Serum Analysis of PCB/PBDE/OCP

Page 1 of 20

Analysis of Polychlorinated Biphenyls (PCBs),
Polybrominated Diphenyl Ether (PBDE) and
Organochlorine Pesticides (OCPs) in Serum

Analyte:

PCB congeners (comprehensive), PBDE congeners (comprehensive) and
organochlorine pesticides (OCPs)

Matrix:

Serum (applicable to plasma and whole blood); applicable to fat with some
modifications to extraction

Method:

High Resolution Gas Chromatography-High Resolution Mass spectrometry
(HRGC/HRMS); High Resolution Gas Chromatography-Low Resolution Mass
Spectrometry (LRGC/MS) and Electron Capture Detection (GC/ECD)

Method No:
Performed by: Organic Chemical Biomonitoring Laboratory, Wadsworth Center, New York
State Department of Health
Contact:

Dr. K. Kannan, [email protected]

Information for users: The biomonitoring laboratory at Wadsworth Center periodically refines
these laboratory methods as new technologies become available. It is the responsibility of the
user to contact the person listed on the title page of each write-up before using the analytical
method to find out whether any changes have been made.
Reviewed by: _________________________
Date reviewed: _________________________

Created 01.31.11 by Sehun Yun

Page 2 of 20

Analysis of Polychlorinated Biphenyls (PCBs),
Polybrominated Diphenyl Ether (PBDE) and
Organochlorine Pesticides (OCPs) in Serum
1. History:

2. Summary:

Polybrominated diphenyl ethers (PBDEs) are used in large quantities as flameretardant additives in polymers, especially in the manufacture of a great variety of
electrical appliances, including televisions, computers, building materials and
textiles. Polychlorinated biphenyls (PCBs) are legacy environmental pollutants.
Organochlorine pesticides (OCPs) including DDT, HCH isomers, chlordanes and
hexachlorobenzene are persistent and bioaccumulative compounds. PBDEs have
a structural similarity to other environmental chemicals with known toxic effects,
such as polychlorinated biphenyls (PCBs), polybrominated biphenyls (PBBs) and
dioxins indicating that PCBs and PBDEs can be harmful to health.
A 2 g-serum sample is spiked with internal standard 13C isotopic analytes (13CPBDEs, 13C-PCBs and 13C-OCPs – details of the internal standards are given in 7)
at the beginning of the procedure at a pre-determined concentration (10 pg each
for PBDEs, 10 pg each for PCBs and OCPs and 10 pg for CB30 and 204) and
prepared. Two g of the serum sample/fat sample is homogenized with 20 g of
anhydrous sodium sulfate (pre-baked at 450ºC). Transfer 10 ml of 15%
dichloromethane in hexane to the sample and shake for 30 minutes. Transfer the
solvent to a round bottom flask and extract the sample 3 times with 10 ml of 15%
dichloromethane in hexane by shaking. Pool all the solvents and rotary-evaporate
to 5 ml. An aliquot (4 ml) is treated with sulfuric acid (3 ml) and further cleaned
using the automated Rapid Trace instrument. The clean-up column is then packed
with silicagel/sulfuric acid silicagel and readied. The Rapid Trace is purged using
a pre-determined method. The concentrated sample is then transferred to a Rapid
Trace tube and placed in the proper slots on the Rapid Trace. The instrument then
runs a pre-determined clean-up method. The elution fractions are then combined
and blown down to a final volume of 50 µl. The extract is then run on a HRGCHRMS to determine target analyte levels.
Target analytes are:
PCBs – as many congeners as possible: importantly, #8, 18, 28, 44, 49, 52, 66, 74,
87, 93, 99, 101, 105, 110, 114, 118, 126, 128, 130, 137, 138, 146, 149, 151, 153,
156, 157, 167, 170, 172, 177, 178, 180, 183, 187, 189, 194, 195, 196, 201, 206,
209.
PBDEs - Tri-to deca-BDE (#28, 47, 49, 66, 85, 99, 100, 153, 154, 183, 196, 197,
203, 207, 209)
Reviewed by: _________________________
Date reviewed: _________________________

Created 01.31.11 by Sehun Yun

Page 3 of 20

Analysis of Polychlorinated Biphenyls (PCBs),
Polybrominated Diphenyl Ether (PBDE) and
Organochlorine Pesticides (OCPs) in Serum

OCPs – Hexachlorobenzene (HCB), alpha-HCH, gamma-HCH, beta-HCH, deltaHCH, Oxychlordane, T-nonachlor, T-chlordane, C-nonachlor, C-chlordane, p,p'DDT, o,p'-DDT, p,p'-DDE, o,p'-DDE, o,p'-DDD, and p,p'-DDD, Dieldrin,
Dechlorane, Toxaphene (Parlar 26 & 50)
3. Sample Tracking:

Each sample contains a unique identifier specific for the sample. This identifier
will be used to track the samples from reception to analysis to final reporting.
4. Equipment:

