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BioOrganic Analytical Chemistry Laboratory
Division of Environmental Health Sciences
Wadsworth Center
Department of Health
State of New York
CLIA: 33D0654341
Standard Operating Procedure
Determination of Polychlorinated Biphenyls and Pesticides in Human
Serum by Solid Phase Extraction and Gas Chromatography/Selected
Ion Monitoring Mass Spectrometry
Approved: ___________________________________________________
Laboratory Supervisor
Robert Jansing, Ph. D.
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Distribution
Dr. Kurunthachalam Kannan, Chief
Laboratory of Organic Analytical Chemistry
Dr. Robert Jansing, Deputy Chief,
Laboratory of Organic Analytical Chemistry
Laboratory Supervisor, Gas Chromatography Laboratory
Mr. Anthony Bucciferro, Quality Manager
Laboratory of Organic Analytical Chemistry
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Revision Record
Rev
Date
Responsible Person
1
11/15/07
Li Zhang, Buu Tran,
Robin Storm and Robert
Jansing
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Description of Change
Initial Release
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The following laboratory staff has read this Manual.
A copy of this page will be distributed to the employee training record file .
Name
Title
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TABLE OF CONTENTS
Page
Abbreviation listing
6
1. Scope and Application
7
2. Summary of Method
8
3. Definitions
9
4. Interferences
10
5. Safety and Waste Management
11
6. Pollution Prevention
12
7.Competency and Assessment Training
12
8. Sample Collection, Preservation, and Handling
14
9. Equipment and Supplies
16
10. Reagents and Standards
18
11.Daily GC/MSD Performance Test
20
12. Calibration
21
13. Automated SPE Procedure
1922
14. Quality Control
24
15. Data Assessment
29
16. Corrective Actions and Contingencies for Out of Control Data
29
17. References
29
18. Tables and Demonstration Data
30
Appendix A – SPE Cartridge Preparation
45
Appendix B – Specific Worksheet
47
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ABBREVIATION LISTING
C18
octadecyl bonded silica gel
CAS
chemical abstract service
CCC
continuing calibration check standard
CCS
calibration check standard
DFTPP
decafluorotriphenylphosphine
EPA
Environmental Protection Agency
GC
gas chromatography
IDA
initial demonstration of accuracy
IDC
initial demonstration of capability
IDP
initial demonstration of precision
IC
internal calibration
IS
internal standard
IUPAC
International Union of Pure and Applied Chemistry
LCL
lower confidence limit
MB
method blank
MDL
method detection limit
MRL
minimum reporting level
MS
matrix spike
MSD
mass selective detector
OCP
organochlorine pesticide
PCB
polychlorinated biphenyl
PDS
primary dilution standard
POP
persistent organic pollutant
PTS
performance test solution
QC
quality control
R
correlation coefficient
2
R
determination coefficient
RB
reagent blank
RF
response factor
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RSD
relative standard deviation
RT
retention time
S
standard deviation
SB
solvent blank
SIM
selected ion monitoring
SOP
standard operating procedure
SPE
solid phase extraction
t
Student’s statistical test value
TIC
total ion chromatogram
UCL
upper confidence limit
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1. SCOPE AND APPLICATION
1.1 Human populations worldwide have been exposed to a broad range of synthetic organic industrial and
agricultural chemicals known collectively as persistent organic pollutants (POPs) since the early part of the
twentieth century. Polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) are persistent,
bioaccumulative POPs that were disseminated in the environment decades before their potential for
involvement in adverse health effects in humans was identified.
Measurable levels of PCBs and OCPs were detected in human serum samples obtained in the 1940s and
comparable levels of these POPs were detected in serum specimens analyzed in 2002. Peak human serum
levels of PCBs and several OCPs occurred in the late 1970s prior to the enactment of legislation banning
their production and use. Results from our laboratory indicate that human serum levels are declining,
however passive human exposure to PCBs and some OCPs still occurs by inhalation of dust or ingestion of
food. It is important from a public health standpoint therefore to determine if serum levels of PCBs and
OCPs are decreasing over time.
1.2 The proposed method describes the procedure for the analysis and quantitation of PCB congeners , 4,4’DDE, 4,4’-DDT and mirex in newborn calf serum by solid phase extraction and gas chromatography with
mass selective detection in the selected ion mode (GC/MSD/SIM). The Con2 standard comprising 44 PCB
congeners, 4,4’-DDE, 4,4’-DDT and mirex is used to establish the calibration curve while the Calibration
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Check Standard (CCS) comprising 21 congeners representing the homologues from tri- to deca-chloro
biphenyls is used verify the accuracy of the calibration standard and for the method development
1.3 Polychlorinated biphenyl (PCB) congeners, 4,4’-DDE, 4,4’-DDT and mirex are extracted using the
Biotage Rapid-Trace solid phase extraction (SPE) system.
1.4 Minimum Reporting Limit (MRL) - Defined as the lowest concentration of an analyte that can be reported
based on the results of the method detection limit study.
1.5 Method Detection Limit (MDL) is defined as the statistically calculated concentration that can be measured
with 99% confidence that the reported value is greater than zero. The MDL study for the target analytes can
be found in the Table 9.
1.6 This SOP should be followed as written. Sometimes, however, deviations from the SOP are unavoidable.
Any deviation for the SOP must be documented and approved by the QC holder.
2. SUMMARY OF METHOD
This work describes the analysis of polychlorinated biphenyls (PCBs) 4,4’-DDE, 4,4’-DDT, and mirex in
human serum using gas chromatography / isotope dilution mass spectrometry. Serum specimens are spiked
with 8-13C PCB congeners and 13C 4,4’-DDE as internal standards. Newborn calf serum is spiked with a
standard mixture containing 44 PCB congeners, 4,4’-DDE, 4,4’-DDT, mirex, and the isotopically labeled
internal standards. The serum specimens the spiked new born calf serum(Matrix Spike) are deproteinized
with 88% formic acid, and extracted using an automated solid phase extraction (SPE) system equipped with
silica-based C18 cartridges. Target analytes are eluted with 15%/85% dichloromethane/hexane (v/v) and
lipids are removed by passing extracts through a silica gel/acid silica gel column. The eluate is reduced to
0.1 mL in hexane before a GC/MSD analysis. Extracts are analyzed in SIM mode at ion source temperature
of 250oC.
Matrix matched calibration at 0.1 0.5,1.0, 2.5, 5.0 and 10 ng/g was used to assess detector
linearity.
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3. DEFINITIONS
3.1 ANALYSIS BATCH – A set of samples analyzed during a 24-hr period using the same instrumentation,
calibration standards and quality control samples. Each analysis batch begins and ends with the analysis of
the appropriate Continuing Calibration Check (CCC) Standard.
3.2 CALIBRATION STANDARD – A solution of method analytes prepared from working standard solutions
used to calibrate the instrument response with respect to analyte concentration.
3.3 CONTINUING CALIBRATION CHECK STANDARD (CCC) – One of the mid level calibration
standards used to verify the acceptability of an existing calibration. A CCC must be analyzed at the
beginning and end of each analysis batch.
3.4 CALIBRATION CHECK STANDARD (CCS) – A solution of method analytes prepared from a second
stock standard source that is different from the source used to prepare calibration standards. The CCS
verifies that the original calibration is acceptable by measuring the measures the accuracy of calibration
standard using a second source standard. The CCS need not be qualitatively identical to the Calibration
Standard but should contain congeners present in each homologue group and at least one pesticide.
3.5 INTERNAL STANDARD (IS) – A pure compound added to a sample, or standard solution in known
amount that is used to measure the relative responses of target analytes. In this study, a 13C PCB mixture,
composed of congeners from each homologue group, CL 1 through CL 10, and 13C 4,4’-DDE were used as
internal standards.
3.6 MATRIX SPIKE (MS) – An aliquot of new born calf serum spiked with known quantities of target
analytes is extracted and analyzed like a sample. The MS is used to calculate recoveries of target analytes
and to determine if the sample matrix contributes any bias to the sample results.
3.7 METHOD BLANK (MB) – An aliquot of new born calf serum that is treated the same as a sample. The
MB is taken through all the same method procedures and exposed to the same glassware, equipment,
preservatives, solvents, reagents, and internal standards as a sample. The MB determines if target analytes
or other interferences are present in the laboratory environment, reagents, or equipment.
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3.8 METHOD DETECTION LIMIT (MDL) – The statistically calculated minimum concentration of an analyte
that can be measured with 99% confidence that the value is greater than zero.
3.9 MINIMUM REPORTING LIMIT (MRL) – The minimum concentration that can be reported for a target
analyte. This limit is based on quality control criteria measured in the MDL study and can be no lower than
the lowest calibration standard. The MRL is typically 3-5 times the MDL.
3.10 PERFORMANCE TEST – The performance test is composed of IUPAC 58 and IUPAC 186 and is
analyzed before each batch to check the reproducibility of the retention time and integrated abundances
(peak areas) of the analytes of interest.
3.11 PRIMARY DILUTION STANDARD SOLUTION (PDS) – A solution of method analytes prepared from
stock standard solutions (SSS) and diluted as necessary to prepare calibration standards or other necessary
analyte solutions.
3.12 REAGENT BLANK (RB) - An aliquot of pure solvent subjected to the same analytical or measurement
process as a normal sample. The RB determines if target analytes or other interferences are present in
laboratory reagents or glassware.
3.13 SOLVENT BLANK - An aliquot of pure solvent analyzed prior to analysis batch, after analytical standards
and after sample extracts showing high levels of target analytes.
3.14 STOCK STANDARD SOLUTION (SSS) – A concentrated solution of target analytes prepared in the
laboratory from referenced and certified standards or a concentrated solution of target analytes purchased
directly from a referenced and certified source.
4. INTERFERENCES
4.1 Method interferences may be caused by contaminants in solvents, reagents, glassware, and other sample
processing apparatus that lead to discrete artifacts or elevated chromatographic baselines. MB and RB must
be run to verify that these types of interferences are not present.
