removal of lipids, H2O, sulfur and co‐contaminants

– Maintain clean injection port, gases (change of gas filters), frequent change of inlets, septa

– Pressure programming, good gas pressure and no leaks– Frequent checks of column resolution

• E.g. peak separation 28/31E.g. peak separation 28/31• good peak shape and consistent response factor for p,p’DDE

– Column duty cycle – may need to change column 2 – 3 x per year

Instruments ‐ Detectors

• GC‐ECD GC‐MSD (LR, EI ‐ SIM)‐high sensitivity (detection 0.05 pg/g) ‐moderate sensitivity (DL 0.3 pg/g)

‐moderate selectivity ‐high selectivity few co‐eluting peaks‐useful for peak identification

‐simple maintenance and robust ‐can be finicky, frequent autotuningvery consistent baseline & response required, response can be variable

ease of training and software use moderate training required‐ease of training and software use ‐moderate training required

‐low cost ($30 – 45 k) ‐moderate cost ($90 – 130 k)

‐limited use for other analytes ‐useful for OPs and other pesticides

Quality Assurance

• Maturity of PCB/OC methods requires adopting sound QA measures with results reportingU f tifi d l ti l t d d• Use of certified analytical standards

• Surrogate spike recoveries• Laboratory blanks

– clean vegetable oil or suitable matrix• Certified reference materials (or in‐house reference homogenates)homogenates)

• Use of quality control charts• Participation in inter‐laboratory comparisons

Accuracy of CRM Analyses

• Mean of replicates (n=7) concentration measured in CRM not

significantly different then certified value (t‐test)g y ( )

• QUASIMEME

– Inter‐laboratory variation 15‐20% for PCB concentrations are common

• European Commission

t bl PRC/OC ( l ) i CRM f ll– acceptable PRC/OCs (mean values) in CRM as follows– Conc. Range < 1ng/g ‐50% to + 20% certified value– 1 – 10 ng/g ‐30% to + 10% certified value– > 10 ng/g ‐20% to + 10% certified value

POPs Training WorkshopJanuary, 2009

Chromatographic Techniques,Limits of Detection

and Calibration

Presentations by C. Metcalfe

Introduction toChromatographic Techniques

Chromatography

• Separation of individual substance from mixtures • Extension of “partitioning theory”

• Applications in environmental analytical chemistry:

• Preparatory - Preparation of samples for analysis

• Analytical - Identification and quantitation of individual “analytes”

Partitioning Theory

• Partitioning between two phases reaches equilibrium• Partition coefficient (K) = conc in phase 1/conc in phase 2

solidgas

liquidliquid

liquidgas

solidliquid

Phase 2Phase 1

No analytical applications

Partitioning• Multiple partitioning of multiple solutes (1, 2, 3, 4, etc.)

between two immiscible phases• Separation of solutes occurs because of differences in

partition coefficients:• a) Solute 1: K1 = 2• b) Solute 2: K2 = 1• c) Solute 3: K3 = 0.5• etc.

Elution Chromatography

ColumnPacking = Stationary Phase(solid)

Solvent=Mobile Phase(liquid)

Eluate

SoluteA&B

Solute B

Solute A

Chromatogram• Detector signal vs. time

0

50

100

150

200

250

300

0 5 10 15 20 25 30time

concentration

AB

Characteristics of a Chromatogram• retention time: identifies substance• peak area: quantifies substance• resolution: separation of peaks• peak shape (width, sharpness): ideal peak is narrow and sharp

Retention time [min]5 64

4.3

5.2

Solute A

Solute B

Classification of Chromatographic Methods• Nature of the mobile phase

• Gas chromatography• Liquid chromatography

• Physical configurations• Planar - Paper chromatography, thin-layer

chromatography• Column – Packed column, open tubular column

• Sample development – Elution chromatography (only type used in analytical applications)

• Application• Preparative – prepare samples for analysis• Analytical – identification and quantification

• Retention mechanism• Adsorption• Partition• Ion exchange• Size exclusion

Stationary phase = solid

Adsorption ChromatographyMainly used in preparative

Applications(e.g. Silica gel, Florisil)

Stationary phase = liquid

Partition ChromatographyMainly used in analytical Applications (GC, HPLC)

Stationary phase – Cation or anion exchange resin

Ion Exchange ChromatographyPreparative applications (e.g. solid phase extraction),

Analytical applications (ion chromatography)

Mobile anions held near cations covalently bound to stationary phase

Anion Exchange Resin

Stationary phase = Porous Gel

Size Exclusion ChromatographyMainly preparative applications

(e.g. gel permeation chromatography)

Smallmolecules are retained

Analytical ChromatographyGas Chromatography

• Used for analytes that are volatile at high temperatures (200-300oC)

• Stationary phase: liquid adsorbed to a “solid support”• Mobile phase: inert gas (nitrogen, helium)• Main separation factor: temperature• Typical column: open tubular (capillary)• Common analyte selective detectors

– Flame photometric (FPD) – e.g. Organophosphate pesticides

– Electron capture (ECD) – e.g. PCBs and OC insecticides

– Mass selective (MSD) – e.g. range of analytes

Gas Chromatography

Open Tubular (Capillary) Column

Length – 10-60 m

Support coated open tubular column(SCOT)

Wall coatedOpen tubular column(WCOT)

Gas flow

Silica column

Determination of PCB congenersby GC-ECD

Cl Cl

209 congeners

Cl

Analytical ChromatographyHigh Performance (or Ultra High Pressure)

Liquid Chromatography• Used for analytes that are not volatile, or not suitable for GC

analysis (e.g. heat labile, polar, ionic)• Stationary phase: liquid adsorbed to a “solid support”• Mobile phase: liquid solvent• Main separation factor: pressure• Typical column: packed column • Common detectors

– Ultraviolet– Fluorescence - e.g. aromatic hydrocarbons – Mass selective (MSD) – e.g. range of analytes

• LC-MS• LC-MS/MS: High sensitivity and selectivity

Liquid Chromatography

Three different separation modesMobile Phases/Stationary Phases

• Normal phase high performance-liquid chromatography (HPLC)– Mobile phase: non-polar solvent– stationary phase: polar (SiO2, Al2O3)– used for: polar analytes

• Reversed-phase HPLC– mobile phase: polar solvent– stationary phase: non-polar (end-capped SiO2/Al2O3, organic

polymers)– used for: non-polar analytes

• Ion chromatography– mobile phase: water (buffer)– stationary phase: ion exchange resin (cation exchange, anion

exchange)– used for: cations (Na+, K+, Ca++), anions (Cl-, F-)

HPLC-Fluorescence: PAH analysisanthracene

pyrene

PAHs = polycyclic aromatic hydrocarbons(environmental carcinogens)

Reverse phase mode

Determination of Anions by Ion Chromatography with Conductivity

Detector (IC-CD)

Limits of Detection

Detection TheoryIf we analyze many replicates of a sample,

we will generate concentration data that are distributed (statistically) normally, and we can calculate a mean concentration and a standard deviation about the mean value. Variability about the mean is caused by:

• Instrumental variability:– Detector signal “noise”– Injection volume variations– Temperature program variations, etc.

• Method variability:– Recoveries during sample prep– Dilution errors during sample prep– Calibration errors during analysis– Matrix effects during analysis, etc.