5. Supplies:

Biological Hood
Chemical Hood
NANOpure Diamond (Barnstead)
Ultrasonic Cleaner (BRANSON 2510)
Scales (OHAUS/METTLER TOLEDO AG245)
Pipettors (Eppendorf Research)
Oven (PRICISION/SYBRON)
Corning Hot Plates (PC-400D)
Vortex
Orbital shaker (Eberbach Corp.)
Centrifuge (Eppendorf 5804)
RapidTrace Automated SPE Extraction Instrument
TurboVap LV Evaporator (Zymark)
Multivap 113 Nitrogen Evaporator (OA-SYS)
Rotary evaporator (BUCHI R-200 with B-490 heating bath)
Agilent 6890N GC/ECD
Thermo-Finnigan HRGC-HRMS MAT95XT or DFS
Sodium Sulfate, Anhydrous, Granular Power (Na2SO4)
Pestle and mortar
Acetone
n-Hexane 95%
Dichloromethane (DCM)
Methanol (MeOH)
Conical Polypropylene (PP) tubes (15 and 50 ml)
Round Bottom Centrifuge Tubes 35mL (29 X 100mm)
Reviewed by: _________________________
Date reviewed: _________________________

Created 01.31.11 by Sehun Yun

Page 4 of 20

Analysis of Polychlorinated Biphenyls (PCBs),
Polybrominated Diphenyl Ether (PBDE) and
Organochlorine Pesticides (OCPs) in Serum

Empty SPE Tubes, 3cc polypropylene with 3cc, 20u polyethylene frits
(Phenomenex)
Sepra C18-E (50um, 65Å) bulk packing sorbent 100g (Phenomenex)
Silicagel (Sigma-Aldrich, Grade 644, 150Å, 100-200 mesh)
Sulfuric Acid (H2SO4), AR®(ACS) 96-98% (Mallinckrodt Chemicals)
Silicagel/Sulfuric Acid (2:1 w/w)
5-3/4" and 9" disposable glass transfer pasteur pipets
Small Latex Droppers 1mL
Finntips (Finntip-20, -250 Universal, and -1000 Ext)
Disposable Culture Tubes (16 X 100 mm)
Disposable Aluminium Dish
15 mL Screw top tube with cap
Target DP Amber ID 2 ml vial, cap, and septa
150 µL or 50 uL glass inserts with standard polyspring
Stainless steel spatula
Purple nitrile gloves
50 and 100 hole vial rack
6. Data Handling:
The software (Xcalibur) is interfaced with the HRGC-HRMS and capable of
collecting, recording and storing chromatographic data. A computer, which is
dedicated to the instrument, is used to store and perform analysis on the data.
7. Reagents and Standards:
Solvents: Ultra-high purity Methanol (MeOH), Hexane (lot certified to be free of
contaminants), HPLC water, dichloromethane (DCM), Acetone, 88% Formic
Acid, 0.1N hydrochloric Acid (HCl), concentrated sulfuric acid.
Standards: 13C-labeled analyte specific isotopic internal standards
Mass labeled PBDE standard
MBDE-MXFS; Wellington Laboratories, Guelph, Ontario, Canada
MBDE-MXFS
Isomer #
28
47

Concentration
(ng/ml)
2000
2000
Reviewed by: _________________________
Date reviewed: _________________________

Created 01.31.11 by Sehun Yun

Page 5 of 20

Analysis of Polychlorinated Biphenyls (PCBs),
Polybrominated Diphenyl Ether (PBDE) and
Organochlorine Pesticides (OCPs) in Serum
100
99
154
153
183

2000
2000
2000
2000
2000

Running Stock#1 (100 ppb) = > 1.2ml + 22.8ml isooctane
Running Stock#2 (10 ppb) = > 1ml (stock#1) + 9 ml isooctane
Mass-labeled PCB standard:
EC-4058 (Cambridge Isotope Laboratories, [CIL] Andover, MA) - Surrogate
Stock Solution (5 ug/ml; 5ppm) 3ml
7 compounds; PCBs #: 28, 52,101, 138, 153, 180, 209
Running Stock#1 (2 ppm) => 3ml + 4.5ml isooctane
Running Stock#2 (200ppb) => 1ml (stock#1) + 9ml isooctane
Mass-labeled OCP standards
4,4'-DDT(13C12) 100ug/ml in Nonane, 1.2ml (CIL) Cat. Number: CLM-1281-S
4,4'-DDE(13C12) 100ug/ml in Nonane, 1.2ml (CIL) Cat. Number: CLM-1281-S
Beta-BHC(13C6) 50ug/ml in Nonane, 1.2ml Ⅹ2 (CIL) Cat. Number: CLM-3623-S
Native standards for all compounds were prepared for instrumental analysis to
generate external calibration curves. The native standards consisted of the
following compounds.
BDE-MXF Native PBDEs Stock Solution (2000 ng/ml; 2ppm) 1.2ml Ⅹ 2 for
Calibration; contained the native PBDE congeners 28, 47, 66, 100, 97, 85, 154,
153, and 183, from Wellington Laboratories.
BDE-196, -197, -203, -207, -209 was purchased from AccuStandard, New Haven,
CT.
PCB calibration solution was C-CCSEC-R PCB Calibration Check Solution (100
ug/mL in Acetone; 100 ppm), from AccuStandard. This contained the following
congeners: BZ#
8,18,28,44,52,66,77,101,105,118,126,128,138,153,170,180,187,195,201,206,209