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4.2 Glassware must be thoroughly cleaned. After use, rinse all glassware with last solvent used, wash with hot
water and detergent, rinse with tap water and reagent water, and heat in an oven to dry at 100-200 oC for a
minimum of one hour. Allow glassware to come to room temperature. Rinse three times with nanograde
hexane, save last rinse, concentrate to 0.05mL and analyze by GC/MSD to determine if interferences are
present.
4.3 Interferences may occur when a sample containing low concentrations of analytes is analyzed following a
sample containing high concentrations of analytes. If this type of interference is believed to have occurred,
solvent blanks must be run through the system until contamination is eliminated and then the affected
sample must be reanalyzed to verify results.
5. SAFETY AND WASTE MANAGEMENT
5.1 PCBs and pesticides should be treated as potential health hazards and exposure to these compounds should
be reduced to the lowest possible level. The laboratory is responsible for maintaining a current awareness
file of current regulations regarding the safe handling of chemicals used in this method. All personnel
handling serum will be encouraged to receive the hepatitis B vaccine. Lab coat, protective gloves, and
safety glasses are to be worn while working in the biocontainment and chemical hoods. Serum samples will
be handled in a biocontainment hood in accordance with NYSDOH Safety Manual (Number 11.2, issued
01/86, and revised 12/04).
5.2 Always follow guidelines listed in material safety data sheets (MSDS) for proper storage, handling, and
disposal of solvents, reagents, and standards. MSDSs are located within the laboratory in a labeled, yellow
binder.
5.3 Handle solvents in an area with adequate ventilation, such as a chemical fume hood. Flammable solvents
should be used away from sources of ignition.
5.4 Solvents are stored in yellow safety storage cabinets. No more than 2 bottles of solvent are allowed to
remain on benches overnight in each laboratory room.
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5.5 In case of chemical spills, evacuate the area. While wearing a chemical respirator, drop disposable pads
over the spill. Place pads inside a chemical fume hood until dry before disposal in the proper waste
container. The area should be cleaned according to the Material Safety Data Sheets (MSDS).
5.6
Laboratory trash is segregated from normal trash. All discarded and disposable glass and gloves are
placed in separate, plastic lined containers supplied by the Safety Office. When ready for collection, seal
the container and label with the designation “Sharps” along with your name, telephone number and room
number. The box is left outside the door for collection.
5.7
All disposable glassware being exposed to serum will be disposed of in aluminum stockpots that are
clearly marked “Biohazard.”
5.7.1 The room number, date and types of biological materials (if known), blood products and body fluids, and
others must be listed on the tag attached to each bucket.
5.7.2 Stockpots must not be filled more than three-quarters full. There should be a minimum of ½ inch of
water in the bottom.
5.7.3 The top of the autoclave bag must be folded inward and tape must be used to secure the lid.
5.7.4 Radioactive or chemical waste must not be placed in the stockpot.
5.8
Non-disposable glassware will be submerged in 10% Clorox overnight. Liquid solvent waste will be
disposed of in red organic-waste containers.
5.9
Dispose of solvent waste in appropriately marked containers. Solvents, except dichloromethane, are
disposed of in red 5-gallon waste containers. Yellow tags are filled out completely with no abbreviations
and tied to the container. Dichloromethane is collected separately in an appropriately labeled 4L glass
solvent bottle. The Safety Office is called to pick up the container.
6
POLLUTION PREVENTION
6.1 Solvent, chemical, reagent, and standard use are minimized whenever possible to reduce the amount of
hazardous waste generated.
6.2 All waste is disposed of in appropriately labeled containers for proper handling and future treatment (see
section 5.0).
7
COMPETENCY AND TRAINING ASSESSMENT
7.1 Any employee learning a new procedure will go through an initial training period. At the end of the training
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period they will be observed and evaluated on the tasks required of them. After being evaluated and proven
competent a technician will be observed and evaluated once a year performing the required tasks by either
the lab supervisor or another competent technician.
7.2 Methods for evaluating competency may include, but are not limited to:
7.2.1 Direct observation of routine test performance
7.2.2 Use of lab equipment, performance and documentation of instrument use and maintenance
7.2.3 Review of worksheets or QC records
7.2.4 Proficiency tests and internal blind specimens
7.2.5 Written quizzes
7.3 INITIAL TRAINING
7.3.1
New employees will be trained and evaluated in the Biological Organic Analytical Chemistry (BOAC)
lab by a previously trained and competent technician.
7.3.2
Training will consist of reading SOPs, direct observation, receipt of samples, preparation with blind
samples, analyzing the samples and preparing a report for data review with minimal supervision.
7.3.3
All work will be documented on BioOrganic Analytical Chemistry Laboratory Demonstration of
Capability Record (Appendix C). If certain criteria have not been met, the Lab Supervisor will meet
with the technician and discuss improvement goals. The new employee would go through the
evaluation process again. Once completing the training and showing competency the technician will be
allowed to work independently with clinical samples.
7.4 Competency: All employees will be assessed once a year for competency. A competent technician or lab
supervisor will observe and evaluate the technician and document these observations on the BioOrganic
Analytical Chemistry Laboratory Demonstration of Capability Record (Appendix C).
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7.5 Continuing Education : Also a minimum of twelve hours of continuing education must be provided to
laboratory staff and is documented. Acceptable forms of continuing education include in-service,
professional meetings or industry sponsored training/workshop programs.
7.6
The lab supervisor will keep the Demonstration of Capability Record, a copy of Proficiency Tests
that the technician has preformed within the year and Continuing Education records.
8
SAMPLE COLLECTION, PRESERVATION, AND HANDLING
8.1 Most persistent organic pollutants are extremely resistant to degradation in the environment and would not
be expected to degrade even if samples are not properly stored. However, a sample may be considered
unsuitable for analysis when there is evidence of damage to a sample container, when the sample volume is
incorrect, or when documentation accompanying a sample is incomplete. The client sending the sample
will then be notified that the sample was rejected. The laboratory will record the reason why the sample
was rejected.
8.2 Samples will be received and accessioned in Room D-634 of the Wadsworth Center. The sample
submission form should contain detailed information regarding collection of the sample and any relevant
observations made by the sample collector. If chain of custody forms are required, they should contain
abbreviated sample information and signatures describing the actual custody of the sample from time of
collection until receipt by the laboratory. Personnel using proper protective measures will open sample
containers only in the biocontainment hood. Samples not processed immediately will be stored in the
BOAC laboratory freezer located in the D-level alcove area.
8.2.1 Freezers are monitored 24/7 by in house technical personnel employed by Johnson Controls. Each
freezer is required to have a yellow “Equipment Information” card prominently displayed that contains
emergency contact information in the event of a malfunction. Johnson Controls also maintains spare
freezers that can be used to store specimens until the primary freezer is repaired or replaced.
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8.2.1.1 In the event of a freezer malfunction, Johnson Controls will immediately contact the appropriate
personnel and arrangements will be made to preserve sample specimens.
8.2.1.2 Document the malfunction on a Nonconformance / Correction Action Report (FORM QS – 11).
8.3 Below is an outline of the sample process.
8.3.1
Sample Collection
8.3.2
Sample Receipt/Tracking
8.3.3
Sample Storage
8.3.4
Sample Preparation
8.3.5
Sample Analysis
8.3.6
Sample Reporting
8.4 Human Serum Collection
8.4.1 The blood is collected in 5-mL or 7-mL red top vacutainers.
8.4.2 A successfully drawn tube should contain less than 1 cm of headspace.
8.4.3 Sample tubes should stay at room temperature at least 1 hour and then spin down the blood cells by
centrifugation at 1500 RPM, and take out the top layer, which is the serum.
8.4.4 Care must be taken to insure that samples are kept at refrigerator temperature during storage and
shipping. Samples should be kept cool during the whole shipping process. Also, special care should be
taken when packing samples to prevent breakage during the shipping process.
8.4.5 Once samples are received at the Wadsworth Center facility, they are logged in, chain of custody
documents are generated (if needed), and the samples are brought to the BioOrganic Analytical
Chemistry lab to be stored at –20 C until analysis.
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8.4.6 Criteria for unacceptable specimens would be: low volume samples; leaks; broken or cracked tube;
headspace requirements; failure to maintain proper temperature; suspected contamination. Reasons for
rejection should be recorded. Reports will be generated for all samples received, whether or not analysis
is performed.
8.5 Sample Receipt/Tracking Protocol
8.5.1
Each sample, even rejected samples, will be assigned a unique NYSDOH identifier at accession time.
This identifier will be recorded on the sample accession form and will be used to track the samples
from reception to analysis to final reporting. If the specimen has been assigned an identifier by the
collector, this identifier will be recorded on the sample accession form. Immediately notify the client
of any and all labeling discrepancies and document the nonconformance using a
Nonconformance/Corrective Action Report (FORM QS-11) according to SOP QS-11
‘Nonconformance / Corrective Action Procedure’.
9. EQUIPMENT AND SUPPLIES
9.1 SOLID PHASE EXTRACTION (SPE) APPARATUS
9.1.1
SPE extraction and SPE clean-up cartridges used for PCB extraction are described in the
Appendix A.
9.1.2
Extraction apparatus: Biotage RapidTrace Automated Workstation (Serial # RT0336N6984) or
equivalent. RapidTrace SPE Workstation software installed on a laptop computer with Windows
NT is used to control the Workstation.
9.1.3
Extract concentration system – Extracts are concentrated by blowdown with nitrogen using
Zymark Turbo Vap LV Concentration Workstation (Serial #TV0332N11869) or equivalent.
9.2 GC/MSD INSTRUMENTATION
9.2.1
Agilent Technologies Gas Chromatograph – Mass Selective Detector (GC/MSD)
9.2.1.1 Gas Chromatograph, model # 6890N (serial # US10633032) - Gas chromatograph
capable of reproducibly injecting down to 2 µL aliquots, and performing binary linear
gradients at a constant flow rate of helium at 1 mL/min (average velocity of 37 cm/min.).
The GC must be equipped for split/splitless injection, autosampler and be capable of
temperature programming from 35 to 350 oC.