Concentration

Mean

-4 4-2 20

+/- 3 SD

99% confidence limits about the meancan be calculated statistically (i.e. students t values), or approximated as 3x the standarddeviation about the mean.

Chromatographic Detection Limits

“Noise”

Analyte “signal”

Detection limit: The smallest signal (i.e. lowest concentration) of a specific analyte that is statistically different from background ”noise”.

All electronic instruments are susceptible to noise as a result of variability inAC current, electromagnetic fields, etc. Noise will vary with the instrument and with chromatographic retention time, and may vary over time and with the operator.

Chromatographic Detection Limits• Instrumental detection limit (IDL): The smallest signal of a particular

analyte that can be distinguished from background noise for a particular instrument.

Typically determined by replicate injections of an analytical standard dissolved in an appropriate carrier solvent into the chromatographic instrument.– IDL should always be below the MDL

• Method detection limit (MDL): The smallest signal of a particular analytethat can be distinguished from background noise for a particular instrument using a particular analytical method.

Typically determined by spiking an analytical standard into an environmental matrix (e.g. water, wastewater, tissues, sediments, soil) and preparing an extract that is injected in an appropriate carrier solvent into the chromatographic instrument:– Limit of detection (LOD): Defined statistically– Limit of quantitation (LOQ): Defined statistically

Note: LOD and MDL are often used synonymously

Chromatographic Detection LimitsSignal to Noise Ratio (S/N): The relative strength of an analytical signal (S) relative to the average strength of the instrument noise (N).

Max

Min

Mean

Convenient method for determining S/N: Divide the arithmetic mean of the signal for a series of replicate analyses by the standard deviation of the replicate results- tells you how much the noise is affecting your results

Chromatographic Detection Limits

• Limit of Detection (LOD): The lowest concentration that can be determined to be statistically different from a blank with a specific level of confidence (i.e. 99%). LODs are typically matrix, method and analyte specific.

Above the LOD, the analyte can be reported as detected< LOD = report analyte as “not detected” (nd); note that we can never

say that the concentration = zero• Limit of Quantitation (LOQ): The concentration above which

quantitative results may be reported with a specific level of confidence (i.e. >99%). LOQs are typically matrix, method and analyte specific.

Above the LOQ, the analyte can be quantified> LOD and < LOQ = report analyte as “present” or “detected”> LOQ = report analyte concentration in the sample matrix (e.g. mg/L)

Determining the LOD Method 1: Replicate Analyses

• The calculation of an LOD is based upon the variability (i.e. precision) observed between replicate analyses (e.g. n = 7) of an identical concentration of the analyte spiked into an appropriate sample matrix.

• Since precision will vary for different concentrations of the analyte, the initial spike level selected for LOD determination is important.

• The spike level should be selected as equivalent to an instrumental S/N in the range of 2.5 to 5.

• At least one method blank (i.e. unspiked) should be analyzed with each set of samples used to determine the LOD.

• In order for the LOD to be valid, the recoveries of the analyte using the analytical method should be “reasonable” (i.e. >75%) and reproducible.

Method 1: Replicate AnalysesPROCEDURE

• Estimate an appropriate spiking level by determining the analyte concentration that corresponds to an instrument S/N of 2.5 to 5.

• Spike the analyte into an appropriate sample matrix1 (e.g. water, tissue, sediment), and prepare at least 7 aliquots for analysis using an appropriate method.

• Analyze the replicate samples and determine the mean and the standard deviation (SD; for n -1 d.f.) for the concentrations determined in the samples.

• Computation method 1a:LOD = SD x 3LOQ = SD x 10

• Computation method 1b (US EPA):LOD = t (n-1, 0.99) x SD; where t = Students’ t value for 99% level confidenceLOQ = LOD x 3.3

1) If possible, choose a sample matrix for spiking that has non-detectable levels of the target analyte.


Analysis of lindane spiked into fish tissue at 0.21 ng/g wet weight (n=9):

Sample # Conc (ng/g) % Recovery1 0.23 1102 0.21 1003 0.24 1144 0.19 905 0.18 866 0.23 1107 0.22 1058 0.17 819 0.16 76

Mean = 0.20 ng/gSD = 0.029S/N = 6.89

Mean recovery = 97%

Computational method 1a:LOD = 0.029 x 3 = 0.087 LOQ = 0.029 x 10 = 0.29

Computational method 1b:LOD = 2.896 x 0.029 = 0.084LOQ = 0.084 x 3.3 = 0.28

Students’ t values at 99% confidence level:n n-1 t-value7 6 3.1438 7 2.9989 8 2.89610 9 2.82111 10 2.764


The five point check:• Does the spike level exceed 10x the

LOD? If so, the spike level was too high.• Is the LOD higher than the spike level? If

so, the spike level was too low.• Is the S/N in the appropriate range (i.e.

<10)?• Are the recoveries of the analytes

reasonable? • Does the LOD meet regulatory

requirements (e.g. fish consumption advisories)?

Lindane spiked into fish at 0.21 ng/g:Mean = 0.20 ng/gSD = 0.029S/N = 6.89

Mean recovery = 97%Range = 76-114%

Computational method 1a:LOD = 0.087 LOQ = 0.29

Computational method 1b:LOD = 0.084LOQ = 0.28

Determining the LOD Method 2: Serial Dilutions

• The calculation of an LOD is based upon analysis of serial dilutions of a standard of the target analyte spiked into an appropriate sample matrix.

• Choose an upper limit of the dilution series that is at least 10x (i.e. one order of magnitude) greater than the IDL.

• Continue the dilution series until the target analyte can no longer be detected (i.e. S/N = 1).

• Analyze all points in the dilution series at least 3 times.• At least one replicate of the method blank (i.e. unspiked) should be

analyzed.• In order for the LOD to be valid, the recoveries of analyte using the

analytical method should be “reasonable” (i.e. >75%) and reproducible.

Method 2: Serial DilutionsPROCEDURE

Spiking Concentration

Sign

al MethodBlank

LOD using Method 1:Lowest concentration that Is statistically different from the blank

LOD using Method 2:Concentration at which there is an asymptote (change in slope) in the serial dilution regression

0 10 20

LOQ = 3.3 x LOD

Uses of Serial Dilution Method

• Confirm that the LODs and LOQs determined using the Replicate Analysis method are reasonable.

• Estimate the LODs when it is not possible to find a matrix blank that is not contaminated with significant quantities of the target analyte, such as:– marine mammal blubber– human blood serum– domestic wastewater

The Sample Matrix• “Matrix effects” are defined as signal suppression or enhancement as

a result of co-extractives that are present in the sample .• The composition of some sample matrices are highly variable (e.g.

domestic wastewater, blood serum), and theoretically, new MDLsshould be determined for every new set of samples.

• However, for practical reasons, it is usually assumed that the MDLsdetermined for a representative sample matrix can be applied to all samples of a similar type.

• Gas chromatography tends to be less susceptible to matrix effects because of the high degree of sample clean-up and because only volatile compounds are analyzed.

• Liquid chromatography with some detection systems (e.g. fluorescence, MS) is more susceptible to matrix effects.

• Internal standards using stable isotope surrogates (e.g. deuterated or 13C-labelled analytes) can be used to compensate for these effects.

Analytical Calibration

Calibration Procedures• External standards: Known concentrations of analytes are analyzed

separately from the sample for quantitation of the analytes in the samples. – Reference standards can be purchased for most contaminants under regulatory

control (e.g. POPs)

– Certified standards are not available for “emerging contaminants” and must be made up gravimetrically by researchers

• Internal standards: Known concentrations of surrogate analytes that do not interfere with the analytes are analyzed with the sample.]