Reviewed by: _________________________
Date reviewed: _________________________

Created 01.31.11 by Sehun Yun

Page 6 of 20

Analysis of Polychlorinated Biphenyls (PCBs),
Polybrominated Diphenyl Ether (PBDE) and
Organochlorine Pesticides (OCPs) in Serum

The OCP mix was Z-014C-R Pesticides Mix#2 (2.0 mg/mL in Toluene; 2000
ppm) from AccuStandard. This mixture contained the following compounds:
Aldrin, α-BHC, β-BHC, γ-BHC, δ-BHC, α-chlordane(c-chlor), γ-chlordane (tchlor), 4,4'-DDD, 4,4'-DDE, 4,4'-DDT, Dieldrin. We also prepared a target OCP
mixture that contained the following compounds: HCB, Oxychlordane, o,p'DDE, c-chlor, t-nonachlor, p,p'-DDE, o,p'-DDD, c-nonachlor, o,p'-DDT
8. Sample Preparation- Extraction:
1. The analyst will prepare a specific worksheet/or describe in lab notebook for
each sample to be analyzed
2. The samples will be removed from the freezer and placed within the
biological safety hood to allow for thawing
3. The analyst will then weigh 2 g of the sample and transfer this onto sodium
sulfate (20 g) placed in pestle and mortar
4. The samples are to be handled in such a way that the unique label for each
sample should be accompanied in all procedures with all equipment/tools.
The mortar will be labeled with the unique sample identifier
5. Grind the sample in pestle and mortar until it becomes a dry, freely flowing
powdery material.
6. Transfer the dry powdery, homogenized sample into a 50 ml polypropylene
(PP) tube using a solvent rinsed stainless steel spatula. Make sure that the
sample is not lost by spill. Label the PP tube with unique sample
identification code.
7. The sample is spiked with mass-labeled standards and surrogate: 10 pg for
each PBDE congeners, 10 pg PCB congeners, 10 pg OCP congeners, and 10
pg for unlabeled CB30 and 204.
8. Add 10 ml of 15% dichloromethane in hexane to the 50mL PP tube or 35mL
Centrifuge Tube.
9. Shake the PP tube for 30 minutes in an orbital shaker for 30 min.
10. Centrifuge the PP tube at 4000 rpm for 5 min.
11. Transfer the solvent to a round bottom flask (100 ml) using solvent cleaned
Pasteur pipettes.
12. Add another 10 ml of 15% dichloromethane in hexane to the PP tube
containing residual sample (this is second extraction) and shake for 30
minutes as above.
13. Centrifuge the PP tube and transfer the solvent to the round bottom flask
containing the first extract.
14. Repeat the extraction 3 times.
Reviewed by: _________________________
Date reviewed: _________________________

Created 01.31.11 by Sehun Yun

Page 7 of 20

Analysis of Polychlorinated Biphenyls (PCBs),
Polybrominated Diphenyl Ether (PBDE) and
Organochlorine Pesticides (OCPs) in Serum

15. Combine all three extracts (30 ml total) and concentrate using a rotary
evaporator set at 40ºC.
16. Transfer the extract to the 15mL PP tube to cleanup.
17. Evaporate to 5 ml and aliquot 4 ml for PBDEs, PCBs and OCPs analysis; 1 ml
is set aside for lipid analysis.
18. To the 4 ml of the extract, add 3 ml of concentrated sulfuric acid.
19. Treatment with HPLC water (Milli-Q water) to remove sulfur.
20. Remove the supernatant (organic layer) to a glass tube for cleanup using
Rapid Trace.
9. Sample Clean Up:
1. The analyst will then pack a two layered clean up column with one frit on
bottom, ~1.0g of silicagel/sulfuric acid (2:1 w/w), a frit added, ~0.1g silicagel,
and a frit placed on top (Refer to figure 1)
2. The Rapid Trace extraction instrument is purged using method ENDOCL1
(Refer to Appendix B)
3. Three Rapid Trace tubes are labeled with the unique sample identifier
4. The sample is transferred from the concentration conical tube to one of the
Rapid Trace tubes
5. The Rapid Trace tubes are loaded onto the extraction tray
6. The prepared clean up columns are placed into the turret on the Rapid Trace
extraction instrument
7. The sample is cleaned up using the method ENDOCL1.
8. The elution fractions, 15% dichloromethane in hexane (6ml x2 each), are
combined into one conical tube labeled with the unique sample identifier
9. The conical tube placed into the TurboVap and concentrated to a final volume
of 50ul.
10. The sample is transferred to an amber vial with remarked 50ul level at 150ul
glass insert with the unique sample identifier capped and stored at -20ºC until
analysis
10. Sample Analysis:
1. Sample is analyzed using a Thermo-Finnigan HRGC-HRMS (MAT95XT or
DFS) and an Agilent GC/ECD.

