RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2005; 19: 1815–1821

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.1990

Analysis of organochlorines in harbor seal

(Phoca vitulina) tissue samples from Alaska
using gas chromatography/ion trap mass
spectrometry by an isotopic dilution technique
Dongli Wang1, Shannon Atkinson2,3, Anne Hoover-Miller3 and Qing X. Li1*
1
Department of Molecular Biosciences and Bioengineering, University of Hawaii, Honolulu, Hawaii 96822, USA
2
School of Fisheries and Ocean Sciences, University of Alaska, Fairbanks, Alaska 99664, USA
3
Alaska SeaLife Center, Seward, Alaska 99664, USA
Received 20 March 2005; Revised 15 April 2005; Accepted 1 May 2005

A gas chromatography/ion trap mass spectrometry (GC/ITMS) method was developed for the deter-
mination of organochlorine pesticides (OCPs), polychlorinated biphenyls (PCBs) and polychlori-
nated naphthalenes (PCNs) in harbor seal (Phoca vitulina) tissues. Tissue samples were
homogenized, lyophilized and fortified with 13C-PCBs 28, 123, 169 and 170, and then extracted
with an accelerated solvent extractor with a mixture of hexane and methylene chloride (1:1, v/v).
After lipid removal using a 40% H2SO4-modified silica gel column, all organochlorines were col-
lected in one fraction and further fractionated with an activated carbon/silica gel (1:20) column
into a first fraction containing OCPs, non-coplanar PCBs and 13C-PCBs 28, 123 and 170, and a sec-
ond containing PCNs, coplanar PCBs and 13C-PCB 169. Prior to GC/MS/MS analysis, 13C-PCB
169 was added into the first fraction as an injection standard and 13C-PCB 170 into the second frac-
tion to calibrate the recoveries of the fortified internal standards. This method can effectively elim-
inate matrix interferences, and has high selectivity and sensitivity. Recoveries averaged 45–86% for
OCPs with relative standard deviations (RSDs) of 2–14%, 52–137% for PCBs with RSDs of 3–29%
and 36–152% for PCNs with RSDs of 7–29% from lard and chicken heart samples, which were used
as alternative matrices to harbor seal samples in recovery studies. The limits of detection for OCPs,
PCBs and PCNs were 0.7–1.9, 1.5–8.9 and 0.5–10 pg/g dry weight, respectively. This method can be
used to analyze OCPs, PCBs and PCNs in harbor seal blubber, liver and kidney samples. Copyright
# 2005 John Wiley & Sons, Ltd.

Organochlorine pesticides (OCPs), polychlorinated biphe-
nyls (PCBs), and polychlorinated naphthalenes (PCNs),
collectively referred to as organochlorines (OCs), are among
the most prevalent pollutants in various environmental com-
partments,1– 7 although their use has been banned in most
countries.8,9 These compounds, being highly persistent and
lipophilic, have been transported to the Arctic by atmo-
spheric and oceanic vectors10 and accumulated at high con-
centrations in top marine predators.
Several environmental factors may impact the biology of
harbor seals (Phoca vitulina), resulting in poor survival and
reproductive rates. One suspected factor is exposure to OCs
including OCPs, PCBs and PCNs, which are probably
associated with endocrine and immune system impair-
ment,11–13 and can interfere with normal physiology and
*Correspondence to: Q. X. Li, Department of Molecular
Biosciences and Bioengineering, University of Hawaii, Honolulu,
Hawaii 96822, USA.
E-mail: qingl@hawaii.edu
Contract/grant sponsors: Alaska SeaLife Center; National
Marine Fisheries Service; Exxon Valdez Oil Spill Trustee
Council.
biochemistry.14–17 PCBs can compete for binding sites on the
transport proteins for the thyroid hormones, affecting early
development or later reproductivity.18 Eco-toxicological
research of OCs on harbor seals requires reliable data
obtained with highly reproducible, selective and sensitive
methodologies.
High-resolution gas chromatography (HRGC) with elec-
tron-capture detection (ECD) has been a widely used
technique for the analysis of OCs because of its high
selectivity, sensitivity and low cost. However, ECD is unable
to differentiate PCBs from coeluting interferences and
resolve PCB congener pairs,19,20 and suffers from non-linear
response within a PCB homologue group.21,22 The reference
analytical method of PCBs in water, soil, sediment, and
tissue23 comprises multi-step isolation, extensive clean-up,
and use of hyphenated HRGC/high-resolution mass spectro-
metry (HRMS). Due to the high cost and difficult main-
tenance of HRGC/HRMS systems, ion trap mass
spectrometry (ITMS) has been used as a low-cost alternative
to HRMS.24,25 The results from the analysis of complex
matrices such as milk, fish and fly ash by HRGC/ITMS26
showed a remarkable reliability as compared with those by