– Mass spectrometric detectors: Can use stable isotope labelled surrogates (i.e. deuterated or 13C-labelled compounds).

– Other detectors: Can use structurally similar surrogates that are not detected in environmental samples.

• Standard additions: Known concentrations of the analytes are added to the sample – a technique rarely used for chromatographic analysis.

External Standard CalibrationChoice depends on:• Linear range of response of the detector over a range of

analyte concentrations:– GC-ECD: Narrow linear range– GC-MS: Wide linear range

• Time required for analysis (e.g. some chromatographic runs may take >60 min).

A) Single Point Calibration:Assumes that the response is linear at concentrations below

the calibration point, and that the calibration line passes through the origin.

Concsample = Concstd x Response sample

Response std

ConcR

espo

nse

Calibration stdat upper limitof linear range

Unknown

External Standard CalibrationB) Two Point Calibration:• High concentration – Near upper limit of linear

range• Low concentration – Near LOQ

C) Multi-point Calibration:Three to six calibration points over the linear range,

with the lowest near the LOQ

Concentrations of analytes are determinedby interpolating from a linear regressionline.

Conc

Res

pons

e

Unknown

Internal Standard CalibrationStandard added to the sample that does not interfere with detection of

the analyte, but has similar or identical chromatographic properties as the analyte (i.e. analytical surrogate):

• Typically added at a constant concentration to all samples; used in conjunction with external calibration method

• Can be added to the sample:– Before sample preparation – “Method” IS that compensates for

recoveries and sample preparation errors– After sample preparation – “Instrument” IS that compensates for

instrumental variability (e.g. injection volumes)– Should report whether data are adjusted for detection of the

internal standards.

How to Validate Your Method•Approaches to Method Validation•Approaches to Method Validation•Frequency•Troubleshooting

Nargis Ismail

GLIER Laboratory – University of Windsor 1

Nargis IsmailGlier

Why Do We Need To Validate A Method?

• Method validation provides evidence that anl ti l th d h i d th h thanalytical method when carried through the

whole process in exactly the same mannerproduces results that fit for the purpose.

• Establish specificity, range and linearity of thetechnique.E t bli h Q tit ti li it IDL d MDL


• Establish Quantitation limit; IDL and MDL

Validation Protocol

By carrying at least 6 replicates of asuitable clean Matrix* spiked with thesuitable clean Matrix spiked with thetarget Analytes atleast three levels ofconcentration (or a Reference Sample)carried through all the sampleprocessing steps.


Frequency of Method Validation• When a new method developed in a lab or if the

method is imported from elsewhere. A j h i i ti th d• Any major changes in existing methods – Change of Solvents/ Absorbent– Change in instrumentation or major parts replacement– Trending failure of QA/QC and intervention

• e.g. suspected interferences or contamination

• Periodic method validation and documentation


• Periodic method validation and documentation– Re-affirm performance characteristics (range and

linearity)

Analytical Method Development• Analytical standard

– commercial availability (Certified standard solution, neat sample, purity)

– commission synthesis; isolation & extraction from env. sample

• Chromatography and peak detection for analytical standards

– evaluate relative purity of standard – Examine for co-elution between analytes within the standard

» Similar number of peaks as indicated on certification papers


p p» Peak quality (peak shape and tailing)» Confirm by GC-MSD (SCAN mode and SIM mode of

quantitation ion/ molecular ion or with library match)

Analytical Method Development Cont.)• Chromatography and peak detection for analytical

standards– Qualitative peak identification for each analyte in standard

» Documented chromatograph from standard supplier or with neat std.

» GC-MSD confirmation based on Molecular Ion or fragmentation pattern

– Confirm standard concentrations » Use of independent std. » Response factors of reference compounds from different

std.– Establish Linear response range of instrument for analytes

» Dilution series of standards that vary ~1000 fold» Determine IDL and/or Instrument Quantitation Limit


» Determine IDL and/or Instrument Quantitation Limit– Examine for co-elution issues between standard components

and other analytes co-examined in the lab

– Establish appropriate spiking recovery standard » Native or labelled Internal recovery standard

Analytical Method Development Cont.)


Figure 1: GC trace of Pesticide Mix 1 std. recorded on GC/MSD


Figure 2: GC trace of Pesticide Mix 1 std. recorded on GC/ECD


Figure 3: GC trace of Quebec Certified PCB std. recorded on GC/ECD

Machine Calibration

• Figure 2.ppt


Performance Parameters1. Accuracy (%E)

expressed is a measure of the Bias from the target values.

2. Precision (CV)• (repeatability) is determined by analyzing at least 6 replicates of a

suitable clean Matrix* spiked with the target Analytes at three levels of concentration (or a Reference Sample) carried through all sample processing steps calculate the Standard Deviation (σ).

• It is defined by the following formula:% CV = (Standard Deviation/Mean Measured Value) x 100


% CV of individual analytes is acceptable when it is <30% (EPA method 1668)

Performance Parameters (Cont.)3. Recovery• As % Recovery (% R) of recovery internal standard is

determined by analyzing at least 6 spiked samplesdetermined by analyzing at least 6 spiked samples (with Surrogate or fortified with Analyte Standard) carried through all sample processing steps and calculate mean measured value. It is defined by the following formula:% R = (Mean Measured Value/Reference Value) x 100% R of internal recovery standard is acceptable when it is ±40%


% R of internal recovery standard is acceptable when it is ±40%

Performance Parameters (Cont.)4. Detection Limit (MDL), is determined by analyzing at least 6 replicates of

a suitable clean Matrix (Trioleine, corn oil or a low level sample) spiked with the target Analytes at a low Concentration level (which must be 10 times higher than the lowest level of the Calibration Standard) carriedtimes higher than the lowest level of the Calibration Standard) carried through all sample processing steps, calculate the Standard Deviation and evaluate the MDL using the following formula:

MDL = t(n-1) x σσ = the Standard Deviation of replicate analysis (ng/mL) at the lowest levelt(n-1) = the student’s distribution value for 99% (or 95%) confidence


Level with (n-1) degrees of freedomn = the number of replicate analysis performed

• Determination of MDL includes adjustments of sample size and final extract volume to optimize recovery of some Analytes.

Validation Data – AIEA-435Tuna Fish Homogenate

• Figure.ppt


Troubleshooting• Identify Isomers (o,p’ e,g.,

DDE, DDD, DDT) based on elution time from the column.

Coeluting PCBs Number of Chlorine atom

Table 1 :

• Read GC trace very carefully and check for any background peak that might be present in reagent blank or in method blank; overlapped with peak of interest.

28, 31 3 66, 95 4, 5 56, 60 4 71, 41, 64 4 84, 89, 101, 90 5 117, 87, 115 5 77, 110 4, 5


• Co-elution of PCB congeners on 5% phenyl phase; DB-5 column are presented in Table 1.

123, 139, 149, 118 5, 5, 6, 5153, 132, 105 6, 6, 5 164, 163, 138 6 158, 129 6

How to Validate Your Method•Approaches to Method Validation•Approaches to Method Validation•Frequency•Troubleshooting

Nargis Ismail


Nargis IsmailGlier

Why Do We Need To Validate A Method?