Reviewed by: _________________________
Date reviewed: _________________________

Created 01.31.11 by Sehun Yun

Page 8 of 20

Analysis of Polychlorinated Biphenyls (PCBs),
Polybrominated Diphenyl Ether (PBDE) and
Organochlorine Pesticides (OCPs) in Serum

Frit
0.1g Activated
Silicagel at 250ºC

1g Silicagel/Sulfuric acid (2:1)

Frit

Figure 1 Clean Up Column
11. % Lipid Determination (if and when needed)
1. Take one round bottom flask per sample and label each with the perspective sample number.
Add a few boiling chips and weigh the flask to acquire the initial weight of the flask and
chips. Record this information on the sample sheets.
2. Weigh out 0.5g of serum, in the Biocontainment Hood, using an ANDEK200g balance with
printer, which can measure weights out to two decimal places; i.e. (0.00g).
3. Add equal amount of MeOH to the sample to denature the protein. (Biocontainment Hood)
4. Extract serum with 15ml of 50% ether/hexane in three aliquots of 5ml. (Chemical Hood)
5. Evaporate extracted solvent to dryness in a dessicator.
6. An ANDHR-60 balance is used in weighing the sample in lipid determination. The balance
can measure out to four decimal places; i.e. (0.0000g).
Reviewed by: _________________________
Date reviewed: _________________________

Created 01.31.11 by Sehun Yun

Page 9 of 20

Analysis of Polychlorinated Biphenyls (PCBs),
Polybrominated Diphenyl Ether (PBDE) and
Organochlorine Pesticides (OCPs) in Serum

Weight of the flask + lipid + chip – Weight of the flask + chip = weight of lipid
28.6959g - 28.6934g = 0.0025g
is the weight of the lipid in the sample
Then, divide the weight of lipid in the sample by the starting weight, multiply by 100; this will
give the percent lipid in the sample.
X=

0.0025g
______ (100) = 0.50%
0.5g

12. Gas Chromatographic-Mass spectrometric Analysis:
The Thermo GC is typically fitted with 30-m x 0.25-mm internal diameter fused silica capillary
column with a 0.25-μm film thickness bonded phase. DB-5 (5% phenyl-, 95% methylpolysiloxane) or equivalent is suitable column phases. Suggested GC and inlet conditions are as
follows (other columns and/or conditions may be specified in project plans):
Injection port temperature 270 °C
Detector temperature
300 °C
Initial oven temperature
100 °C
Initial hold
1 min
Ramp 1 rate
8 °C /min to 160°C (hold 1 min)
Ramp 2 rate
2.1 °C /min to 260° (hold 1 min)
Ramp 3 rate
10 °C /min to 300°C (hold 1 min)
Carrier gas flow
1 mL/min (helium)
The Agilent GC/ECD is connected with DB-5ms (8m x 0.25mm x 0.1um).
Injection port temperature 240 °C
Detector temperature
300 °C
Initial oven temperature
100 °C
Initial hold
1 min
Reviewed by: _________________________
Date reviewed: _________________________

Created 01.31.11 by Sehun Yun

Page 10 of 20

Analysis of Polychlorinated Biphenyls (PCBs),
Polybrominated Diphenyl Ether (PBDE) and
Organochlorine Pesticides (OCPs) in Serum
Ramp 1 rate
Ramp 2 rate
Ramp 3 rate
Carrier gas flow
Makeup (N2)

6 °C /min to 190°C (hold 2 min)
6 °C /min to 240° (hold 1 min)
5 °C /min to 300°C (hold 5.67 min)
2 mL/min (helium)

12.1. Mass Spectrometer
A Thermo Finnigan Trace GC Ultra gas chromatograph / MAT95XP high resolution mass
spectrometer (HRGC-HRMS) is used for the identification and quantification of all target
compounds except PBDE-196, -197, -203, -207, -209, for which GC with electron capture
detection (ECD) is used. Measurements are carried out at a resolution of R >9000-10000.
PBDE congeners are monitored using the two most abundant masses of the ion clusters [M+2]
(m/z 405.8026 and 407.8026) for tri-BDE, [M+4], (m/z 485.7106 and 483.7126) for tetra-BDE,
[M-2Br], (m/z 403.7865 and 405.7845) for penta-BDE, [M-2Br] (m/z 481.6970 and 483.6950)
for hexa-BDE, and [M-2Br], (m/z 561.6055 and 563.6035) for hepta-BDE. A eight-point
calibration curve (0.1 - 20 ng/mL) is prepared every time when samples were measured. The
analysis of 2,2’,3,3’,4,4’,5,6’-octabromodiphenyl ether (BDE-196), 2,2’,3,3’,4,4’,6,6’octabromodiphenyl ether (BDE-197), 2,2’,3,4,4’,5,5’,6-octabromodiphenyl ether (BDE-203),
2,2’,3,3’,4,4’,5,6,6’-nonabromodiphenyl ether (BDE-207) and 2,2’,3,4,4’,5,5’,6octabromodiphenyl ether (BDE-203), and decabromodiphenyl ether (BDE-209) is performed
using an Agilent Technologies 6890N gas chromatograph / electron capture detector (GC/ECD).
Quantification of PBDE congeners is based on isotope-dilution method. An external calibration
curve is prepared for each congener, to determine the recoveries of target compounds in samples.
The quantification and confirmation ions used in the analysis of selected PCB and PBDE
congeners are based on most intense ions of the molecular ion cluster. If possible, ion groups
should be selected so that no more than 20 ions are monitored in a single group. It should be
noted that as the number of ions scanned per group increases and the individual dwell time
decreases, sensitivity will also decrease. Each ion in a group (and each ion between groups)
should have identical dwell times to ensure that correct ion ratios are preserved.
Prior to sample analysis, the elution order of the analytes of interest must be determined and/or
verified. This is performed by analyzing the analytes individually or in combination with
analytes having known pre-determined retention orders. The elution order and retention times
will be verified with each set of samples by analyzing a mixed congener standard that contains
all target congeners. Quantification of dioxin-like congeners PCB77, 81, 123, and 126 may be
affected by mass fragment contributions from congeners PCB110, 87, 149, and 129 when
Reviewed by: _________________________
Date reviewed: _________________________