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9.2.1.2 Injector, model # 7683B (serial # US63010507) or equivalent,
9.2.1.3 Autosampler, model # N10149 (serial # US62815638) or equivalent,
9.2.1.4 Mass Selective Detector (MSD), model # 5975B (serial # US62723834) or equivalent.
The mass spectrometer must be capable of electron ionization at a nominal electron
energy of 70 V and of scanning from 35 to 550 amu with a complete scan cycle of 1.5 sec
or less. The MSD must be equipped for selected ion monitoring (SIM) mode to acquire
the mass of the analytes, and the ion source is capable of operating up to 300 oC.
9.2.1.5 GC/MSD Data System, MSD ChemStation Software version D.03.06.611 (Agilent
Technologies).
9.2.1.6 Capillary column, J&W P/N 121-1232, DB-XLB 30m,x 0.18mm ID, 0.18um film or
equivalent.
9.3 REAGENT AND STANDARD PREPARATION EQUIPMENTS
9.3.1
Analytical balance – Ohaus, model # AP250D, serials # 1127211982, capable of weighing from
0.00001 to 10 g.
9.3.2
Sonicator – Branson, model 5510, serial # RNB020497109E.
9.3.3
Vortex – Scientific Industries Inc., model G-560, serial #2-280415.
9.3.4
Glassware – All glassware used in this study must be meticulously cleaned. Wash glassware with
detergent and tap water, rinse with tap water, followed by reagent water. Non-volumetric
glassware can be heated in an oven at 105 oC for 24 hr. Volumetric glassware should not be heated
above 120 oC.
9.3.5
Graduated cylinders – Kimble USA, various sizes.
9.3.6
Volumetric flasks – Kimble, USA, Class A, various sizes.
9.3.7
Micro syringes – Hamilton, various sizes.
9.3.8
Beakers - various sizes.
9.3.9
Erlenmeyer flasks - various sizes.
9.3.10
15 mL disposable culture tubes - Kimble Glass Inc., catalog # 73500 16100.
9.3.11
1.5 mL amber screw top autosampler vials - National Scientific Company, catalog # C4000- 1W.
9.3.12
Disposable Pasteur pipettes.
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10. REAGENTS AND STANDARDS
10.1 GASES, REAGENTS AND SOLVENTS - Reagent grade or better chemicals should be used. Unless
otherwise indicated, it is intended that all reagents shall conform to the specifications of the Committee on
Analytical Reagents of the American Chemical Society, where such specifications are available.
10.1.1
Nitrogen, Northeast Gas Technologies, Ltd. (grade 5.0) purity 99.999% or equivalent,
10.1.2
Helium, Northeast Gas Technologies, Ltd. (grade 5.0); purity 99.999% or equivalent,
10.1.3
Acetone, Mallinckrodt, purity 99.5 % or equivalent,
10.1.4
Dichloromethane (JT Baker), purity 99.9 % or equivalent,
10.1.5
Hexane (J.T. Baker), purity 99.5 % or equivalent,
10.1.6
Hydrochloric Acid (J.T. Baker), 36.5-38 % or equivalent, ASC reagent
10.1.7
Methanol (JT Baker), ultra high purity, HPLC grade or equivalent,
10.1.8
Reagent Water (Barnstead), HPLC grade or equivalent,
10.1.9
5% methanol/acidic H2O Solution - In a 1L Pyrex bottle, add 10 mL of MeOH, 190 mL reagent
water, and 200 mL of 0.1N HCl, and mix the solution using an ultrasonic bath for 5 minutes. The
pH of the solution should be in the range of 1-1.3
10.1.10 15% dichloromethane/hexane solution - In a 1 L Pyrex bottle add 60 mL of dichloromethane and
340 mL of hexane, and mix the solution by an ultrasonic bath for 5 minutes.
All solvents should be prechecked before using
10.2 STANDARD SOLUTIONS
10.2.1 Stock Standard Solution (SSS)
10.2.1.1 Isotopically labeled Internal Standards:Cambridge Isotope Laboratories, Inc.
13
C labeled
PCBs, 50 ug/ml in nonane,.IUPAC, 28, 52, ,118, ,153, 180, 194, 206, 209, 13C 4, 4’_DDE,
100 ug/ml in nonane
10.2.1.2 Analyte Standard Solution – The PCB congener analytical standard mixture (Con2) is
composed of 44 PCB congeners obtained from AccuStandard or Ultra Scientific. These
congeners are most often found in the highest concentrations in human serum specimen and
their concentrations reflect the extent of human exposure.
The concentration of each
component in stock standard solution is 100 ug/mL each. 4,4’-DDE, 4,4’-DDTand mirex
were obtained from Ultra Scientific, stock standard solution of each is 100ug/ml in methanol
(Table 1A).
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10.2.1.3 Calibration Check Standard (CCS) – The calibration check standard mixture (AccuStandard,
C-CCSREC-R, composed of 21 PCB congeners), was used to develop this method and are
included as an example. The concentration of each component in calibration check standard
solution is 100 ug/mL each (Table 1B).
10.2.2 Primary Dilution Standard (PDS)
10.2.2.1 IS primary dilution standard - The IS primary dilution standard is prepared by adding 0.5 mL
of each of the IS stock standard solution to a 10 mL volumetric flask partially filled with
acetone to make a final concentration at 5 µg/mL when filled to volume with acetone. The IS
primary dilution standard is stored in 1 mL amber ampoules, and has been shown to be
stable for 6 months when stored at 4 °C or less.
10.2.2.2 Analyte primary dilution standard solution
10.2.2.2.1 The analyte primary dilution standards contain all the target analytes of interest listing
in the Table 1. The primary dilution standard
Con2
is used to establish the
calibration curve.
10.2.2.2.2 The analyte primary dilution standard are prepared by adding 1 mL of each of the
analyte stock standard solution (SSS) to a 100 mL volumetric flask partially filled with
acetone to make a final concentration at 1 µg/mL when filled to the mark with acetone.
The analyte primary dilution standard is stored in 1mL amber ampoules, and has been
shown to be stable for 6 months when stored at 4 °C or less.
10.3 MSD TUNING SOLUTION
10.3.1
MSD tuning solution is used to tune and evaluate the mass spectrometer performance.
10.3.2
MSD tuning solution contains decafluorotriphenylphosphine (DFTPP) at the concentration of 5
ug/mL in dichloromethane (DCM).
10.3.3
MSD tuning solution is prepared by adding 1.25 mL of 100 ug/mL DFTPP stock solution (Ultra
Scientific, IST-341-1) to a 25 mL volumetric flask partially filled with dichloromethane to make a
final concentration at 5 ug/mL when filled to volume with dichloromethane.
10.3.4
The MSD tuning solution is stored in 1 mL ampoules, and has been shown to be stable for 6
months when stored at 4 °C or less.
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10.4 PERFORMANCE TEST SOLUTION (PTS)
10.4.1
The performance test solution (PTS) contains IUPAC 58 and IUPAC 186 at the concentration of
50 ng/mL in hexane.
10.4.2
The PTS solution is prepared by adding 0.1 mL of each of the stock standard solution of IUPAC
58 (Ultra Scientific, RPC-128S) and IUPAC 186 (Ultra Scientific, RPC-116S) at the concentration
of 100 ug/mL each to a 200 mL volumetric flask partially filled with hexane to make a final
concentration at 50 ng/mL when filled to volume with hexane.
10.4.3
The PTS is stored in 5 mL ampoules, and has been shown to be stable for 6 months when stored at
4 °C or less.
11. GC/MSD PERFORMANCE TEST
11.1 DFTPP TUNING
11.1.1
At the beginning of the batch that analytes are to be performed, the GC/MSD system must be
calibrated with the tuning solution and procedures prescribed by the manufacturer criteria.
11.1.2
DFTPP is the compound specified by the EPA to be used to verify that mass spectrometer voltages
are set correctly [1, 2]. DFTPP tuning allows to adjust mass spectrometer parameters to meet
relative abundance criteria defined by EPA methods 625 [3].
11.1.3
Analyze the tuning solution (Section10.3) containing 5 ug/mL DFTPP in DCM using GC/MSD
operating conditions shown in the Table 2A.
11.1.4
When the DFTPP is then injected the instrument tuning parameters are stored in DFTPP.U file.
11.1.5
When a target tune is finished, the evaluation of DFTPP tuning is performed from the View menu
in the Instrument Control, Manual Tune. The fragmentation pattern in the mass spectrometer
should pass the ion ratio criteria specified in the Table 2B.
11.1.6
If one of the listed criteria is not achieved, the analyst must retune the mass spectrometer and
repeat the test until all criteria are achieved. The DFTPP key masses and criteria must be achieved
before any samples, blanks or standards are analyzed.
11.1.7
Over time the ratios will start to fail. It is necessarily to retune the mass spectrometer.
11.2 PERFORMANCE TEST
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The performance test is used to test the performance of the GC/MSD after the DFTPP tuning
procedure and before each analyze batch.
11.2.2
The performance test is carried out by analyzing 5 replicates of the PTS samples using analysis
conditions described in Table 3 for PCB target analytes.
11.2.3
The evaluation of performance test is described in the section 14.1
12. CALIBRATION
12.1 INITIAL CALIBRATION
12.1.1
Selected ion monitoring (SIM) mode is used to acquire the masses of the target analytes and IS.
The specific ions characterized for each target analyte were selected to establish a SIM table for all
target analytes and IS. The GC/MSD operating conditions for the PCB analysis are summarized in
Table 3, and the SIM data of each target analyte constituted Con2 mixture, 4,4’-DDE, 4,4’-DDT
and mirex are listed in the Table 4.
12.1.2
Isotopically labeled Internal Standard: PCB 28, 52, 118, 153, 180, 194, 206, 209, 50ug/ml in
nonane and 13C-labeled 4,4’-DDE and 13C-labeled mixex 100ug/ml in nonane (Cambridge Isotope
Laboratories).