Copyright # 2005 John Wiley & Sons, Ltd.

1816 D. Wang et al.
HRGC/HRMS. In some cases tandem mass spectrometry
(MS/MS) has been more selective than HRMS,27 whilst, in
others,28 HRMS gave more accurate results. Both techniques
are regarded as complementary.29 Recently, HRGC/ITMS has
proven to be effective for trace analysis of OCs in environ-
mental and biological samples.24,30–34 The aim of this work
was to develop a sensitive HRGC/ITMS method for the
determination of OCPs, PCBs and PCNs in harbor seal tissues.
EXPERIMENTAL
Reagents, solvents and standard solutions
Methylene chloride (GC resolve), n-hexane, toluene, isooc-
tane (Optima), and concentrated sulfuric acid were pur-
chased from Fisher Scientific (Fair Lawn, USA). Anhydrous
sodium sulfate (Mallinckrodt Baker, Philipsburg, USA) was
baked overnight at 4508C. Activated carbon (MCB, Norwood,
OH, USA) was washed with toluene. Acidified silica was a
mixture of 67 g of concentrated H2SO4 and 100 g of silica
gel, and was activated overnight at 1608C (330–400 mesh, Sig-
ma Chemical Co., St. Louis, USA). Activated carbon/silica
was a mixture of activated carbon and silica gel (1:20, w/w).
PCB (C-WNN-PCB) and Halowax 1014 standard solutions
(AccuStandard, New Haven, USA) were used at a concentra-
tion of 10 mg/mL each PCB and 0.1 mg/mL in total PCNs,
respectively. OCP standard solution (Supelco, St. Louis,
USA) was used at a concentration of 0.1 mg/mL each OCP.
13
C-PCBs 28, 123, 169 and 170 (Wellington Laboratories,
Guelph, Ontario, Canada) were each used at 50 mg/mL. The
PCB (C-WNN-PCB) stock solution containing 28 PCB
congeners was each diluted to 100 ng/mL in isooctane, and
PCN (Halowax 1014) stock solution to 1000 ng/mL in total
PCNs in isooctane, which were used for recovery studies.
Three sets of working standard solutions contained 0.5, 1, 5,
20, 100, 500 and 2000 ng/mL of each OCP, and 0.5, 1, 5, 20, 100,
500 and 1000 ng/mL of each PCB, and 100, 500, 1000, 5000,
and 10000 ng/mL of Halowax 1014 technical mixture,
respectively. Each working solution also contained 100 ng/
mL of individual 13C-PCB. The concentration and composi-
tion of Halowax 1014 were the same as those referred to in
previous studies.35 One 13C-PCB solution was prepared to
contain 100 ng/mL of 13C-PCBs 28, 123 and 170 each in
isooctane, and another 100 ng/mL of 13C-PCB 169 alone. All
standard solutions were stored in the dark at
48C.
Sample collection and storage
Lard and chicken heart samples were purchased in a local
supermarket in Honolulu, Hawaii. Harbor seal liver, kidney,
and blubber samples were collected by subsistence hunters
from southcentral Alaska during 2000 and 2001, and pro-
cessed through the Alaska Native Harbor Seal Commission
Biosampling Program. A sub-sample was taken from inner
tissue, while frozen, with a knife cleaned with acetone. The
frozen 8–15 g samples were wrapped in aluminum foil,
placed in plastic bags, and delivered to the laboratory, where
they were then stored at
258C until analysis.
Extraction, clean-up and fractionation
The samples were homogenized with a blender, and lyophi-
lized for 48 h. The lyophilized samples were mixed with a
Copyright # 2005 John Wiley & Sons, Ltd.
three-fold amount of anhydrous sodium sulfate, and then