Created 01.31.11 by Sehun Yun

Page 11 of 20

Analysis of Polychlorinated Biphenyls (PCBs),
Polybrominated Diphenyl Ether (PBDE) and
Organochlorine Pesticides (OCPs) in Serum

analyzed on a DB-5 column and may therefore result in unreliable results. PCB77, 81, 123, and
126 can therefore not be reliably analyzed on a DB- 5 column without pre-separation such as a
Carbon Column Chromatography from the other congeners. PCB118 coelutes with PCB106
(same level of chlorination) on the DB-5 column and those are reported as coeluters (PCB106 is
a minor congener relative to PCB118 in Aroclor and environmental samples).
Quantification of OCPs is based on external calibration standard, because labeled congeners are
not available for all OCPs.
12.2. GC/MS Mass Calibration (Tuning) (If applicable)
Prior to the analysis of analytical standards and/or samples the mass spectrometer must be tuned.
This procedure utilizes perfluorotributlyamine (PFTBA) to maximize the sensitivity of the
instrument. The technical acceptance criteria for tuning the mass spectrometer are as follows:
Mass 69 100%, Base peak, Mass 219 40-65% of mass 69, Mass 502 4-12% of mass 69
12.3. Calibration and Standardization
Demonstration of a linear initial calibration is required prior to the analysis of samples. The
GC/MS or HRGC/HRMS must be calibrated to determine the retention time of each target
congener and to determine the instrument sensitivity and linearity of the response of the target
compounds. If the response factor (RF) for any compound is constant (less than 25% RSD) over
the calibration range, an averaged response factor may be used for that compound; otherwise, the
complete calibration curve for that compound shall be used over the calibrated range.
12.4. Identification of Target Compounds
When using SIM mode in GC/MS or HRGC/HRMS techniques, an analyst competent in the
interpretation of mass spectra identifies the compounds in a sample by comparing the ratio of the
target ion and the confirmation ion with that provided in this SOP. Comparison is also made to
other reference standards such as the instrument control check (ICC) sample, and/or other project
specific samples. Two criteria must be satisfied to verify the identifications of target compounds:
1. Elution of the sample analyte within GC retention time window
2. Correspondence of the sample analyte and calibration standard ion ratios
GC Retention Time. The sample component retention time (RT) should be within ± 0.1 min (6
sec) of the standard component.
Reviewed by: _________________________
Date reviewed: _________________________

Created 01.31.11 by Sehun Yun

Page 12 of 20

Analysis of Polychlorinated Biphenyls (PCBs),
Polybrominated Diphenyl Ether (PBDE) and
Organochlorine Pesticides (OCPs) in Serum

Mass Spectra. For each target analyte, the corresponding ions listed in Attachment 5 must be
present and the relative intensities must agree to within ±20 percent.
Signal to Noise Ratio. A quantifiable analyte peak should exhibit a signal to noise ratio of
approximately 3:1 or greater.
12.5. Quantification of Target Compounds
Target PCB, PBDE congeners and pesticide (OCP) compounds are quantified by the internal
standard method, using the average response factor from the initial calibration or response
factors from the single-point calibration, unless otherwise specified in the project protocols. The
average response factor (RF) from the initial calibration is used to calculate the concentration in
the sample for the fully calibrated analytes. The response factors from the single-point
calibration are used to quantify the other target analytes. Secondary ion quantification is allowed
only when there are sample interferences with the primary ion. If secondary ion quantification is
performed, the reason must be documented in the case narrative.
The equation for calculating the concentration of chlorinated pesticides, PCB congeners and
homologues in a sample is as follows:
Ca = ((Aa/RFi) XD)/Va
Where,
Ca = Concentration target analyte
Aa = Area quantification ion for target analyte
RFi = Average RF for analyte determined from initial calibration or single-point calibration
D = Dilution factor if applicable
Va = Sample size
Sample size may refer to sample volume or sample wet/lipid weight. The project protocol will
specify reporting criteria.
13. Quality Control
Glassware: All Solvents should be high purity and should be tested for contamination. All
glassware and tools that come in contact with samples should be rinsed with acetone and hexane
(see Appendix B)
Procedural blank: Procedural blank contains 1-2 ml water in lieu of serum/plasma sample. All
solvents and reagents used in the analysis will be analyzed. Any contamination in procedural
Reviewed by: _________________________
Date reviewed: _________________________

