PRESENTATIONS Training Workshop on Laboratory Methods and ...

 

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  Documentation •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 results how 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  p demonstrate 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 knowledge People 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 requirements measurement 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 samples unknown 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

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

Principles (Cont.):  • Objectivity of Results – Test results are mainly based on measurable quantities with  repeatable measurement characteristics repeatable measurement characteristics

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

• Traceability of Measurement T bilit f M t – 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  System  System • Management Requirements • Technical Requirements • Revision History

• Quality System Procedures (QSP) – Second level document detailing instructions &  procedures in QSM procedures  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) ‐Each sub‐section reflects a stand‐alone document subject to revision ‐Describes Purpose, Scope and Authorities and Responsibilities for tasks 

– Technical Requirements – Personnell – Controlled Environment and  Facilities – Method Validation and  Calibration – Equipment q p – Measurement Traceability – Handling of Test Items – Quality Assurance of Test  Results – Results Reporting

2) Quality System Procedures (QSP) (Focus on Technical Requirements) 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 laboratory productivity, competency of technical personnel/training, safety,  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

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

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

• Environmental Conditions – Facilities are adequate to carry out testing F iliti d t t t t ti – Descriptions of  • Laboratory space  – Criteria for lighting, temperature, humidity, air quality

• Safety considerations  – Use/availability of fumehoods, venting, solvent cabinets,  chemical storage, emergency showers/eyewash stations, first  h i l t h / h t ti fi t aid kits, emergency exits, other institutional safety procedures

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

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

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

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

• Test Method and Method Calibration – Validation  V lid ti • 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

2) Quality System Procedures (QSP) (Focus on Technical Requirements) • Equipment – Equipment 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

2) Quality System Procedures (QSP) (Focus on Technical Requirements) • 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  ( (sample/standard) storage areas, gas chromatographs l / d d) h h

– 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

2) Quality System Procedures (QSP) (Focus on Technical Requirements) • 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 Control Charting and evaluation of control chart data 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

2) Quality System Procedures (QSP) (Focus on Technical Requirements) • 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 Control Charting and evaluation of control chart data 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

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

• Test Reports Test Reports – QA signoff – Flagged results – ND, 

 2 year time period

•Short term storage of biological samples (95% of PCB/OCs in biological samples contained within  neutral lipids (triglycerides) of samples with > 1% total lipid  content • Expression of lipid normalized POP residues useful to  i f li id li d O id f l 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) (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

•Total lipids sometimes determined for other  purposes, i.e. to establish health and energy  density of animal •For tissues with low lipid content (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 Analyses PROCEDURE • •

• •

•

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 3 LOQ = SD x 10 Computation method 1b (US EPA): LOD = t (n-1, 0.99) x SD; where t = Students’ t value for 99% level confidence LOQ = LOD x 3.3 1) If possible, choose a sample matrix for spiking that has non-detectable levels of the target analyte.

Method 1: Replicate Analyses PROCEDURE Analysis of lindane spiked into fish tissue at 0.21 ng/g wet weight (n=9): Sample # 1 2 3 4 5 6 7 8 9

Conc (ng/g) 0.23 0.21 0.24 0.19 0.18 0.23 0.22 0.17 0.16

% Recovery 110 100 114 90 86 110 105 81 76

Mean = 0.20 ng/g SD = 0.029 S/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 = 0.084 LOQ = 0.084 x 3.3 = 0.28

Students’ t values at 99% confidence level: n n-1 t-value 7 6 3.143 8 7 2.998 9 8 2.896 10 9 2.821 11 10 2.764

Method 1: Replicate Analyses PROCEDURE Lindane spiked into fish at 0.21 ng/g: Mean = 0.20 ng/g SD = 0.029 S/N = 6.89 The five point check: • Does the spike level exceed 10x the Mean recovery = 97% LOD? If so, the spike level was too high. Range = 76-114% • Is the LOD higher than the spike level? If so, the spike level was too low. Computational method 1a: • Is the S/N in the appropriate range (i.e. LOD = 0.087 75%) and reproducible.

Method 2: Serial Dilutions PROCEDURE LOQ = 3.3 x LOD

Signal

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

Method Blank

0 Spiking Concentration

LOD using Method 2: Concentration at which there is an asymptote (change in slope) in the serial dilution regression 10

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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 MDLs should be determined for every new set of samples. • However, for practical reasons, it is usually assumed that the MDLs determined 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 Calibration Choice 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).

Calibration std at upper limit of linear range

Concsample = Concstd x

Response sample Response std

Response

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.

Unknown Conc

External Standard Calibration B) Two Point Calibration: • High concentration – Near upper limit of linear range • Low concentration – Near LOQ

Concentrations of analytes are determined by interpolating from a linear regression line.