12.1.3
At least five calibration concentrations are required to prepare the initial calibration curve
spanning a 100-fold concentration range. Larger concentration ranges require more calibration
points. New born calf serum is spiked at 0.1;0.5;1.0;2.5;5.0; 10 ng/g of PCBs 4,4’-DDE, 4, 4’DDT and mirex. The IS mixture is added to the calibration standard at a concentration of 10
ng/mL. The lowest concentration on the curve must be at or below the MRL, which may depend
on system sensitivity.
12.2 CONTINUING CALIBRATION CHECK STANDARD (CCC)
12.2.1
The CCC standards are analyzed at the beginning and at the end of each analysis batch to verify
the initial calibration during analysis by injecting a mid-level calibration standard.
12.2.2
The CCC standards are analyzed under the same GC/MSD conditions used in the initial
calibration (Table 3).
12.2.3
The evaluation of CCC is described in the section 14.4 of the quality control.
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12.3 DATA CALCULATION
The linear calibration curve of each analyte is established by Agilent ChemStation software that is based
on integrated abundance (or peak area) - concentration data pair obtained from each of the calibration
points. The origin is not included in the curve. The following parameters were calculated:
12.3.1 The response factors (RFx) of each analyte was given by:
RFx
Ax C IS
AIS C x
[Equation 1]
where:
Ax = Integrated abundance (peak area) of the quantitation ions of the analyte,
AIS = Integrated abundance (peak area) of the IS quantitation ions,
Cx = concentration of analyte,
CIS = concentration of internal standard.
12.3.2 Linear Correlation Coefficient - R
The linear correlation coefficient between integrated abundance or peak area (A) and concentration
(C) of each analyte is calculated by the following mathematical form:
n CA C A
R
n
C C
2
2
n
A A
2
2
[Equation 2]
where n is the number of calibration points.
12.3.3 Coefficient of Determination - R2
The coefficient of determination (R2) of each analyte represents the percentage of the data being
the closest to the line of best fit. It is used to measure how well the regression line represents the
linear correlation data.
13. AUTOMATED SPE PROCEDURE
The PCB congeners and pesticides in the matrix spike sample are extracted using the Biotage
Trace SPE workstation. The sample is allowed to equilibrate to ambient temperature in a
biocontainment hood before being spiked with the IS. A specific worksheet (Appendix B) should
be filled in for each sample, and the collection tube is labeled with a unique sample identifier
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before extraction procedure.
13.1 SAMPLE EXTRACTION
13.1.1
A 1 g sample is dispensed into 15 mL disposable culture tube and spiked with 10 uL of a
100ng/mL IS spiking solution. The tube is capped and allowed to equilibrate for 1 hr. Add 1 g of
88% formic acid, vortex the sample for 10 sec. and add 1 mL methanol, vortex for 10 sec and
sonicate for 10 min at room temperature. After diluting with 1 mL of reagent water, the sample is
then vortexed again for 10 sec.
13.1.2
Purge the cannula tubes of the RapidTrace workstation using Tlpurge method (Table 5A).
13.1.3
Place C18 cartridge, sample tubes and collection tubes in their position in the RapidTrace
workstation. Run Tmethdhx method (Table 5B) for PCB extraction. Concentrate the extract in each
collection tube from 12 mL to about 1 mL using a Turbo Vap concentrator. The water bath
temperature is set at 40 oC.
13.2 SAMPLE CLEAN-UP
The co-extracted lipid in the extract above was removed using automated SPE system by the
following steps:
13.2.1
Purge the cannula tubes of the RapidTrace workstation using CLPUR704 method (Table 6A).
13.2.2
Place the extracted samples, collection tubes, and clean-up cartridges in their position in the
RapidTrace workstation. Run CLEANPCB method (Table 6B) to clean up the sample. The
extracts are collected in the culture tubes at the end of the clean up procedure.
13.2.3
Concentrate each extract from 12 mL to less than 1.0 mL using a Turbo Vap concentration
workstation. The water bath temperature is set at 40 oC. Transfer the extract from the collection
tube to 0.3 mL GC vial insert and reduce the extract volume with a gentle stream of nitrogen.
Rinse the collection tube twice with 0.5 mL of hexane, transfer to the insert and reduce volume to
0.1mL for GC/MSD analysis.
13.3 SAMPLE ANALYSIS
13.3.1
The specimen extracts are analyzed by GC/MSD in the same conditions as described previously
in initial calibration (section 12.1.1).
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The acceptance criteria for a qualitative identification for each analyte are described in the section
14.7.2
13.3.3
Calculation of analyte concentration - The concentration of each analyte (Cx) in extract is
calculated using the response factor (RFx) determined from Equation 1
Cx
(ng / g )
Ax mIS
AIS RFx m x
[Equation 3]
where:
Ax = Integrated abundance (peak area) of the quantitation ions of the analyte,
AIS = Integrated abundance (peak area) of the IS quantitation ions,
mIS = amount of IS added to each sample (ng),
mx = amount of sample (g).
14. QUALITY CONTROL
The quality control requirements of this method consist of an initial demonstration of capability, and ongoing
quality control requirements that must be met when preparing and analyzing the samples. The acceptance
criteria established for each of the quality control requirements in this section allow the analyst to determine if
the results of analyses meet the performance characteristics of the method.
14.1 PERFORMANCE TEST
14.1.1
The performance test allows checking the precision of the retention time (RT) and integrated
abundances (peak area counts) of IUPAC 58 and IUPAC 186, which are used in this study.
14.1.2
The retention times (RT) and integrated abundances of these compounds are determined from 5
replicate analyses.
14.1.3
The RSD of RT of these compounds should be less than 1%, and the RSD of integrated
abundances of these compounds should be less than 10%. The RSD of the integrated abundances
for each compound must be less than 10 % of its average value.
14.1.4
If any performance criterion is not met, the analyst should perform routine maintenance
procedures such as changing the autosampler syringe and/or injection port liner and seal. Cleaning
the mass spectrometer source can also result in higher abundances for performance test
compounds. A new performance test must be performed after any routine maintenance.
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14.2 INITIAL CALIBRATION
14.2.1
The RSD of all of the response factors should be less than 20%.
14.2.2
The RSD of the IS area counts must be less than 20% of the average value of the initial
calibration.
14.2.3
The correlation coefficient of the calibration curve for each analyte must be greater than 0.995
before any analysis of samples can begin.
14.2.4
If these criteria cannot be met a new calibration curve must be established.
14.3 SOLVENT BLANK (SB)
14.3.1
An aliquot of nanograde hexane analyzed prior to the batch analysis after analytical standards and
samples containing high levels of target analytes. The SB is used to demonstrate that the analytical
system is free of interferents before the analytical sequence is started and to verify that target
analytes were not carried over from an analytical standard to a sample,or from a sample containing
a high level of target analytes to a sample containing low levels of target analytes.
14.3.2
If any PCBs are found in the solvent blank at a level greater than twice the MDL, a new SB must
be analyzed. If any PCBs are still present in the SB, the analyst must check the inlet system
including sample vial, syringe, and injector for possible sources of contamination.
14.4 CONTINUING CALIBRATION CHECK (CCC)
14.4.1
A middle level calibration standard is analyzed at the beginning and end of a daily analysis batch
to verify the acceptability of an existing calibration.
14.4.2
Determine that the absolute area of the IS quantitation ion has not changed by more than ± 20%
from the average area measured during initial calibration.
14.3.3
Calculate the concentration of each analyte in the CCC. The calculated amount for each analyte
for medium level CCC must be within ±20% of the true value.
14.3.4
If these conditions are not achieved, then all data for the existing calibration must be considered
invalid, and remedial action should be taken, which may require recalibration.
14.5 CALIBRATION CHECK STANDARD (CCS)
14.5.1
A middle level CCS is analyzed after CCC to verify the acceptability of an existing calibration.
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Determine that the absolute area of the IS quantitation ion has not changed by more than ± 20%
from the average area measured during initial calibration.
14.5.3
Calculate the concentration of each analyte in the CCS. The calculated amount for each analyte for
medium level CCS must be within ±20% of the true value.
14.5.4
If these conditions are not achieved, then all data for the existing calibration must be considered
invalid and a second CCS must be analyzed. If the second calibration check does not meet the
acceptance criterion then routine maintenance such as changing injection port liner and seal, and
removal of 6 inches from the injection port end of the analytical column must be performed.
14.6 INITIAL DEMONSTRATION OF CAPABILITY
14.6.1
Initial Demonstration of Low System Background in Method Blank and Reagent Blank
14.6.1.1
Used to identify interference that may be present in the sample matrix. It is processed
through the entire procedure. Every analyst must complete an initial demonstration of
capability before the analysis of samples can begin.
14.6.1.2
Determine that the absolute area of the IS quantitation ion in MB sample has not
changed by more than ± 20% from the average area measured during initial calibration.
14.6.1.3
If any PCBs are found in the method blank (newborn calf serum) or reagent blank
(solvent) at greater than twice the MDL, the results obtained for that analyte will not be
reported.
14.6.2 Method Detection Limits (MDL)
14.6.2.1
An MDL must be determined before any specimen can be analyzed. An MDL study
must be done on an annual basis thereafter and when any major method modifications
are instituted.
14.6.2.2
Analyze 8-10 samples that have been spiked with CCS mixture at 0.1 ng/g.
14.6.2.3
Calculate the MDL using the following equation:
MDL = S t (n-1, 1-α = 0.99)
[Equation 4]
where:
S: standard deviation of replicate sample
t
(n-1, 1-α = 0.99):
Student’s t value for the 99% confidence level with n-1 degrees of
freedom
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n: number of replicates
14.6.2.4
The minimum reporting level (MRL) is then determined based on the results of the
MDL study. The MRL must be at least 3-5 times the MDL and equal to or greater than
the lowest calibration standard.
MRL = 3 MDL [Equation 5]
14.6.3 Initial demonstration of precision (IDP) and accuracy (IDA)
14.6.3.1
Analyze 8-10 samples that have been spiked with CCS at midrange concentration (1.0
ng/mL).
14.6.3.2
The RSD of mean recovery for IS must be less than 20%, and the RSD of the
individual recovery for each analyte must be less than 20% of initial value (spiking
level).