Created 01.31.11 by Sehun Yun

Page 13 of 20

Analysis of Polychlorinated Biphenyls (PCBs),
Polybrominated Diphenyl Ether (PBDE) and
Organochlorine Pesticides (OCPs) in Serum

blank should be reported. One procedural blank is analyzed for every 20 samples. If the levels
of contaminants in procedural blanks exceed the detection limits, data should be flagged or the
batch should be repeated.
Matrix spike and matrix spike duplicate (MS and MSD): For every 20 samples a matrix spike
and a matrix spike duplicate is analyzed. The recoveries of matrix spike should be 100±30%.
The RPD of repeated analysis should be <20%. Any deviation from the results should be flagged
or if samples are available, analysis should be repeated.
13

C-labelled internal standards: The recoveries of labeled internal standards of PCBs, PBDEs,
and OCPs should be between 100 ± 30%. Any deviation from the results should be flagged or if
samples are available, analysis should be repeated.
Duplicate analysis: For every 30-40 samples, duplicate analysis of samples is needed. A
randomly selected sample is analyzed in triplicate and precision is calculated. The precision
should be >90%. Any deviation from the results should be flagged or if samples are available,
analysis should be repeated.
Continuing calibration curve: A mid-point calibration curve should be injected after every 10
sample to check instrumental stability and response. If the responses vary by 50%, the batch
should be reinjected with a new calibration curve.

14. Safety:
PCBs, PBDEs and OCPs 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. Serum samples will be handled
in a biocontainment hood in accordance with NYSDOH Safety Manual (Number
II.11, issued 12/90, and revised 9/92). All disposable glassware being exposed to
serum will be disposed of in aluminum stockpots that are clearly marked
“Biohazard.” Non-disposable glassware will be submerged in Clorox overnight.
Liquid waste will be disposed of in red organic-waste containers.
Reviewed by: _________________________
Date reviewed: _________________________

Created 01.31.11 by Sehun Yun

Page 14 of 20

Analysis of Polychlorinated Biphenyls (PCBs),
Polybrominated Diphenyl Ether (PBDE) and
Organochlorine Pesticides (OCPs) in Serum
Cleanup:

All disposable glassware being exposed to blood or serum should be disposed of
in aluminum stockpots that are clearly marked “Infectious Waste.” After soaking
overnight in 50% Clorox solution. Non-disposable glassware should be in a 50%
Clorox solution overnight. Liquid organic waste should be disposed of in
organic-waste containers.
All manipulations of the sample before extractions will be carried out in the
biocontainment hood. Lab coat and latex gloves are to be worn and the blower on
while working in the biocontainment hood. In addition to gloves and lab coat, safety
glasses should be worn while working in the chemical fume hood.
15. Documentation:
The laboratory will maintain a hardbound notebook where any modifications to the
analytical system will be recorded. Modifications such as changing columns, inlet
liners, any routine maintenance performed, and any malfunction that occurs in the
analytical system will be noted. The notebook will also have recorded the location of
all samples and sample extracts. The Rapid Trace automated extraction system will
have a hardbound book to show any maintenance and sample location during
extraction.
Data files (the electronic data) transfer to other computers for data reduction. All
chromatography data are archived including: ChemStation raw data files, processed
data files, and associated calibration and integration files.
REFERENCES
United States Environmental Protection Agency (U.S. EPA), 1999. Chlorinated Biphenyl
Congeners in Water, Soil, Sediment, and Tissue by HRGC/HRMS, Method 1668, Revision A,
Office of Water, U.S. Environmental Protection Agency, Washington, DC.
United States Environmental Protection Agency (U.S. EPA), 1985. Test Methods for
Determination of Pesticides and PCBs in Water and Soil/Sediment by Gas
Chromatography/Mass Spectroscopy, Method 680, Physical and Chemical Methods Branch,
Environmental Monitoring and Support Laboratory, Office of Research and Development, U.S.
Environmental Protection Agency, Cincinnati, Ohio.
Reviewed by: _________________________
Date reviewed: _________________________

Created 01.31.11 by Sehun Yun

Page 15 of 20

Analysis of Polychlorinated Biphenyls (PCBs),
Polybrominated Diphenyl Ether (PBDE) and
Organochlorine Pesticides (OCPs) in Serum