• Method validation provides evidence that anl ti l th d h i d th h thanalytical method when carried through the

whole process in exactly the same mannerproduces results that fit for the purpose.

• Establish specificity, range and linearity of thetechnique.E t bli h Q tit ti li it IDL d MDL


• Establish Quantitation limit; IDL and MDL

Validation Protocol

By carrying at least 6 replicates of asuitable clean Matrix* spiked with thesuitable clean Matrix spiked with thetarget Analytes atleast three levels ofconcentration (or a Reference Sample)carried through all the sampleprocessing steps.


Frequency of Method Validation• When a new method developed in a lab or if the

method is imported from elsewhere. A j h i i ti th d• Any major changes in existing methods – Change of Solvents/ Absorbent– Change in instrumentation or major parts replacement– Trending failure of QA/QC and intervention

• e.g. suspected interferences or contamination

• Periodic method validation and documentation


• Periodic method validation and documentation– Re-affirm performance characteristics (range and

linearity)

Analytical Method Development• Analytical standard

– commercial availability (Certified standard solution, neat sample, purity)

– commission synthesis; isolation & extraction from env. sample

• Chromatography and peak detection for analytical standards

– evaluate relative purity of standard – Examine for co-elution between analytes within the standard

» Similar number of peaks as indicated on certification papers


p p» Peak quality (peak shape and tailing)» Confirm by GC-MSD (SCAN mode and SIM mode of

quantitation ion/ molecular ion or with library match)

Analytical Method Development Cont.)• Chromatography and peak detection for analytical

standards– Qualitative peak identification for each analyte in standard

» Documented chromatograph from standard supplier or with neat std.

» GC-MSD confirmation based on Molecular Ion or fragmentation pattern

– Confirm standard concentrations » Use of independent std. » Response factors of reference compounds from different

std.– Establish Linear response range of instrument for analytes

» Dilution series of standards that vary ~1000 fold» Determine IDL and/or Instrument Quantitation Limit


» Determine IDL and/or Instrument Quantitation Limit– Examine for co-elution issues between standard components

and other analytes co-examined in the lab

– Establish appropriate spiking recovery standard » Native or labelled Internal recovery standard

Analytical Method Development Cont.)


Figure 1: GC trace of Pesticide Mix 1 std. recorded on GC/MSD


Figure 2: GC trace of Pesticide Mix 1 std. recorded on GC/ECD



Machine Calibration

• Figure 2.ppt


Performance Parameters1. Accuracy (%E)

expressed is a measure of the Bias from the target values.

2. Precision (CV)• (repeatability) is determined by analyzing at least 6 replicates of a

suitable clean Matrix* spiked with the target Analytes at three levels of concentration (or a Reference Sample) carried through all sample processing steps calculate the Standard Deviation (σ).

• It is defined by the following formula:% CV = (Standard Deviation/Mean Measured Value) x 100


% CV of individual analytes is acceptable when it is <30% (EPA method 1668)

Performance Parameters (Cont.)3. Recovery• As % Recovery (% R) of recovery internal standard is

determined by analyzing at least 6 spiked samplesdetermined by analyzing at least 6 spiked samples (with Surrogate or fortified with Analyte Standard) carried through all sample processing steps and calculate mean measured value. It is defined by the following formula:% R = (Mean Measured Value/Reference Value) x 100% R of internal recovery standard is acceptable when it is ±40%


% R of internal recovery standard is acceptable when it is ±40%

Performance Parameters (Cont.)4. Detection Limit (MDL), is determined by analyzing at least 6 replicates of

a suitable clean Matrix (Trioleine, corn oil or a low level sample) spiked with the target Analytes at a low Concentration level (which must be 10 times higher than the lowest level of the Calibration Standard) carriedtimes higher than the lowest level of the Calibration Standard) carried through all sample processing steps, calculate the Standard Deviation and evaluate the MDL using the following formula:

MDL = t(n-1) x σσ = the Standard Deviation of replicate analysis (ng/mL) at the lowest levelt(n-1) = the student’s distribution value for 99% (or 95%) confidence


Level with (n-1) degrees of freedomn = the number of replicate analysis performed

• Determination of MDL includes adjustments of sample size and final extract volume to optimize recovery of some Analytes.

Validation Data – AIEA-435Tuna Fish Homogenate

• Figure.ppt


Troubleshooting• Identify Isomers (o,p’ e,g.,

DDE, DDD, DDT) based on elution time from the column.

Coeluting PCBs Number of Chlorine atom

Table 1 :

• Read GC trace very carefully and check for any background peak that might be present in reagent blank or in method blank; overlapped with peak of interest.

28, 31 3 66, 95 4, 5 56, 60 4 71, 41, 64 4 84, 89, 101, 90 5 117, 87, 115 5 77, 110 4, 5


• Co-elution of PCB congeners on 5% phenyl phase; DB-5 column are presented in Table 1.

123, 139, 149, 118 5, 5, 6, 5153, 132, 105 6, 6, 5 164, 163, 138 6 158, 129 6

Quality Control Charts and Evaluation of Data Integrity

• Record keeping• Westgard Rules• Issuing Non‐compliance reports• Corrective Actions

Data Quality Evaluation• Laboratory Supervisor/Analyst Must Inspect Chromatgrams Carefully for Analysis Anomolies

• Qualitative/Subjective Evaluations – Important!– Experience counts!– Chromatography Peak Shape

• Peak Co‐Elutions and interferences• Excessive peak tailing

– septa leak, dirty inlet, degraded column, matrix artifacts

• Double peaks (column overload)p• Retention time drift (dirty sample – e.g. Excess lipids)• Interference peaks (matrix artifact)• Negative Baseline (matrix artifact – e.g. Hydrocarbons)

– Peak Integration Area • Computer Autocalculation Vs Manual Baseline Set

• Quantitative Assessment of data is the role of the Quality Manager

Data Quality Evaluation

• Censoring Rules– Rules of thumb to guide decisions

• Blank Adjustment Procedures:– Sample peak area 0 ‐ 30% batch blank, perform blank correction– Sample peak ares >30% batch blank, indicate INT (interference)

• Acceptable Spiking Recovery Range: e.g. (70% ‐ 130%)• Below Detection Limit: Peak < MDL or MQL, indicated <MDLBelow Detection Limit: Peak MDL or MQL, indicated MDL

• Statistical Approaches– Interpretation of Control Charts– Westgard Rules

Control Charting• Control charts provide temporal history of QA measurements

Aid i k d d i i– Aids in source track down and intervention strategies

• Database used to establish statistical criteria on which to judge batch to batch QA performance– Detect out‐of‐control samples while considering normal variability in method output

Control Charts – What to Collect?• Any Quality Control parameter can be used to establish database. Examples include:– Equivalent signal to analyte peak in sample blanks– Spiking standard recoveries in blanks, samples, CRMs– %Difference in analytical results between sample duplicates run within batches

– Deviation of lab result from certified value of analytesin SRMs

• Other parameters of potential interest:Other parameters of potential interest:– Detector response (i.e. peak areas) for standards– Instrument calibration results (e.g. Slopes and intercepts)

• Databases – ‘Build as you go’– Excel, Access, other formsDatabase generally valid when > 15 points are

Control Charts – What to Collect?

– Database generally valid when > 15 points are included

– Initial data set should be evaluated to ensure qualitative acceptability of performance measures

• Censor Rules

– Continual developmentL t d h t t d t d• Long term and short term records generated.