14.6.3.3
The entire procedure must be repeated if these criteria are not achieved.
14.7 MATRIX SPIKE (MS) / HUMAN SERUM SAMPLE
14.7.1
The matrix spike sample is used to evaluate the effect of the matrix on the recovery of the target
analytes. The reference matrix, newborn calf serum is spiked with the target PCBs at a final
concentration of 0.5, 1.0, 10ng/g. Recovery pertains to the extraction efficiency of an analytical
method within the limits of variability. Recovery to the analyte need not be 100%, but the extent
of recovery of an analyte and of the internal standard should be consistent, precise, and
reproducible.
14.7.2
Determine that the recovery of internal standards in MS sample must be within 70-130% of the
average value of the initial calibration, and the recovery of each target analyte must be within 70130% of the initial value (spiking level).
14.7.3
Qualitative identification: For both human serum and MS samples, the absolute RT of target
analyte must be within ± 0.05 min. of the RT for that analyte in the Initial Calibration, and the ion
ratios between target ion and qualified ion must be within ±20% of those established for target
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analyte in the SIM table (Table 4).
14.8 INSTRUMENT DETECTION LIMIT (IDL)
14.8.1
Prepare 7 to 10 replicates using standard calibration solutions at a concentration at 2-5 times the
noise level.
14.8.2
Analyze these samples under GC/MSD conditions and analyze with the same conditions used
during the initial calibration (Section 10.1).
14.8.3
Calculate the IDL using the following equation:
IDL = S t (n-1, 1-α = 0.99)
[Equation 6]
where:
- S: standard deviation of replicate sample
- t (n-1, 1-α = 0.99) : Student’s t value for the 99% confidence level with n-1 degrees of freedom
- n: number of replicates
14.8.4
The lower confidence limit (LCL) and upper confidence limit (UCL) are derived from IDL at
95% confidence level:
LCL = 0.64 IDL
[Equation 7]
UCL = 2.2 IDL
[Equation 8]
14.9 EXTERNAL PROFICIENCY TEST
The laboratory participates in the Arctic Monitoring and Assessment Program that tests proficiency
analyzing human serum for PCB congeners, pesticides and pesticide metabolites. The test is administered
quarterly by the Institut national de santé publique, Centre de toxicologie, 945, Ave Wolfe, Quebec
Canada, G1V 5B3.
15. DATA ASSESSMENT
15.1 All analytical batches must meet all quality control criteria described in section.
15.2 These quality control criteria must be documented and archived.
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15.3 If any analytical batch contains quality control data that fails any of the criterion, then the relevant data
should be reported as “suspect”.
16. CORRECTIVE ACTIONS AND CONTINGENCIES FOR OUT OF CONTROL DATA
Corrective actions are different for each quality control criterion. Section 14 (Quality Control) describes
all of the necessary quality control and procedures to follow if any data is deemed out of control.
17. REFERENCES
[1]
Eichelberger. J.W., Harris L.E., and Budde W.L. Reference Compound to Calibrate Ion Abundance
Measurements in Gas Chromatography-Mass Spectrometry Systems. Anal. Chem. 1975, 47 (7), 995.
[2]
EPA Method 625. Base/Neutrals and Acids. Genium Publishing Corporation 1996, Schenectady, NY
12304. July 1991.
[3]
Agilent Technologies Publication number 05171 (http://www.chem.agilent.com). Using Target Tune to
Tune for DFTPP.
[4]
Agilent Technologies Publication number 5989-5669EN (http://www.chem.agilent.com). Strategies for
Developing Optimal Synchronous SIM-Scan Acquisition Methods – AutoSIM/Scan Setup and Rapid
SIM.
[5]
Sjodin A., Jones R.S., Lapeza C.R., Focant, J-F., McGahee, E.E., Patterson, D.G. Semiautomated HighThroughput Extraction and Cleanup Method for the Measurement of Polybrominated Diphenyl Ethers,
Polybrominated Biphenyls, and Polychlorinated Biphenyls in Human Serum. Anal. Chem. 2004, 76,
1921-1927.
18. TABLES AND DEMONSTRATION DATA
TABLE 1A
POLYCHLORO BIPHENYLS IN CON2 MIXTURE, 4,4’-DDE, 4,4’-DDT AND 13C INTERNAL
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STANDARDS
Symbol
IUPAC 28
IUPAC 28*
IUPAC 44
IUPAC 52
IUPAC 52*
IUPAC 66
IUPAC 74
IUPAC 77
IUPAC 99
IUPAC 101
IUPAC 105
IUPAC 110
IUPAC 118
IUPAC 118*
IUPAC 126
IUPAC 128
IUPAC 138
IUPAC 146
IUPAC 153
IUPAC 153*
IUPAC 156
IUPAC 167
IUPAC 170
IUPAC 172
IUPAC 177
IUPAC 178
IUPAC 180
IUPAC 180*
IUPAC 183
IUPAC 187
IUPAC 194
IUPAC 194*
IUPAC 195
IUPAC 199
IUPAC 201
IUPAC 203
IUPAC 206
IUPAC 206*
IUPAC 209
IUPAC 209*
IUPAC 49
IUPAC 87
Compound Name
2,4,4'-Trichlorobiphenyl
2,4,4'-Trichlorobiphenyl
2,2',3,5'-Tetrachlorobiphenyl
2,2',5,5'-Tetrachlorobiphenyl
2,2',5,5'-Tetrachlorobiphenyl
2,3',4,4'-Tetrachlorobiphenyl
2,4,4',5-Tetrachlorobiphenyl
3,3',4,4'-Tetrachlorobiphenyl
2,2,4,4',5-Pentachlorobiphenyl
2,2',4,5,5'-Pentachlorobiphenyl
2,3,3',4,4'-Pentachlorobiphenyl
2,3,3',4',6-Pentachlorobiphenyl
2,3’,4,4',5-Pentachlorobiphenyl
2,3’,4,4',5-Pentachlorobiphenyl
3,3’,4,4',5-Pentachlorobiphenyl
2,2',3,3',4,4'-Hexachlorobiphenyl
2,2',3,4,4',5'-Hexachlorobiphenyl
2,2',3,4',5,5'-Hexachlorobiphenyl
2,2',4,4',5,5'-Hexachlorobiphenyl
2,2',4,4',5,5'-Hexachlorobiphenyl
2,3,3',4,4',5-Hexachlorobiphenyl
2,3',4,4',5,5'-Hexachlorobiphenyl
2,2',3,3',4,4',5-Heptachlorobiphenyl
2,2',3,3',4,5,5'-Heptachlorobiphenyl
2,2',3,3',4',5,6-Heptachlorobiphenyl
2,2',3,3',5,5',6-Heptachlorobiphenyl
2,2',3,4,4',5,5'-Heptachlorobiphenyl
2,2',3,4,4',5,5'-Heptachlorobiphenyl
2,2',3,4,4',5',6-Heptachlorobiphenyl
2,2',3,4',5,5',6-Heptachlorobiphenyl
2,2',3,3',4,4',5,5’-Octachlorobiphenyl
2,2',3,3',4,4',5,5’-Octachlorobiphenyl
2,2',3,3',4,4',5,6-Octachlorobiphenyl
2,2',3,3',4,5,5’,6'-Octachlorobiphenyl
2,2',3,3',4,5',6,6'-Octachlorobiphenyl
2,2',3,4,4',5,5',6-Octachlorobiphenyl
2,2',3,3',4,4',5,5',6-Nonachlorobiphenyl
2,2',3,3',4,4',5,5',6-Nonachlorobiphenyl
2,2',3,3',4,4',5,5',6,6'-Decachlorbiphenyl
2,2',3,3',4,4',5,5',6,6'-Decachlorbiphenyl
2,2',4,5'-Tetrachlorobiphenyl
2,2',3,4,5'-Pentachlorobiphenyl
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CAS number
7012-37-5
7012-37-5
41464-39-5
35693-99-3
35693-99-3
32598-10-0
32690-93-0
32598-13-3
38380-01-7
37680-73-2
32598-14-4
38380-03-9
31508-00-6
31508-00-6
57465-28-8
38380-07-3
35065-28-2
51908-16-8
35065-27-1
35065-27-1
38380-08-4
52663-72-6
35065-30-6
52663-74-8
52663-70-4
52663-67-9
35065-29-3
35065-29-3
52663-69-1
52663-68-0
35694-08-7
35694-08-7
52663-78-2
52663-75-9
40186-71-8
52663-76-0
40186-72-9
40186-72-9
2051-24-3
2051-24-3
41464-40-8
38380-02-8
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3,4,4’,5-Tetrachlorobiphenyl
2,2’,3,5,5',6-Hexachlorobiphenyl
2,2’,3,4’,5',6-Hexachlorobiphenyl
2’,3,4,4’,5-Pentachlorobiphenyl
2,3,4,4’,5-Pentachlorobiphenyl
2,3,3',4,4',6-Hexachlorobiphenyl
2,3,3',4,4',5’-Hexachlorobiphenyl
3,3’,4,4’,5,5’-Hexachlorobiphenyl
2,2',3,3',4,4',5,6’-Octachlorobiphenyl
2,3,3’,4,4’,5,5’-Heptachlorobiphenyl
1,1'-(Dichloroethenylidene)bis(4-chlorobenzene)
1,1'-(Dichloroethenylidene)bis(4-chlorobenzene)
1,1a,2,2,3,3a,4,5,5,5a,5b,6-dodecachloroMirex
octohydro-1,3,4-metheno-1H-cyclobuta[cd}
pentalene
1,1a,2,2,3,3a,4,5,5,5a,5b,6-dodecachloroMirex*
octohydro-1,3,4-metheno-1H-cyclobuta[cd}
pentalene
1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene
4,4’-DDT
dichlordiphenyldichlorethylene
* 13C12 Internal Standard
IUPAC 81
IUPAC 151
IUPAC 149
IUPAC 123
IUPAC 114
IUPAC 158
IUPAC 157
IUPAC 169
IUPAC 196
IUPAC 189
4,4’-DDE
4,4’-DDE*
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Rev. No. 1
Page 31 of 49
70362-50-4
52663-63-5
38380-04-0
65510-44-3
74472-37-0
74472-42-7
69782-90-7
32774-16-6
42740-50-1
39635-31-9
72-55-9
72-55-9
2385-85-5
2385-85-5
50-29-3
TABLE 1B
POLYCHLORO BIPHENYLS IN CCS MIXTURE
Symbol
Compound Name
CAS number
IUPAC 8
2,4'-Dichlorobiphenyl
34883-43-7
IUPAC 18
2,2',5-Trichlorobiphenyl
37680-65-2
IUPAC 28
2,4,4'-Trichlorobiphenyl
7012-37-5
IUPAC 44
2,2',3,5'-Tetrachlorobiphenyl
41464-39-5
IUPAC 52
2,2',5,5'-Tetrachlorobiphenyl
35693-99-3
IUPAC 66
2,3',4,4'-Tetrachlorobiphenyl
32598-10-0
IUPAC 77
3,3',4,4'-Tetrachlorobiphenyl
32598-13-3
IUPAC 101
2,2',4,5,5'-Pentachlorobiphenyl
37680-73-2
IUPAC 105
2,3,3',4,4'-Pentachlorobiphenyl
32598-14-4
IUPAC 118
2,3’,4,4',5-Pentachlorobiphenyl
31508-00-6
IUPAC 126
3,3’,4,4',5-Pentachlorobiphenyl
57465-28-8
IUPAC 128
2,2',3,3',4,4'-Hexachlorobiphenyl
38380-07-3
IUPAC 138
2,2',3,4,4',5'-Hexachlorobiphenyl
35065-28-2
IUPAC 153
2,2',4,4',5,5'-Hexachlorobiphenyl
35065-27-1
IUPAC 170
2,2',3,3',4,4',5-Heptachlorobiphenyl
35065-30-6
IUPAC 180
2,2',3,4,4',5,5'-Heptachlorobiphenyl
35065-29-3
IUPAC 187
2,2',3,4',5,5',6-Heptachlorobiphenyl
52663-68-0
IUPAC 195
2,2',3,3',4,4',5,6-Octachlorobiphenyl
52663-78-2
IUPAC 201
2,2',3,3',4,56,6'-Octachlorobiphenyl
40186-71-8
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IUPAC 206
IUPAC 209
Rev. No. 1
2,2',3,3',4,4',5,5',6-nonachlorobiphenyl
2,2',3,3',4,4',5,5',6,6'-Decachlorbiphenyl
Page 32 of 49
40186-72-9
2051-24-3
TABLE 2A
GC/MSD OPERATING CONDITIONS FOR DFTPP TUNING
1. GC OPERATING CONDITIONS
- Injection volume:
1 uL
- Inlet temperature:
250 oC
- Temperature program:
Oven Ramp
Rate
C/min.