Appendix A (If applicable)
Sample Preparation and Extraction
1. Sample weight (1g) into a 15mL test tube.
2. Sample spiked with 10ul of 13C-labeled surrogate standards at 10ng/mL and wait
1hr
3. Sample Preparation:
a) 88% Formic acid is then added to the sample in a 1:1 ratio (1g)
b) The sample was then vortexed (10s) and sonicated with no heat (10min)
c) HPLC H2O was added to the sample at a 1:1 ratio (2g) and vortexed
(10s)
d) The sample is transferred into the Rapid Trace tube
4. SPE using the Rapid Trace Automated System
a) Column packed with 1.3g of C18-Sepra Sorbent
5. Instrument extraction method (ENDOEXT1 method)
6. Concentrate to 1mL
(If applicable)
Condition
Condition
Condition
Condition
Load
Rinse
Dry
Dry
Purge-cannula
Collect1
Collect2
Purge-Cannula
Purge-Cannula

MeOH
5%MeOH
DCM
5%MeOH
Sample
5%MeOH
N2
N2
DCM
30%DCM/Hex
30%DCM/Hex
DCM
H2O

3mL 3mL/min
3mL 3mL/min
3mL 3mL/min
3mL 3mL/min
5mL
0.40mL/min
1mL
3mL/min
Time = 20min
Time = 20min
3mL 3mL/min
6mL 0.40mL/min
6mL 0.40mL/min
3mL
3mL/min
3mL
3mL/min

Reviewed by: _________________________
Date reviewed: _________________________

Created 01.31.11 by Sehun Yun

Page 16 of 20

Analysis of Polychlorinated Biphenyls (PCBs),
Polybrominated Diphenyl Ether (PBDE) and
Organochlorine Pesticides (OCPs) in Serum
Appendix B

SPE Packing: 0.1g Silica gel (SG) + 1g SG/Sulfuric
Acid (2:1) + 0.1g SG
Condition1
Hexane
5ml
5ml/min
Condition2
15%DCM/Hex
5ml
5ml/min
Load
Sample
2.5ml 0.38ml/min
Purge
15%DCM/Hex
3ml
5ml/min
Collect1
15%DCM/Hex
6ml
0.38ml/min
Collect2
15%DCM/Hex
6ml
0.38ml/min
Purge
15%DCM/Hex
3ml
5ml/min
Appendix C
Glassware Cleaning
1.
2.
3.
4.
5.
6.

Wash glassware in hot soapy water using brushes that are specific for BOAC use
Thoroughly rinse glassware with copious amounts of hot water
Rinse glassware again with copious amounts of organic-free water
Oven dry glassware @ 105 C for at least 1.5 hours
Remove glassware from oven and allow too cool to room temperature
After cooling, rinse glassware 3x with nano-grade hexane (pre-checked lot), saving third
rinse. (Note: use tongs to hold glassware while rinsing)
7. Concentrate combined rinses to 1ml, then analyze by GC
8. If there are any contaminant is detected, the glassware is to be re-rinsed and re-checked
before using. If a contaminant still appears, then the glassware is placed in the washtub to
start from the beginning
Vial and Insert Cleaning
Cleaning glass insert:
1.
2.
3.
4.
5.

Remove plastic feet from inserts
Place vials or inserts into BOD bottle or flask
Add acetone until all inserts are completely filled
Soak for at least 15 minutes
Discard acetone and add hexane
Reviewed by: _________________________
Date reviewed: _________________________

Created 01.31.11 by Sehun Yun

Page 17 of 20

Analysis of Polychlorinated Biphenyls (PCBs),
Polybrominated Diphenyl Ether (PBDE) and
Organochlorine Pesticides (OCPs) in Serum
6.
7.
8.
9.

Soak for at least 15 minutes
Discard hexane and let vials dry in crystallizing dish covered with foil
When solvent is completely gone put into 105 C oven, uncovered, overnight
When completely cooled, feet can be put back on insert vials and both vials are stored
in foil-covered beakers

Reviewed by: _________________________
Date reviewed: _________________________

Created 01.31.11 by Sehun Yun

Page 18 of 20

Analysis of Polychlorinated Biphenyls (PCBs),
Polybrominated Diphenyl Ether (PBDE) and
Organochlorine Pesticides (OCPs) in Serum
Appendix D

Method Detection Limits (MDL) for PCB, PBDEs, and OCP congeners
PCBs
IUPAC No.
PCB18
PCB28
PCB44
PCB49
PCB52
PCB66
PCB74
PCB87
PCB99
PCB101
PCB118
PCB128
PCB138
PCB146
PCB149
PCB151
PCB153
PCB156
PCB157
PCB167
PCB170
PCB172
PCB177
PCB178
PCB180
PCB183
PCB187
PCB189
PCB194
PCB195
PCB196
PCB201
PCB206
PCB209

PBDEs
MDL (ng/g, wet weight)
<0.0032
<0.0032
<0.0032
<0.0032
<0.0032
<0.0032
<0.0032
<0.0032
<0.0032
<0.0032
<0.0032
<0.0032
<0.0032
<0.0032
<0.0032
<0.0032
<0.0032
<0.0032
<0.0032
<0.0032
<0.0032
<0.0032
<0.0032
<0.0032
<0.0032
<0.0032
<0.0032
<0.0032
<0.0032
<0.0032
<0.0032
<0.0032
<0.0032
<0.0032