• Database continuously updated with ‘in‐control’ data as it is created

Example Control Chart: TBB Spike Recoveries (GLIER GC‐ECD SOP)

Example Control Chart: Reference Tissue (GLIER GC‐ECD SOP)

Evaluations using Control Charts

• The control chart database should be set up to provide an updated sample mean and standard deviation for tested protocol

• Levey‐Jennings Chart Rules:– For a given batch of samples, the QA parameter is examined to see if it

falls in the range of mean ± 3*SD of control chart value– Depending on goals, values of ±2*SD or ±1*SD may be used

• less common in Environmental Analytical Laboratories

• Example: – Control Chart Spike TBB Recovery: 95 ± 8 %p y– Sample Recovery = 120%– Control Chart Acceptable Range: 71 – 119 %– Sample recovery > acceptable range: therefore reject sample QA

Evaluations using Control Charts• The evaluation is based on t‐test statistic. Since the standard deviation is derived from data generated by the lab this means that approximately 1 in 20the lab, this means that approximately 1 in 20 samples will be rejected for QC!!!

• QC Parameters which are measured more frequently (e.g. spike recoveries performed in blanks, every sample and CRM analyzed) will fail QC more often than QC parameters measured less frequent (i.e. CRM recoveries)recoveries)

• Failures in QC are subject to corrective actions. The steps associated with corrective actions must be documented but will vary depending on degree of seriousness of the failure

Statistical Formulae’s

• Mean:

• Microsoft Excel formulae: =Average(cell range)• Microsoft Excel formulae: =Average(cell ‐ range)

• Standard Deviation

Mi ft E l Std ( ll )• Microsoft Excel: = Stdev(cell range)

• Example: = Stdev(A4..A120) or = average(a4..a120)

Westgard Rules• Multi‐rule QC procedure

– Provides high rigor in error detection and limits excessive false error hits– Provides trending interpretation of QC measures from control chart data

• Implemented across consecutive QC measurements and/or across multiple p Q / pbatches of samples– 1 x 2SD Rule

• QC in any given sample is within 2xSD of control chart mean – Sample is ‘In Control’– 1 x 3SD Rule and nested rules

• QC in any given sample is outside 3xSD of control chart mean – Reject, sample is out of control

– 22S Rule• 2 Consecutive values are outside of 2xSD – Run is out of control

– R4S Rule• The Difference (Range) between 2 controls within a batch of samples exceeds 4xSD – Run

is out of control– 41S Rules

• 4 consecutive control values on one side of the mean > 1 SD from the mean– 10x Rule

• 10 consecutive control values are on one side of the mean for the control chart

Westgard RulesQC in any given sample is> Mean± 3SD

QC in 2 consecutive sample> Mean± 2SD

The range of QC values among 4 consecutive measures exceeds 4 SD

QC values for 4 consecutive measuresare all on one side of the mean + are > mean± 2 SD

QC values for 10 consecutive measuresare all on one side of the mean

Non‐Conformance/Corrective Action Report

• Failure of any laboratory QC rule (e.g. i t l h ti W t d)censoring, control charting or Westgard)

should be followed up with a Non‐Compliance Report

• Non‐compliance report is filled out and filed in conjunction with the analytical reportj y p

Corrective Actions

• Vary depending on the severity of the QC measure and up to the discretion of themeasure and up to the discretion of the laboratory supervisor

• 13S – rule and acceptable within batch results:– QC issue communicated to client and accepted by client as acceptable measurement

– Data re‐examined for transcription errorsData re examined for transcription errors– Chromatogram re‐examined for peak anomolies– Sample re‐extracted and failed data discarded

Corrective Actions

• Failure of trending data and multiple QC t ithi d b thmeasurements within and across bathes as

per Westgard or other censoring rules– Systematic analyst error? Re‐train/re‐test analyst

– Procedural/method bias?• Examine instrument calibration & performance

• Cross validate analytical standards – ‘working std.’ issue

• Full method validation

Micro‐extraction Technique• Developed in‐house at GLIER for PCB/OC‐pesticides

– Reduced sample size requirements (0.15 – 1.0 g)• Less pooling required for small samples (e.g. Invertebrates)p g q p ( g )• Omit GPC step by extracting < 0.15 g lipid from sample

– Reduced solvent volume utilization• Cheaper consumable costs (especially when omitting GPC!)• Less time watching roto‐evaporators!

– Higher throughput• Batch of 6 sampels (+ blank, CRM) can be full extracted, clean‐Batch of 6 sampels (+ blank, CRM) can be full extracted, cleanup in 1 day

– Disadvantages• Less mass extracted, so mostly the ‘major’ OC‐pesticides/PCBs picked up

Materials‐SPE Manifold with PTFE valves (12 port unit)

‐Customized glass reservoirs (40 mL)Customized glass reservoirs (40 mL)‐Customized reservoir rack

‐25 mL glass luer‐lock syringes1 μm Glass fiber syringe filters‐1 μm Glass fiber syringe filters‐small mortar & pestles (4 oz)‐micro‐scale (5 digit) for lipids

Setup costs: ~$3000 USD

Procedure• 0.15‐1 g of sample is ground up with ~ 10 g Na2SO4 in small mortar and pestle

• Homogenate poured into 25 mL syringe containg 15 mLh DCM (1 1 / ) ( l l d)hexane:DCM (1:1 v/v) (valves closed)

• Spiking recovery std spiked into syringe and allowed to stand 1 hr

• ‐Valves opened and eluant drained • by gravity at ~ 3 drips/second into reservoirsy g y p /• ‐after solvent reaches bedding, add• 5mL additional extraction solvent & elute• ‐Apply vacuum until columns are dry

Clean‐up of POPs from Biological Samples

GPC

Florisil

Silica Gel

Clean‐up• Several clean‐up protocols available for PCB/OC‐pesticides

• Common ones include:

– Gel permeation chromatographyGel permeation chromatography

– Florisil

– Silica Gel

– Alumina

• Often GPC + one more clean‐up step included for high fat biological samples

• This presentation – detailed methods for GPC, florisil and Silica Gel.

– Not intended to be proscriptive of individual labs

Gel Permeation Chromatography• Size exclusion chromatography• Biobeads wet are packed in solvent within a chromatography

column• Beads contain pores on surface with MW cutoff of 1000• Beads contain pores on surface with MW cutoff of 1000

– Molecules > 1000 g/mol are excluded from pores. They elute from the column via the mobile phase by passing in between beads. Stationary phase is inert relative to these compounds.