placed in a 33 mL accelerated solvent extraction (ASE) cell.
The remaining volume of the cell was filled up with Ottawa
sand. The sample cell was loaded into a Dionex ASE 200 sys-
tem. The extraction was performed with a mixture of hexane
and methylene chloride (1:1, v/v) at 20 mPa and 1008C for
three static cycles, a flush volume of 60% of the cell volume
and N2 purge time of 5 s.
The extract was concentrated to 8.0 mL using a rotary
evaporator and a gentle stream of N2 at 408C. An aliquot
(1.0 mL) was used to determine lipid contents by the
gravimetric method. The remaining extract was spiked with
13
C-PCBs 28, 123, 169 and 170, and further concentrated to 2–
3 mL. For recovery efficiency, 13C-PCBs 28, 123, 169 and 170
were fortified in the lard and chicken heart samples prior to
extraction. OCPs, PCBs and PCNs in the concentrated
extracts were eluted with 50 mL of hexane through an acidic
silica gel column (9 g; 40 cm length, i.d. 10 mm). The eluate
was evaporated to 2 mL, and further fractionated into two
fractions with a 5-mL disposable glass pipette which was
packed with a portion of 250 mg of activated carbon/silica gel
(1:20) between two layers of 100 mg of silica gel. The first
fraction was eluted with 15 mL of 40% methylene chloride in
hexane, and then concentrated to 200 mL for the analysis of
OCPs and non-coplanar (ortho-substituted) PCBs. The
second fraction was back-flushed with 15 mL of toluene,
and concentrated to 20 mL for the analysis of coplanar (non-
ortho-substituted) PCB congeners (PCBs 77, 81, 126, and 169)
and PCNs. 13C-PCB169 and 13C-PCB170, the injection
internal standards, were added to the first and second
fractions, respectively.
GC/ITMS analysis
All sample analyses were performed on a Varian 3800 GC/
Saturn 2000 ion trap mass spectrometer (Varian, Walnut
Creek, CA, USA). The gas chromatograph was equipped
with a 60 m ZB-1 column (0.25 mm i.d., 0.25 mm film thick-
ness) with a constant flow rate (2 mL/min) of carrier gas
(helium). The oven temperature was programmed from 120
to 2808C at 48C/min, and held for 15 min at 2808C. The injec-
tor temperature was 2808C. Sample extracts (2 mL) were
injected in splitless mode with an AS 8400 autosampler.
The ion trap was operated in MS/MS mode, and the transfer-
ring line and ion trap temperatures were 280 and 2008C,
respectively. Data were analyzed with Saturn GC-MS Work-
station version 5.4 software. Parent (precursor) ions and GC
retention time segments for the analytes were selected by
separate injections of the OCPs, C-WNN-PCB and Halowax
1014 standard solutions in full-scan mode. The parent ions
selected were the most abundant ions in the full-scan mass
spectrum of each congener, and were subject to MS/MS ana-
lysis in resonant excitation mode. MS/MS conditions were
optimized with four parameters, excitation amplitude (EA),
stability parameter (qz), excitation time (ET) and isolation
time (IT), in multiple standard runs. EA was optimized by
the automated method development (AMD) tool, for which
the optimization was performed with the maximum number
of cycles through different voltages across each peak. AMD
allowed incremental changes of collision-induced dissocia-
tion (CID) voltage on a scan-by-scan basis for up to 10 scans
Rapid Commun. Mass Spectrom. 2005; 19: 1815–1821