0
20
o
Initial
Ramp 1
Final Temperature
o
C
90
300
Holding Time
minutes
2
2
Run time
minutes
2
14.5
2. MSD OEPRATING CONDITIONS
- Acquisition Mode:
SCAN
- Electron energy:
70 V (nominal)
- Mass spectrometer source temperature:
300 oC
- Mass spectrometer quadruple temperature:
150 oC
- Mass Range:
35-550 amu
- Scan time:
At least 5 scans/peak but not exceed 7 seconds/scan.
TABLE 2B
Target Mass
51
68
69
70
127
197
198
199
275
365
441
DFTPP KEY MASSES AND ABUNDANCE CRITERIA
Relative to Mass
Lower Limit %
198
20
69
0
198
0
69
0
198
40
198
0
442
40
198
5
198
10
198
1
443
1
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Upper Limit %
60
3
100
2
60
1
100
9
40
100
100
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443
442
Rev. No. 1
Page 33 of 49
17
23
TABLE 3
GC/MSD OPERATING CONDITIONS FOR PCB/PESTICIDE ANALYSIS
1. GC OPERATING CONDITIONS
- Injection volume:
2 uL
- Inlet temperature:
250 oC
- Temperature program:
Oven Ramp
Rate
C/min.
0
30
2
5
50
o
Initial
Ramp 1
Ramp 2
Ramp 3
Ramp 4
Final Temperature
o
C
90
150
244
280
300
Holding Time
minutes
1
0
0
0
2
Run time
minutes
1
3
50
57.2
59.6
2. MSD CONDITIONS
- Acquisition Mode:
SIM
- Electron energy:
70 V (nominal)
- Mass spectrometer source temperature:
300 oC
- Mass spectrometer Quadruple temperature:
150 oC
- Mass Range:
35-550 amu
- Scan time:
Select a dwell time that yields 15-20 cycles across a
peak for each analyte [4].
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Page 34 of 49
TABLE 4
TARGET ION AND QUALIFICATION ION MASSES
Analyte
Target ion
Qual ion
Qual ion
Target ion
(relative ratio) (relative ratio)
IUPAC 28*
257.9
255.9 (94.5)
268.1
IUPAC 52*
291.9
289.9 (79.7)
303.9
IUPAC 49
219.9
222 (71.5)
291.9 (115.4)
IUPAC 44
291.9
289.9 (75.5) 219.8 (96.0)
IUPAC 74
291.8
289.9 (47.8) 293.9 (78.7)
IUPAC 66
291.8
289.9 (77.5) 293.8 (48.9)
IUPAC 101
325.8
327.8 (64.9) 323.9 (59.1)
IUPAC 99
325.8
323.8 (62.4) 327.8 (59.0)
IUPAC 87
325.9
327.9 (66.7) 255.9 (68.0)
4,4'-DDE*
246
248 (63.0)
258
IUPAC 110
325.8
323.8 (62.8
327.8 (63.4)
IUPAC 81
291.9
289.9 (80.8) 293.9 (51.2)
IUPAC 151
359.9
289.9 (73.6) 361.9 (80.1)
IUPAC 77
291.8
289.9 (71.0) 293.9 (47.1)
IUPAC 149
359.9
361.9 (82.0) 289.9 (68.0)
IUPAC 123
325.9
327.9 (65.2) 323.9 (64.2)
IUPAC 118*
325.9
327.9 (65.2)
337.9
IUPAC 146+114**
359.8
361.8 (82.0)
325.9
IUPAC 153*
359.8
361.8 (82.5)
371.9
IUPAC 105
325.8
323.8 (63.8) 327.8 (62.0)
IUPAC 138+4,4’-DDT**
359.8
361.8 (77.4)
235
IUPAC 178+158**
393.8
395.8 (96.1)
359.9
IUPAC 187
393.8
395.8 (96.4) 397.8 (52.0)
IUPAC 183
393.7
395.7 (96.3) 397.8 (51.6)
IUPAC 126
325.8
323.8 (58.2) 327.8 (56.4)
IUPAC 128
359.8
289.8 (79.2) 361.8 (83.9)
IUPAC 167
359.8
357.8 (50.8) 361.8 (78.1)
IUPAC 177
393.7
395.7 (96.9) 397.7 (52.6)
IUPAC 201
429.7
427.7 (89.7) 431.6 (63.3)
IUPAC 156
359.8
357.8 (45.1) 361.8 (75.5)
IUPAC 172+157**
393.8
395.8 (95.3)
359.9
IUPAC 180*
393.7
395.8 996.0)
405.9
IUPAC 170
393.8
395.7 (98.7) 397.7 (58.5)
IUPAC 199
429.7
427.7 (87.8) 431.7 (67.4)
IUPAC 169+(196&203)**
362
363.9 (56.4
429.9
IUPAC 189+195**
393.9
395.9 (99.0)
429.8
IUPAC 194*
429.7
427.6 (71.9)
439.9
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Qual ion
(relative ratio)
269.9 (91.0)
301.9 (78.3)
260 (65.0)
339.9 (62.8)
323.9 (64.2)
373.9 (79.9)
237 (64.3)
361.9 (80.1)
361.9 (77.1)
407.9 (92.1)
427.8 (95.5)
427.8 (87.5)
441.9 (112)
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Mirex
271.70
273.90(94.60) 269.80(57.1)
IUPAC 206*
463.8
461.7 (72.9)
IUPAC 209*
497.7
499.7 (82.9)
The analytes are classified following the order of the retention time
Relative ion ratio between qualified ion and target ion
* Native and IS coelute, choose two ions for each
** Coelute, choose two ions for each analyte
Page 35 of 49
475.9
509.8
473.9 (85.1)
511.8 (80.8)
TABLE 5A
Step
1
2
3
4
5
6
7
8
9
10
11
AUTOMATED EXTRACTION PROCEDURE –
TLPURGE PURGE METHOD
Description
Flow Rate, ml/min
Purge Cannula with 4 mL of Dichloromethane
30
Purge Cannula with 4 mL of Dichloromethane
30
Purge Cannula with 4 mL of Methanol
30
Purge Cannula with 4 mL of Methanol
30
Purge Cannula with 4 mL of 5% MeOH/Acidic H2O
30
Purge Cannula with 4 mL of 5% MeOH/Acidic H2O
30
Purge Cannula with 4 mL of H2O
30
Purge Cannula with 4 mL of H2O
30
Purge Cannula with 4 mL of 15% Dichloromethane /Hexane
30
Purge Cannula with 4 mL of 15% Dichloromethane /Hexane
30
Purge Cannula with 4 mL of H2O
30
Output
Canula
Canula
Canula
Canula
Canula
Canula
Canula
Canula
Canula
Canula
Canula
TABLE 5B
AUTOMATED EXTRACTION PROCEDURE –
TMETHDHX PCBS EXTRACTION METHOD
Step
1
2
3
4
5
6
7
8
9
10
11
12
Description
Condition Cartridge with 3 mL of Methanol
Condition Cartridge with 3 mL of 5% MeOH/Acidic H2O
Condition Cartridge with 3 mL of Dichloromethane
Condition Cartridge with 3 mL of Methanol
Condition Cartridge with 3 mL of 5% MeOH/Acidic H2O
Load 5mL of Sample onto Cartridge (*)
Rinse Cartridge with 1.5 mL of 5% MeOH/Acidic H2O
Dry Cartridge with Nitrogen gas for 40 minutes
Purge Cannula with 3 mL of 15% Dichloromethane /Hexane
Collect Fraction 1 with 12mL of 15% Dichloromethane /Hexane
Purge Cannula with 3 mL of 15% Dichloromethane /Hexane
Purge Cannula with 3 mL of water
Flow Rate, ml/min
5
5
5
5
5
0.38
5
Output
Organic solvents waste
Organic solvents waste
Organic solvents waste
Organic solvents waste
Organic solvents waste
Organic solvents waste
Organic solvents waste
5
0.38
5
5
Cannula
Fract 1
Cannula
Cannula
(*) A sample volume larger than the actual sample size is entered to ensure that whole sample is used.