• Mostly biogenic materials – e.g. Lipids, hydrophobic proteins that can be discarded

– Molecules < 1000 g/mol can ‘fit’ within pores. They exhibit a tortuous diffusive path, moving in out of pores of beads and between beads. They are eluted from the column slowly. Stationary exhibits i t ti ithinteractions with

– Yields analyte fraction

• Manual, semi‐automated, automated systems available• Since stationary phase is inert for both analyte and biogeneic

materials, the GPC column can be re‐used refused provided blank and calibration checks are routinely performed

GPC – Decision Rules (GLIER SOP)

• GPC clean‐up required when– % lipid content of a sample > 3.75 % in a 4 g sample

– Total extracted lipid content in sample > 0.15 g

• Limitations– % lipid content of sample > 16.25% in a 4 g sample

R d t ti i ht f l• Reduce extraction weight of sample

• Split sample and run across multiple GPCs

– GPC removes up to 0.5 g lipid in extracted sample• Total lipid extracted in sample should not exceed 0.65 g

MaterialsGPC Columns-50 cm x 2.2 cm i.d. Pyrex column-250 mL pressure equalizing Separatory funnel (as reservoir)Separatory funnel (as reservoir)-50 g BioBeads (S-X3, BioRad), wet packed in 50% Hexane:DCM (V/V)

We find it practical to set up a bank of 8 GPC columns. Enables full sample batch to be GPC processed simultaneouslyGPC processed simultaneously

Approx. Costs to set up bank of GPC: ~$4000.00 (glassware + Biobeads)

GPC – Maintenance and Calibration(See GLIER‐ Related Procedures SOP 02.005.1)

• Maintenance– GPC columns remain active as long as the beads are kept covered in

solvent.

– The packing solvent should be similar to the extraction solvent derived from the sample extraction process

• Calibration (Preliminary)– Dilute 0.2 g vegetable oil into 4mL hexane/DCM (1:1 v/v)

– Extracts are transferred to GPC column

– Before beads dry, the initial container containing extracts are rinsed with 3 x 4 mL (12 mL total pre measured solvent) rinses ofwith 3 x 4 mL (12 mL total pre‐measured solvent) rinses of hexane/DCM (1:1 v/v) and pipetted onto the top of the column

– After final rinsing, separatory funnel is replaced on top of GPC column and reservoir filled with 288 mL hexane:DCM

– Column eluant is collected in 20 mL fractions and each fraction evaporated until dryness

– Cummulative volume containing 100% lipids is determined

• Validation (at least once per year)– Use of NIST‐SRM 1588a (cod liver oil) or triolein spiked with PCB/OC

d d

GPC – Maintenance and Calibration(See GLIER‐ Related Procedures SOP 02.005.1)

standard

– Proceed as per calibration instructions

– First fraction = cumulative eluant containing lipids. Typically this reflects the first 120 mL of eluant. The fraction is discarded

– Second fraction = remaining column eluant (120‐300 mL). This fraction is concentrated and subject to florisil or silica gel clean‐up

• Alternatives to GPC‐clean‐up (lipid removal)Alternatives to GPC clean up (lipid removal)– Acid silica gel can be used for PCBs

– Several OC‐pesticides are degraded by the acid‐clean‐up

Florisil Chromatography • Florisil – proprietary solid phase (activated MgSiO3) with high surface area

• Polar components are strongly bound to florisil, non‐Polar components are strongly bound to florisil, nonpolar compounds removed using a gradient of non‐polar to moderately polar solvents strengths

• Florisil becomes contaminated and is generally not‐reusable

• Fractionation for PCB‐OcsFractionation for PCB Ocs

– Fully activated florisil• Fraction 1 – Hexanes: PCBs and some OC‐pesticides

• Fraction 2 – 85%Hexane:15% DCM: OC‐pestides

• Fraction 3 – 50%Hexane:50% DCM – OC‐pesticides

• Fraction 4 – Toluene – NO‐PCBs

• Activation Procedure– Analytical grade Florisil purchased (Fisher Scientific)

Florisil Chromatography

Scientific)

– Florisil 60‐100 mesh is kept in drying oven at 130oC over‐night (minimum over‐night)

– One hour prior to use, sufficient quantity of Florisil is removed and placed in a dessicator to cool to room temperaturecool to room temperature

– Note other SOPs call for 1.2% deactivation, by shaking fully activiated florisil with HPLC grade water and storing in a sealed glass jar

MaterialsFlorisil Columns-25 cm x 1 cm i.d. glass column equiped with 250 mL reservoirs and PTFE stopcock-add 2 cm glass wool to bottom of column-wet pack 6 g fully activated florisil in excess of hexane-add ~2cm cap of activated Na2SO4

We find it practical to set up a bank of 8 florisil columns. Enables full sample batch to be processed simultaneously

Approx. Costs to set up bank of GPC: ~$1200.00 (glassware)

Florisil Procedure– Prepare florisil column as described

above

– Hexane is eluted until it is level with the Na2SO4 cap

– Without allowing column to dry, sample extracts (~2mL) are pipetted onto of column & eluted (~1 drip/s)

– Original container with extract rinsed with 3 x 2 mL portions of hexane (pre‐measured 6 mL total)

– Column eluted 44 mL hexane; cummulative hexane eluant = Fraction1

– Column eluted with 50 mL of 15% DCM/85% hexane (v/v) = Fraction 2

– Column eluted with 150 mL 60% DCM/40% hexane (V/v) = Fraction 3

Fractionation Florisil

• Florisil Cleanup (3 fractions)– Fraction 1:Fraction 1:

• PCBs – all congeners (except NO‐PCBs), HCB (F1,F2), Heptachlor (F1, F2), Aldrin (F1, F2), Endosulfan II (f1, f2), DDE (F1, F2 – both isomers), DDD (F1, F2) ; mirex (F1)

– Fraction 2:• HCHs (100 F2), HCB (F1,F2), Heptachlor (F1,F2), heptachlor epoxide (Isomer a) Aldrin (F1,

F2), oxychlordane (F2, F3), trans‐chlordane (F2); Endosulfan I (F1+F2), cis‐chlordane (F2); DDE (F1, F2), DDD (F1, F2); DDT (F2)

– Fraction 3:• Endrin heptachlor epoxide (F2 F3) heptachlor epoxide (isomer B) oxychlordante (F2 F3)• Endrin, heptachlor epoxide (F2, F3) heptachlor epoxide (isomer B), oxychlordante (F2, F3),

Dieldrin (F3), methoxychlor (F3)

• Silica gel used in place of florisil

• Baker Scientific, 60‐200 mesh activated silica gel

Silica Chromatography (Trent Univesity’s Procedure)

, g

• 30 cm x 1 cm i.d. Glass column plugged with glass wool

• 5 g activated silica gel wet packed into column in hexane

• Hexane eluted to column until it reaches silica gel bedding

• Sample extracts added to column with sample rinses

• Fraction 1 = elution with 40 mL hexane– PCBs, some OC‐pesticides

• Fraction 2 = elution with 70 mL hexane (50%):DCM (50%)– Remaining OC‐pesticides

GC‐ Analysis

-Florisil or Silica gel fractions are each

concentrated to 2 mL (or less)

-Sulphur containing materials clean-up

using activated copper prior to addition

to GC-vials

-Each fraction analyzed by GC-ECD

GC Methods and Instrument Maintenance

• Documented Methods for POPs (OCs/PCBs)• Basic Maintenance and Troubleshooting

GLIER - University of Windsor 1

Documented Methods for POPs (OCs/PCBs)

• Injector Temp: 250oC; 63Ni-ECD Detector Temp: 300oC.

Column: 60 m x 0 25 mm I D x 0 10 µm film thickness• Column: 60 m x 0.25 mm. I.D. x 0.10 µm film thickness DB-5 (J&W).

• 1 µl injection volume using splitless injection mode.

• Carrier Gas: (UHP) - He at approximately 22 cm/sec –


( ) pp ydetermined at 90oC (1 mL/min); Column Head Pressure is 22.88 psi.

• Make-up Gas: Ar/CH4 (95%/5%) at 50 mL/min.

Documented Methods for POPs (OCs/PCBs) (Cont.)