Response

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

Unknown Conc

Internal Standard Calibration Standard 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 •Frequency •Troubleshooting Nargis Ismail Glier GLIER Laboratory – University of Windsor

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Why Do We Need To Validate A Method? • Method validation provides evidence that an analytical l ti l method th d when h carried i d through th h the th whole process in exactly the same manner produces results that fit for the purpose. • Establish specificity, range and linearity of the technique. • Establish E t bli h Quantitation Q tit ti limit; li it IDL and d MDL

GLIER Laboratory – University of Windsor

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Validation Protocol By carrying at least 6 replicates of a suitable clean Matrix Matrix* spiked with the target Analytes atleast three levels of concentration (or a Reference Sample) carried through all the sample processing steps.

GLIER Laboratory – University of Windsor

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Frequency of Method Validation • When a new method developed in a lab or if the method is imported from elsewhere. • Any A major j changes h iin existing i ti methods th d – 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 – Re-affirm performance characteristics (range and linearity) GLIER Laboratory – University of Windsor

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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)

GLIER Laboratory – University of Windsor

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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 – 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 GLIER Laboratory – University of Windsor

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Analytical Method Development Cont.)

Figure 1: GC trace of Pesticide Mix 1 std. recorded on GC/MSD GLIER Laboratory – University of Windsor

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Figure 2: GC trace of Pesticide Mix 1 std. recorded on GC/ECD GLIER Laboratory – University of Windsor

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Figure 3: GC trace of Quebec Certified PCB std. recorded on GC/ECD GLIER Laboratory – University of Windsor

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Machine Calibration • Figure 2.ppt

GLIER Laboratory – University of Windsor

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Performance Parameters 1. 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  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 20 the 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) • 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 ‐ Microsoft Excel formulae: =Average(cell range) • Standard Deviation • Microsoft Excel:  = Stdev(cell range) Mi ft E l Std ( ll ) • 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 / p batches 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 Rules QC 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 measures are all on one side of the mean + are  >  mean± 2 SD

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

Non‐Conformance/Corrective Action  Report • Failure of any laboratory QC rule (e.g.  censoring, control charting or Westgard)  i t l h ti W t d) should be followed up with a Non‐Compliance  Report • Non‐compliance report is filled out and filed in  conjunction with the analytical report j y p

Corrective Actions • Vary depending on the severity of the QC  measure and up to the discretion of the measure 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 Data re‐examined examined for transcription errors 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  measurements within and across bathes as  t ithi d b th 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  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  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 • Reduce extraction weight of sample R d t ti i ht f l • 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

Materials GPC Columns -50 cm x 2.2 cm i.d. Pyrex column -250 mL pressure equalizing 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 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 of with 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

GPC – Maintenance and Calibration (See GLIER‐ Related Procedures SOP 02.005.1) • Validation (at least once per year) – Use of NIST‐SRM 1588a (cod liver oil) or triolein spiked with PCB/OC  standard d d – 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 Alternatives to GPC‐clean‐up clean up (lipid removal) (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, non polar compounds removed using a gradient of non‐ polar to moderately polar solvents strengths • Florisil becomes contaminated and is generally not‐ reusable  Fractionation for PCB‐Ocs Ocs • Fractionation for PCB – 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

Florisil Chromatography  • Activation Procedure – Analytical grade Florisil purchased (Fisher  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 temperature cool 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

Materials Florisil 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 Endrin, heptachlor epoxide (F2, F3) heptachlor epoxide (isomer B), oxychlordante (F2, F3),  heptachlor epoxide (F2 F3) heptachlor epoxide (isomer B) oxychlordante (F2 F3) Dieldrin (F3), methoxychlor (F3)

Silica Chromatography  (Trent Univesity’s Procedure) • • • • • • •

Silica gel used in place of florisil , g Baker Scientific,  60‐200 mesh activated silica gel 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

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Documented Methods for POPs (OCs/PCBs) • Injector Temp: 250oC; 63Ni-ECD Detector Temp: 300oC. • Column: 60 m x 0.25 0 25 mm mm. II.D. D x0 0.10 10 µm film thickness DB-5 (J&W). • 1 µl injection volume using splitless injection mode. pp y 22 cm/sec – • Carrier Gas: ((UHP)) - He at approximately determined at 90oC (1 mL/min); Column Head Pressure is 22.88 psi. • Make-up Gas: Ar/CH4 (95%/5%) at 50 mL/min. GLIER - University of Windsor

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Documented Methods for POPs (OCs/PCBs) (Cont.) 5 min

280oC 3oC/ min

2 min

200oC 20oC/ min 1 min 90oC

42 min

Figure 1: GC Oven Temperature Program for Separation of OCs and PCBs. GLIER - University of Windsor

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

Figure 2: GC trace of Quebec Certified PCB std. recorded on GC/ECD GLIER - University of Windsor

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

Figure 3: GC trace of Pesticide Mix 1 Std. recorded on GC/ECD GLIER - University of Windsor

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Basic Maintenance of GC/ECD • UHP Compressed Gases with ith in in-line line filters (moisture and oxygen traps) • GC Injection Port • GC Column • GC Detector

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Basic Maintenance of GC/ECD (Cont.) GC Injection Port • •

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

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Basic Maintenance of GC/ECD (Cont.) GC Column • Fused silica capillary column coated with non non-polar 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. GLIER - University of Windsor

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Basic Maintenance of GC/ECD (Cont.) 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