IUPAC No.
PBDE28
PBDE47
PBDE49
PBDE66
PBDE85
PBDE99
PBDE100
PBDE153
PBDE154
PBDE183
PBDE197
PBDE203
PBDE196
PBDE207
PBDE209

MDL (ng/g, wet weight)
<0.0050
<0.0050
<0.0050
<0.0050
<0.0050
<0.0050
<0.0050
<0.0050
<0.0050
<0.0050
<0.0050
<0.0050
<0.0050
<0.0050
<0.0050

OCPs
IUPAC No.
HCB
α-HCH
β-HCH
g-HCH
δ-HCH
Oxychlordane
Trans-nona
Trans-chlordane
Cis-chlordane
Cis-nona
p,p'-DDT
o,p'-DDT
p,p'-DDE
o,p'-DDE
o,p'-DDD
p,p'-DDD

MDL (ng/g, wet weight)
<0.0085
<0.0100
<0.0098
<0.0100
<0.0100
<0.0460
<0.0150
<0.0086
<0.0273
<0.0452
<0.0391
<0.0132
<0.0100
<0.0100
<0.0100
<0.0100

Reviewed by: _________________________
Date reviewed: _________________________

Created 01.31.11 by Sehun Yun

Page 19 of 20

Analysis of Polychlorinated Biphenyls (PCBs),
Polybrominated Diphenyl Ether (PBDE) and
Organochlorine Pesticides (OCPs) in Serum
Supplies
Ordering:

Krackler Scientific (Contract)
P.O. Box 1849
Tel: (518) 462-4281
#207-C4000-2W-PK Target DP Amber ID Vials 100/pk $17.74/pk
#207-C4012-530-PK 150 L Standard Polyspring Glass Inserts 100/pk $46.26/pk
#207-C4000-53G Target DP Caps and Septa 100/pk $25.25
#6-73500-16100 Disposable Culture Tubes 16x100mm 1000/CS $54.93
#343-V349-1000 300 Series Amber Boston Round Bottom Flask 12/CS $55.57
# 11-0128-05 Formic Acid, 2.5L, 88%
$64.97
#11-3891-05 Sodium Sulfate, Anhydrous, Granular Powder 2.5kg (4/CS) $378.20
#3-352096-CS 15mL PP Tubes (500/CS)
$86.47
#3-352070-CS 50mL PP Tubes (500/CS)
$97.81
#4-9590-3-PK Universal range pH Strips (100/PK) $18.00

Ordering:

Phenomenex (Contract)
411 Madrid Ave
Torrance, CA 90501
Tel: (310) 212-0555
# AH0-7001 Empty SPE Tubes, 3cc (50/Box)
# AH0-7007 Frits for 3cc SPE Tubes, 20u (100/Bag)
# 04G-4348 Sepra Bulk C18 (100g)

Ordering:

$ 32.00
$27.25
$190.00

Sigma-Aldrich
PO Box 14508, St.Louis, MO 63178
Tel: 800-325-3010
#236829-1KG Silicagel, 100-200mesh, Grade644, Pore 150Å $229.50
Silica Gel/Sulfuric Acid Procedure (2:1 w/w)

1. Washing the Silica Gel

Reviewed by: _________________________
Date reviewed: _________________________

Created 01.31.11 by Sehun Yun

Page 20 of 20

Analysis of Polychlorinated Biphenyls (PCBs),
Polybrominated Diphenyl Ether (PBDE) and
Organochlorine Pesticides (OCPs) in Serum

a) 200 g of Silica gel is weighed into a Pyrex-Plus Heavy Wall Filter Flask.
dichloromethane (DCM) is added to cover the weighed out powder and a slurry is
made and washed around
b) The apparatus is placed into a vacuum oven with no heat to evaporate the DCM off
the Silica gel over the weekend
2. Silica Gel/ Sulfuric Acid (2:1 w/w)
a) 100 g of washed Silica gel is weighed out into a Amber Boston Round Bottom Bottle
b) 50 g (or ~28ml) of Sulfuric Acid is added to the bottle
c) The bottle is capped with a Teflon cap or tape and placed on the roller overnight to
give a homogenous mixture.
Acid Water Procedure
1.
2.
3.
4.
5.

190 mL of HPLC water is placed into a Pyrex bottle
10 mL of MeOH is added
200 mL of 0.1N HCl is added
Mix well
The solvent should test at a pH of 1-1.3
30% DCM/Hex Procedure

1. In a Pyrex bottle add 180mL of dichloromethane
2. Add 420 mL of Hexane
3. Mix well
15% DCM/Hex Procedure
1. In a Pyrex bottle add 850mL of dichloromethane
2. Add 150 mL of Hexane
3. Mix well

Reviewed by: _________________________
Date reviewed: _________________________

Created 01.31.11 by Sehun Yun