280oC5 min

90oC

200oC

20oC/ min

3oC/ min

1 min

2 min


42 min

Figure 1: GC Oven Temperature Program for Separation of OCs and PCBs.






Figure 3: GC trace of Pesticide Mix 1 Std. recorded on GC/ECD

Basic Maintenance of GC/ECD

UHP Compressed Gases ith in line filters• UHP Compressed Gases with in-line filters (moisture and oxygen traps)

• GC Injection Port• GC Column• GC Detector


• GC Detector

Basic Maintenance of GC/ECD (Cont.)

GC Injection Port• Glass liner should be replace with a new one, depend p , p

on quality and quantity of samples. • Co-Extractives present in samples, often stick to the

cold spot of the injector. The injector’s temp. must keep higher than the oven initial temp.

• Some Pesticides may partially degraded in the injector port liner for e g DDT to DDE Endrin to Endrin


port liner, for e.g., DDT to DDE, Endrin to Endrin Ketone). Use deactivated liner. If the degradation is >15%, replace the liner with a new one.


GC Column• Fused silica capillary column coated with non polar• Fused silica capillary column coated with non-polar

chemically bonded stationary phase; polysiloxanes with outer protective layer of polyimide.

• Do not exceed the upper temp. limit more than the temp. specified from the column manufacturer, this might damage the column coating and destroy the stationary phase.


p• Always keep GC column inside box and close both ends

with septa.• It is a good practice to keep an extra new column in

hand.


GC Detector (ECD)• ECD detector is highly sensitive, the entire GC system

should be leak free otherwise oxidation of 63Ni foil will occur and cause higher noise level, baseline drifting and shorter lifetime of the detector will result.

• Always pre-condition the column out of the detector (capped the detector port), while attached first end of the column to the injector.


• Before installing or removing a column to the ECD, lower the temp. of the detector <100oC to prevent oxidation of 63Ni foil.

Troubleshooting of GC/ECD Machine• Interferences – Sometimes Negative and

positive peaks or spikes appear due to the presence of sulfur, phthalate ester (positive p , p (ppeak) and hydrocarbon (negative peak/spikes).

• If sensitivity of peaks goes down, increase in noise level:

Action - change glass liner, cut column ~10-20 cm from injector side and condition the column overnight


column overnight. This problem is also related to the detector due to incorrect installation distance.

Troubleshooting of GC/ECD Machine (Cont.)

• In Standard mix, if peaks deteriorated and lost resolution – don’t waste time and change column with a new one.co u t a e o e

• Having Noisy Baseline or Column bleeding –Bake the system for an hour; Injector at 300oC, Column at 285 oC and detector at 320 oC.

• Polysiloxane - Stationary Phase in DB-5 Column – destroys in presence of moisture as well as Ni


destroys in presence of moisture as well as Ni foil of EDC detector.

• Samples should be free of moisture.

Data Reporting &Data Reporting &Data Management

Official Data Reports• Standardize cover page should be developed t i t t ti t tto ensure consistent reporting structure

• Pertinent information about method, MDLs, QC results

• Results with appropriate significant digits

• Data flags (i e ND’s INT other)• Data flags (i.e ND’s, INT, other)

• Authorization sign‐offs – Laboratory Supervisor and Quality Manager

Data Reporting

From Muir and Sverko 2006. Paper provided in electronic materials.

• Test Reports


– QA signoff

– Flagged results

– ND, <DL

– Sig. digits

Data management• Apart from logs, control charts and other documents, the

laboratory has a responsibility to maintain data records and review data results at clients requestdata results at clients request

• Large amounts of residual material and data generated in analysis process– Remaining, unextracted sample– Sample extracts– Clean‐up extracts (post‐GC analysis)– Raw data (chromatograms and/or chromatographic files)

Peak Areas and Calc lation Spreadsheets– Peak Areas and Calculation Spreadsheets– Calculation results– Final Report

• Laboratory should develop policy and procedures for retention, storage and eventual destruction of above materials

Data storage and back‐up

• Physical samples and/or extracts kept in secure location. Often refrigerated or frozen.– E.g. Use of vial‐files

• Print‐outs of chromtographs filed with client final report

• Electronic data sets periodically backed upE t l h d d i– External hard‐drive

– Network back‐ups (ideal)

– CD’s Notoriously Insecure

DETERMINATION OF PCBs AND PESTICIDES IN TUNAHOMOGENATE (IAEA-435) - Dec 2008

Tuna Homo

AVG. S.D. RSD IDL MDL

ref. & info Mean Standard Relative Inst. Methodvalues

Compound values (ng/g) stdev n observed Deviation Standard Detection Detection

conc. Deviation Limit Limit (ng/g) (ng/g) (ng/g) (%) (ng/mL) (ng/g) PCB18/17 PCB 18 4.3 4.6 8 0.449 0.0912 20.33 0.192 0.170 PCB 31/28 2.9 1.2 8 1.593 0.1963 12.32 0.101 0.365 PCB 52 4.4 2.5 8 2.919 0.2167 7.42 0.151 0.403 PCB 49 3.1 1.7 8 1.470 0.1155 7.86 0.113 0.215 PCB 44 3.0 2.8 8 0.662 0.1225 18.50 0.093 0.228 PCB 70 4.6 2.1 8 2.010 0.2924 14.55 0.089 0.544 PCB 95 8.3 6.3 8 7.211 0.5224 7.24 0.100 0.972 PCB 101 23.0 10.0 8 15.975 1.1365 7.11 0.097 2.114 PCB 99 16.0 4.1 8 9.614 0.7400 7.70 0.080 1.376 PCB 87 6.9 3.9 8 2.645 0.5317 20.10 0.055 0.989 PCB 110 15.0 8.3 8 4.546 0.8388 18.45 0.063 1.560 PCB 151/82 12.0 2.7 8 1.381 0.0981 7.10 0.069 0.182 PCB 149 29.0 7.1 8 18.816 1.0886 5.79 0.092 2.025 PCB 118 24.0 9.3 8 16.873 0.6717 3.98 0.074 1.249 PCB153 81.0 37.0 8 55.451 4.3924 7.92 0.077 8.170 PCB 105/132 6.7 3.6 8 4.338 0.7997 18.43 0.061 1.487 PCB 138 70.0 32.0 8 44.131 3.2421 7.35 0.057 6.030 PCB 187 31.0 10.0 8 20.330 1.0187 5.01 0.072 1.895 PCB 183 11.0 2.9 8 6.954 0.6662 9.58 0.057 1.239

GLIER Laboratory – University of Windsor

PCB 128 9.5 4.7 8 2.389 0.3980 16.66 0.044 0.740

DETERMINATION OF PCBs AND PESTICIDES IN TUNAHOMOGENATE (IAEA-435) - Dec 2008 (CONT.)