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TABLE 6A
AUTOMATED CLEAN-UP PROCEDURE –
CLPUR704 PURGE METHOD
Step
1
2
3
4
5
6
Description
Purge Cannula with 6 mL of Hexane
Purge Cannula with 6 mL of Hexane
Purge Cannula with 6 mL of Methanol
Purge Cannula with 6 mL of Methanol
Purge Cannula with 6 mL of Reagent water
Purge Cannula with 6 mL of Reagent water
Flow Rate, ml/min
30
30
30
30
30
30
Output
Cannula
Cannula
Cannula
Cannula
Cannula
Cannula
TABLE 6B
AUTOMATED CLEAN-UP PROCEDURE CLEANPCB CLEAN-UP METHOD
Step
1
2
3
4
5
6
7
Description
Condition Cartridge with 5 mL of Hexane
Condition Cartridge with 5 mL of Hexane
Load 2 mL of Sample onto Cartridge (*)
Purge Cannula with 3 mL of Hexane
Collect Fraction 1 with 12 mL of Hexane
Purge Cannula with 3 mL of Methanol
Purge Cannula with 3 mL of Reagent Water
Flow Rate, ml/min
5
5
0.38
5
0.38
5
5
Output
Organic solvents waste
Organic solvents waste
Organic solvents waste
Cannula
Fract 1
Cannula
Cannula
(*) A sample volume larger than the actual sample size is entered to ensure that whole sample is used.
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TABLE 7
INSTRUMENT DETECTION LIMIT (IDL) (*)
Analyte
Mean
SD
RSD%
IDL
LCL
IUPAC 8
0.48
0.025
5
0.07
0.05
IUPAC 18
0.51
0.033
6
0.10
0.06
IUPAC 28
0.45
0.027
6
0.08
0.05
IUPAC 52
0.53
0.018
3
0.05
0.03
IUPAC 44
0.47
0.031
7
0.09
0.06
IUPAC 66
0.42
0.030
7
0.09
0.06
IUPAC 101
0.56
0.029
5
0.09
0.06
IUPAC 77
0.36
0.023
6
0.07
0.04
IUPAC 118
0.50
0.041
8
0.12
0.08
IUPAC 153
0.55
0.037
7
0.11
0.07
IUPAC 105
0.45
0.037
8
0.11
0.07
IUPAC 138
0.57
0.043
8
0.13
0.08
IUPAC 187
0.61
0.035
6
0.11
0.07
IUPAC 126
0.38
0.039
10
0.12
0.08
IUPAC 128
0.55
0.048
9
0.14
0.09
IUPAC 201
0.56
0.047
8
0.14
0.09
IUPAC 180
0.56
0.051
9
0.15
0.10
IUPAC 170
0.53
0.049
9
0.15
0.09
IUPAC 195
0.58
0.034
6
0.10
0.06
IUPAC 206
0.58
0.047
8
0.14
0.09
IUPAC 209
0.58
0.036
6
0.11
0.07
(*) determined from GC/MSD analysis of 0.5 ng/mL of CCS mixture in hexane (n=8)
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UCL
0.16
0.21
0.18
0.12
0.20
0.20
0.19
0.15
0.27
0.24
0.24
0.29
0.23
0.26
0.32
0.31
0.33
0.32
0.22
0.31
0.24
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TABLE 8
CALIBRATION CURVE FOR PCBs/ PESTICIDES IN NEW BORN CAIF SERUM*
ng/g
Analyte
IUPAC 28
IUPAC 52
IUPAC 49
IUPAC 44
IUPAC 74
IUPAC 66
IUPAC 101
IUPAC 99
IUPAC 87
4,4'-DDE
IUPAC 110
IUPAC 81
IUPAC 151
IUPAC 77
IUPAC 149
IUPAC 123
IUPAC 118
IUPAC 146
IUPAC 114
IUPAC 153
IUPAC 105
IUPAC 138
4,4'-DDT
IUPAC 178
IUPAC 158
IUPAC 187
IUPAC 183
IUPAC 126
IUPAC 128
IUPAC 167
IUPAC 177
IUPAC 201
IUPAC 156
IUPAC 172
IUPAC 157
IUPAC 180
0.1
0.13
0.12
0.09
0.10
0.14
0.13
0.12
0.10
0.08
0.28
0.13
0.11
0.09
0.10
0.12
0.09
0.11
0.09
0.09
0.11
0.09
0.09
0.06
0.11
0.10
0.13
0.14
0.07
0.06
0.07
0.10
0.36
0.06
0.04
0.17
0.11
0.5
0.56
0.54
0.41
0.48
0.62
0.64
0.49
0.45
0.38
0.72
0.54
0.44
0.43
0.47
0.53
0.41
0.48
0.41
0.43
0.49
0.43
0.41
0.24
0.58
0.50
0.63
0.62
0.29
0.33
0.40
0.47
1.73
0.33
0.21
0.75
0.48
1
0.82
0.93
0.85
0.79
1.18
1.18
0.84
0.82
0.66
1.21
0.96
0.98
0.81
1.00
0.98
0.81
0.89
0.82
0.84
0.93
0.82
0.78
0.51
1.05
0.98
1.17
1.13
0.60
0.62
0.78
0.88
3.09
0.66
0.40
1.28
0.89
2.5
2.02
2.33
1.87
1.99
2.89
3.01
2.06
1.97
1.61
2.66
2.40
2.48
2.02
2.56
2.51
1.95
2.19
1.98
1.97
2.27
2.05
1.93
1.35
2.55
2.45
2.87
2.79
1.46
1.57
1.96
2.28
8.15
1.72
0.99
3.28
2.18
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5
3.67
4.34
3.58
3.74
5.67
5.81
3.97
4.03
3.21
5.05
4.83
4.81
3.99
4.92
4.89
4.00
4.38
4.02
4.17
4.48
4.13
3.77
2.54
5.23
4.93
5.87
5.77
2.94
3.08
3.88
4.53
15.65
3.42
1.94
6.26
4.43
10
7.71
9.21
7.79
7.83
12.37
12.50
8.21
8.33
6.54
10.09
9.52
10.53
8.01
10.50
9.77
8.25
9.16
8.18
8.68
9.13
8.52
7.67
5.22
10.47
9.99
11.85
11.52
6.17
6.15
8.01
9.08
32.14
6.89
4.03
12.19
9.06
R
0.9993
0.9994
0.9989
0.9996
0.9990
0.9993
0.9998
0.9998
0.9999
0.9999
1.0000
0.9991
1.0000
0.9995
1.0000
0.9998
0.9997
0.9999
0.9996
0.9999
0.9999
0.9999
0.9998
1.0000
1.0000
0.9999
0.9999
0.9997
1.0000
0.9999
1.0000
0.9999
1.0000
0.9998
0.9998
0.9999
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IUPAC 170
0.08
0.37
0.70
1.75
3.57
7.16
0.9996
IUPAC 199
0.20
0.94
1.67
4.34
8.08
16.96
0.9996
IUPAC 169
0.05
0.25
0.48
1.12
2.30
4.69
0.9999
IUPAC 196 and 203
0.22
0.85
1.71
4.26
8.75
17.70
0.9999
IUPAC 189
0.08
0.34
0.64
1.60
3.24
6.65
0.9999
IUPAC 195
0.15
0.74
1.28
3.20
6.11
12.85
0.9996
IUPAC 194
0.12
0.56
0.99
2.47
4.65
9.68
0.9997
IUPAC 206
0.09
0.41
0.86
1.99
3.87
7.96
0.9998
IUPAC 209
0.09
0.44
0.90
2.18
4.35
8.98
0.9999
(*)
New born calf serum spiked at 0.1, 0.5, 1.0,2.5,5.0,10 ng/g Con2 mixture and 4,4’-DDE and 4,4’-DDT
Used the analyte concentration and the ratio of the analyte response to IS response to calculate the coefficient
TABLE 9
METHOD DETECTION LIMIT (MDL) (*) AND
MINIMUM REPORTING LEVEL (MRL)**
Analyte
Mean
SD
RSD%
MDL nn/g MRL ng/g
IUPAC 18
0.11
0.014
13
0.04
0.2
IUPAC 28
0.11
0.008
7
0.02
0.1
IUPAC 52
0.12
0.009
8
0.03
0.15
IUPAC 44
0.11
0.011
11
0.03
0.15
IUPAC 66
0.14
0.006
4
0.02
0.10
IUPAC 101
0.10
0.006
6
0.02
0.10
IUPAC 77
0.14
0.003
2
0.01
0.05
IUPAC 118
0.12
0.006
5
0.02
0.10
IUPAC 153
0.11
0.009
9
0.03
0.15
IUPAC 105
0.11
0.007
6
0.02
0.10
IUPAC 138
0.11
0.004
4
0.01
0.05
IUPAC 187
0.10
0.007
7
0.02
0.10
IUPAC 126
0.12
0.005
4
0.01
0.05
IUPAC 128
0.11
0.008
7
0.02
0.10
IUPAC 201
0.10
0.006
6
0.02
0.10
IUPAC 180
0.11
0.005
5
0.02
0.10
IUPAC 170
0.13
0.003
3
0.01
0.05
IUPAC 195
0.11
0.004
3
0.01
0.05
IUPAC 206
0.11
0.006
5
0.02
0.10
IUPAC 209
0.11
0.007
6
0.02
0.10
4,4'-DDE
0.17
0.009
5
0.02
0.10
Mirex
0.02
0.10
(*)
determined from matrix spike at 0.1 ng/g CCS mixture and 4,4’-DDE (n=9), Mirex=3,
based on GC/ECD analyses.