PCB 195/208 2.2 1.3 8 1.821 0.1910 10.49 0.085 0.355 PCB 194 3.9 1.1 8 2.758 0.1647 5.97 0.041 0.306 PCB 206 2.8 1.4 8 2.143 0.0725 3.38 0.047 0.135 PCB 209 2.0 0.8 8 1.398 0.1108 7.93 0.053 0.206 mirex 8 1.951 0.0773 3.96 0.045 0.144 HCB 2 6 1 0 8 1 813 0 1535 8 46 0 031 0 285HCB 2.6 1.0 8 1.813 0.1535 8.46 0.031 0.285heptachlor 1.1 2.1 8 0.266 0.0222 8.36 0.041 Aldrin 1.1 0.6 8 0.281 0.0454 16.15 0.084 endosulfan I 7.7 6.1 8 0.809 0.1008 12.45 0.191 trans nonachlor 27.0 9.0 8 0.030

' DDE 91 0 58 0 8 116 824 11 5911 9 92 0 030 19 544pp'-DDE 91.0 58.0 8 116.824 11.5911 9.92 0.030 19.544op'-DDE 2.9 2.3 8 8.340 0.3941 4.73 0.733 Endosulfan II 17.0 17.0 8 pp'-DDD 12.0 7.4 8 9.772 0.6563 6.72 0.034 1.221 o,p-DDD 2.5 0.5 8 1.140 a-BHC 0.8 0.7 8 0.029 0.0046 15.75 0.020 0.009 b-BHC 1.3 1.4 8 0.106 0.0222 20.94 0.062 0.041g-BHC 1.1 0.9 8 0.215 0.0296 13.74 0.024 0.055 Trans-chlordane 0.5 0.3 8 0.825 0.0638 7.73 0.027 0.119 cis-Chlordane 8.0 1.4 8 4.751 0.2957 6.22 0.029 0.550cis-nonachlor 13.0 3.5 8 0.028 pp'-DDT 18.0 10.0 8 30.121 2.7117 9.00 0.083 5.044 op'-DDT 7.8 4.8 8 0.742 Heptachloro Epoxide isomer A 2.8 2.5 8 0.979 0.1243 12.70 0.033 0.231Oxychlordane 8 2.130 0.2164 10.16 0.032 0.402 heptachlor epoxide isomer B 8 Dieldrin 3.80 2.1 8 3.161 0.1281 4.05 0.030 0.238 Endrin 12.0 12.0 8 0.390 0.0646 16.58 0.120 methoxychlor

12 50


TBB 12.50 (82.01)

PCB 30 21.04

Table. List of CBs, OCs and PCB’s, providing Instrument Detection Limit (IDL), Calibration (Working) Linear Range and Coefficient of Determination (R2).

Parameter Rt (min) IDL Calibration/Working Range ng/mL R2

ng/mL (Levels):L0; L1; L2; L3; L4; L5; L6

(May/2008)

Hexachlorobenzene (HCB)

11.922 0.022 0 ;0.2; 1.0; 2.0; 10.0; 20.0; 60.0 0.9994

Octachlorosty(OCS)

11.698 0.017 0 ;0.2; 1.0; 2.0; 10.0; 20.0; 60.0 0.9995rene (OCS)

pp’-DDE 19.304 0.019 0 ;0.2; 1.0; 2.0; 10.0; 20.0; 60.0 0.9983

Mirex 19.478 0.028 0 ;0.2; 1.0; 2.0; 10.0; 20.0; 60.0 0.9978

a-BHC 11.698 0.014 0 ;0.2; 1.0; 2.0; 10.0; 20.0; 60.0 0.9985

b BHC 12 273 0 044 0 ;0 2; 1 0; 2 0; 10 0; 20 0; 60 0 0 9996b-BHC 12.273 0.044 0 ;0.2; 1.0; 2.0; 10.0; 20.0; 60.0 0.9996

g-BHC 12.464 0.016 0 ;0.2; 1.0; 2.0; 10.0; 20.0; 60.0 0.9987

oxy-Chlordane

17.054 0.020 0 ;0.2; 1.0; 2.0; 10.0; 20.0; 60.0 0.9971

trans-Chl d

17.903 0.018 0 ;0.2; 1.0; 2.0; 10.0; 20.0; 60.0 0.9981Chlordane

cis-Chlordane 18.515 0.019 0 ;0.2; 1.0; 2.0; 10.0; 20.0; 60.0 0.9989

pp’-DDD 18.715 0.026 0 ;0.2; 1.0; 2.0; 10.0; 20.0; 60.0 0.9984

cis-Nonachlor 21.395 0.018 0 ;0.2; 1.0; 2.0; 10.0; 20.0; 60.0 0.9990

pp’ DDT 22 868 0 026 0 ;0 2; 1 0; 2 0; 10 0; 20 0; 60 0 0 9977pp -DDT 22.868 0.026 0 ;0.2; 1.0; 2.0; 10.0; 20.0; 60.0 0.9977

HC Epoxide 16.996 0.018 0 ;0.2; 1.0; 2.0; 10.0; 20.0; 60.0 0.9971

Dieldrin 17.054 0.017 0 ;0.2; 1.0; 2.0; 10.0; 20.0; 60.0 0.9978

PCB #18/17 12.66/12.72 0.131 0.08-249.20 0.9978

PCB #28/31 13.89/13.94 0.065 0.11-351.10 0.9988PCB #28/31 13.89/13.94 0.065 0.11 351.10 0.9988

PCB #33 14.231 0.077 0.0634-198.2 0.9986

PCB #52 14.977 0.098 0.0634-198.4 0.9986

PCB #49 15.131 0.073 0.0633-198.0 0.9987

PCB #44 15.685 0.060 0.0633-198.0 0.9988


PCB #74 16.964 0.050 0.064-200.0 0.9991

PCB #70 17.084 0.057 0.0634-198.2 0.9994

PCB #95 17 262 0 068 0 0320-100 2 0 9985

(Cont.)

PCB #95 17.262 0.068 0.0320-100.2 0.9985

PCB #101 18.141 0.061 0.0643-201.0 0.9989

PCB #99 18.36 0.050 0.0634-198.0 0.9989

PCB #87 19.238 0.035 0.0638-199.4 0.9988

PCB #110 19.667 0.039 0.0637-199.0 0.9990

PCB #151/82 20.198 0.043 0.0801-250.5 0.9990

PCB #149 20.713 0.056 0.0633-197.8 0.9981

PCB #118 20.791 0.046 0.0641-200.2 0.9988

PCB #153 21.793 0.046 0.0635-198.4 0.9987

PCB # 105/132 21.949 0.038 0.0478-149.5 0.9979

PCB #138 23.098 0.035 0.0645-201.6 0.9990

PCB #158 23.221 0.029 0.0161-50.20 0.9977

PCB #187 23.906 0.043 0.0635-198.4 0.9988

PCB #183 24.152 0.035 0.0633-198.0 0.9961

PCB #128 24.406 0.027 0.0643-200.8 0.9988

PCB #177 25.298 0.037 0.0640-200.0 0.9988

PCB #171/156 25.532 0.027 0.1271-397.4 0.9987

PCB #180 26.398 0.029 0.0638-199.4 0.9986

PCB #191 26.742 0.025 0.0640-200.0 0.9987

PCB #170 27.871 0.032 0.0634-198.0 0.9989

PCB #201 28.328 0.025 0.0480-150.0 0.9988

PCB #195/208 30 05/30 11 0 042 0 1281 400 4 0 9985PCB #195/208 30.05/30.11 0.042 0.1281-400.4 0.9985

PCB #194 31.135 0.023 0.0641-200.2 0.9974

PCB #205 31.425 0.023 0.0638-199.4 0.9985

PCB #206 33.067 0.025 0.064-200.0 0.9983

PCB #209 34 643 0 029 0 064-200 0 0 9984


PCB #209 34.643 0.029 0.064-200.0 0.9984