(**)
Calculated from Equation 5
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TABLE 10
METHOD VALIDATION – PRECISION AND ACCURACY
OF MATRIX SPIKE (*)
Analyte
IUPAC 28
IUPAC 52
IUPAC 49
IUPAC 44
IUPAC 74
IUPAC 66
IUPAC 101
IUPAC 99
IUPAC 87
4,4'-DDE
IUPAC 110
IUPAC 81
IUPAC 151
IUPAC 77
IUPAC 149
IUPAC 123
IUPAC 118
IUPAC 146
IUPAC 114
IUPAC 153
IUPAC 105
IUPAC 138
4,4'-DDT
Mirex
IUPAC 178
IUPAC 158
IUPAC 187
IUPAC 183
IUPAC 126
IUPAC 128
IUPAC 167
IUPAC 177
Max
Min
Recovery% Recovery%
120
94
115
86
104
93
109
88
98
82
98
84
117
86
97
85
101
86
113
93
107
90
99
80
97
85
102
80
100
87
98
83
103
86
97
84
96
80
103
84
95
83
100
86
107
76
79
71
97
84
97
83
99
83
97
83
96
80
101
82
98
80
98
85
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Mean
Recovery%
107
101
99
101
91
93
101
92
95
105
100
89
92
90
94
90
95
91
89
95
91
94
94
75
90
89
91
91
88
92
90
91
SD
RSD%
9.72
10.65
4.76
9.14
6.45
6.63
10.82
4.88
5.91
7.85
7.59
6.77
4.72
8.78
5.16
6.11
6.71
5.32
5.64
6.97
5.71
6.12
13.07
0.39
5.38
5.57
6.59
5.58
6.01
7.17
7.13
5.57
9
11
5
9
7
7
11
5
6
7
8
8
5
10
5
7
7
6
6
7
6
7
14
5
6
6
7
6
7
8
8
6
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IUPAC 201
95
85
89
3.66
4
IUPAC 156
98
83
89
6.28
7
IUPAC 172
99
83
93
6.84
7
IUPAC 157
95
81
87
5.54
6
IUPAC 180
100
82
92
6.94
8
IUPAC 170
100
82
93
7.87
8
IUPAC 199
95
86
90
3.87
4
IUPAC 169
105
88
94
5.71
6
IUPAC 196 and 203
94
80
88
5.75
7
IUPAC 189
101
81
91
7.33
8
IUPAC 195
108
84
95
8.85
9
IUPAC 194
105
86
97
6.68
7
IUPAC 206
103
86
95
6.74
7
IUPAC 209
99
81
92
7.71
8
(*)
New born calf serum spiked at 1 ng/g Con2 mixture and 4,4’-DDE and 4,4’-DDT (n=6),
Mirex , spiked at 10ng/mL n=3
FIGURE 1
TOTAL ION CHROMATOGRAM IN SIM MODE OF CON2 AND PESTICIDES AND IS EXTRACTED
FROM NEW BORN CALF SERUM
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FIGURE 2
TIC IN SIM MODE OF EXTRACTED HUMAN SERUM SPECIMEN
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APPENDIX A
SPE CARTRIDGE PREPARATION
Two types of SPE cartridges have been used for SPE extraction and clean-up PCB [5].
Reagent and Supplies
- SPE polypropylene cartridge (Phenomenex), catalog #AHO-7001
- Polyethylene frits (Phenomenex), catalog #AHO-7007
- C18 sorbent (Phenomenex; 50 um; 65 Å), catalog #04G-4348
- Silica gel (Mallinckrod; 100-200 Mesh; 60 Å), catalog #6447
- Sulfuric acid (Mallinckrodt), catalog #2468
Procedure
A. C18 cartridge (Extraction column)
Pack 1.3 g of C18 sorbent in a polypropylene tube. Two polyethylene frits were placed at the bottom and at the top
of the cartridge (Figure A.1).
B. Silica gel cartridge (Clean-up column)
1. Washing of the silica gel
a)
Weigh 200g of silica gel into a Pyrex heavy wall filter flask,
b) Add dichloromethane to cover the silica gel powder and mix well to form a slurry,
c)
Place the flask into a vacuum oven maintained at room temperature to evaporate the DCM over the weekend.
2. Sulfuric acid treatment silica gel
a)
Weigh 100 g of washed silica gel out into an amber round bottom bottle
b) Add 50 g or 28 ml of sulfuric acid (51% concentration) to the bottle,
c)
Cap the bottle with a Teflon cap and place it on the roller overnight to form a homogenous mixture.
3. Silica Gel Cartridge Preparation
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A clean up cartridge is made by packing two layer sorbents in a polypropylene tube. The first layer (lower
layer) contains 1 g of sulfuric acid treatment silica gel obtained from the section 2 above, and the second one
(upper layer) contains 0.1 g of silica gel. A polyethylene frit is placed between layers (Figure A.2).
Figure A.1: Extraction column
Figure A.2: Clean up column
Polyethylene Frit
Silica gel
Polyethylene Frit
Silica gel/Sulfuric acid
Polyethylene Frit
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Appendix B
BioOrganic Analytical Chemistry
Solid Phase Extraction
Batch Number: ___________
Sample ID# _________________
Sample Type: Serum sample
Project:_____________________
Initials
Date
A. Sample Preparation and Extraction
1. Sample weight__________g.
Balance:___AND EK-200G____
Witness
2. Spike:________uL
13
C12 PCB/DDE Mix @100ng/mL (9-16-10)
______ ______
Let sit for 1 hour
3. Sample Preparation
a) Add 1g 88% Formic Acid,
b) Add 1ml Methanol, vortex 10 seconds
c) Sonicate for 10 min. @ room temp
d) Add 1g DI water, vortex 10 seconds
______
______
______
______
4. Solid Phase Extraction using the Rapid Trace Automated System:
a)
b)
c)
d)
Column: Strata C18
Purge lines method: tlpurge.spe
Extraction method: tmethdhx.spe
Concentrate to 1 mL
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B. Sample Cleanup
a) Column: Silicagel:Sulfuric Acid/silicagel
b.) Purge lines method: clpur704.spe
b) Cleanup method:
cleanpcb.spe
______
C. Concentrate to ___________
______
______
______
D. Final Concentration
PCB and Pesticides
Conc. to __________uL
Date______________
Initials_____________
D. Notes:
Hexane______________
DCM _____________
Methanol___________
Water______________
Formic Acid ________
Sepra C-18 ______________
Silica Gel ______________
Acidified Silica Gel
______________
PCB Analyzed by___________________
Date____________
Reviewed By_______________________
Date____________
APPENDIX C – BioOrganic Analytical Chemistry Laboratory Demonstration of Capability Record
Employee:_____________________________
( __ ) Initial Training Assessment
Sample Receiving and Accessioning
Task
Evaluated by:_____________________________
( __ ) Annual Competency Assessment
Not
Trained
Training
in
Progress
Trained /
Competent
Knowledge of procedure
Follows safety precautions
Follows SOP
Recognizes acceptable specimens
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Employee
Initials /
Date
Evaluator
Initials /
Date
* Method of
Assessment
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Accession and prepares paperwork
Sample Storage
Sample Preparation and Extraction
Task
Not
Trained
Training
in
Progress
Trained /
Competent
Employee
Initials /
Date
Evaluator
Initials /
Date
* Method of
Assessment
Not
Trained
Training
in
Progress
Trained /
Competent
Employee
Initials /
Date
Evaluator
Initials /
Date
* Method of
Assessment
Not
Trained
Training
in
Progress
Trained /
Competent
Employee
Initials /
Date
Evaluator
Initials /
Date
* Method of
Assessment
Knowledge of procedure
Follows SOP
Syringe and balance quality
control
Prepares worksheet
Prepares reagents
Spike Samples
Extraction procedure
Concentration techniques
Proper cleanup
Instrumentation
Task
Follows SOP
Able to perform routine
maintenance
Check calibration
Check instrument for
contamination
Set up sample sequence and
analytical run
Ability to troubleshoot
Data Acquisition and Processing
Task
Knowledge of procedure
Follows SOP
Quantitative and review
integration
Prepare preliminary report for
data review
Safety
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Not
Trained
Training
in
Progress
Trained /
Competent
Employee
Initials /
Date
Evaluator
Initials /
Date
* Method of
Assessment
Not
Trained
Training
in
Progress
Trained /
Competent
Employee
Initials /
Date
Evaluator
Initials /
Date
* Method of
Assessment
Adheres to the Wadsworth Center
safety policies
Continuing Education
Task
Has completed required annual
training for:
Confidentiality
Blood borne pathogens
Hazard communications
Other:
Has completed the minimum
requirement of 12 hours of
continuing education
*KEY: Method of Assessment
DO = Direct Observation
PT = Proficiency Test Performance
W = Written Quiz
Doc = Documentation Review
Action Recommended
Training
( ) Completed
( ) Continue Training
Comments/Developmental Plan:____________________________________________________
_____________________________________________________________________________
_____________________________________________________________________________
_____________________________________________________________________________
Competency
( ) Satisfactory ( ) Unsatisfactory
Comments/Developmental Plan:____________________________________________________
_____________________________________________________________________________
_____________________________________________________________________________
_____________________________________________________________________________
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EmployeeName____________________________Signature______________________ Date________
Trainer/Observer Name______________________Signature______________________ Date________
Supervisor/Director Name____________________Signature_____________________
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Date________
| File Type | application/pdf |
| File Title | DRAFT |
| Author | Kenneth M Aldous |
| File Modified | 2014-10-22 |
| File Created | 2012-05-14 |