per cycle. In this study, EAs were optimized with 0.5, 0.25 or
0.2 V increments in separate runs, while other parameters
were maintained at default values. The stability parameter
(qz) was tuned to modify the position of the ion within the sta-
bility diagram of the trap. ET and IT were also optimized by
multiple sample injections and corresponding parameter
adjustments. The optimum EA gave minimum and maxi-
mum abundances of parent and daughter (product) ions,
respectively, and maintained the correct isotopic ratio
between product ions. Analytes were identified with their
retention times compared to those of the corresponding
authentic standards, signal-to-noise (S/N) ratio of >3 for
the selected ions, and chlorine isotope ratio of two character-
istic ions matching the theoretical values within 20% devia-
tion. In the first fraction, the concentrations of benzene
hexachlorides (BHCs, specifically, a-BHC, b-BHC, g-BHC
and d-BHC isomers), heptachlor and DiCBs, TriCBs and Tet-
raCBs were calculated according to 13C-PCB 28, and p,p0 -
dichlorodiphenyltrichloroethane (pp0 -DDT), p,p0 -dichlorodi-
phenyldichloroethylene (pp0 -DDE), p,p0 -dichlorodiphenyldi-
chloroethane (pp0 -DDD), PentaCBs, HexaCBs, HeptaCBs
according to 13C-PCB 123, and OctaCBs, NanoCBs and Dec-
aCBs according to 13C-PCB 170. The recoveries of 13C-PCBs
28, 123, and 170 in the first fraction were determined by the
injection standard 13C-PCB 169. The concentrations of
PCNs and coplanar PCBs in the second fraction were calcu-
lated by 13C-PCB 169, while the recovery of 13C-PCB 169
was determined by the injection standard 13C-PCB 170.
Quality assurance and quality control
Average recoveries and relative standard deviations (RSDs)
were first obtained to appraise method performance by mul-
tiple analyses of the spiked lard and chicken heart samples. A
solvent blank and a matrix blank were analyzed through the
entire procedure prior to and after every 10 samples. Work-
ing standard solutions of OCPs, PCBs and PCNs were run at
the beginning of sample analysis to determine the relative
response factors and evaluate peak resolutions. The method
detection limits (MDLs), defined as 3-fold of standard devia-
tions, for OCPs, PCBs and PCNs were determined using a
standard mixture at a low concentration (5 ng/g for each
PCB and each OCP, and 2.5 ng/g for total PCNs) in the spiked
chicken hearts.
RESULTS AND DISCUSSION
Extraction, clean-up and fractionation
In general, accumulation of OCs in tissues correlates well
with lipid contents, and the OC concentrations are typically
expressed on a lipid basis. However, the method-related dif-
ferences in the lipid yield may cause discrepancies in the nor-
malized OC concentrations on a lipid basis, even when the
same amounts of the analytes are extracted. Liver and kidney
tissues contain much more highly polar lipids than the other
organ tissues. Therefore, it is important to employ a method
that can nonselectively extract different lipids and the ana-
lytes of interest. ASE is a simple and efficient extraction tech-
nique that utilizes conventional solvents at controlled
temperature and pressures and has been well established
for recovery of OCs from various matrices, showing better
Copyright # 2005 John Wiley & Sons, Ltd.
GC/MS analysis of organochlorines in harbor seals 1817
or comparable results to those obtained with Soxhlet and
other techniques.36,37 Recent studies showed that the ASE
method using an equal volume mixture of hexane and methy-
lene chloride under the conditions adapted in this study effi-
ciently coextracts OCs and lipids.38–40
Gel permeation chromatography,41 concentrated sulfuric
acid digestion,42,43 and H2SO4-modified silica gel columns23
are widely used clean-up techniques for the analysis of OCs
in biological samples. In this study, lipids were removed with
40% H2SO4-modified silica gel columns. The OCPs, PCBs and
PCNs were completely eluted with 50 mL of hexane, and the
eluates were suitable for the subsequent activated carbon/
silica gel column fractionation.
To measure a large concentration range of different OCs,
the analytes were fractionated into several fractions. The
fractionation methods include high-performance liquid
chromatography44,45 and the activated carbon/silica gel
column.5,46,47 In this study, an activated carbon/silica gel
column was used to separate OCPs and non-coplanar PCBs
into the first fraction, and PCNs and coplanar PCBs into the
second fraction. The second fraction was concentrated 5–10
times of the first fraction for the analysis of lower level of
PCNs and coplanar PCBs.
Optimization of GC/MS/MS parameters
In full-scan MS spectra, all molecular ions [M]þ of PCBs and
PCNs were dominant (data not shown), while, in the MS/MS
spectra, [M–Cl]þ and [M–2Cl]þ ions were always predomi-
nant (Fig. 1). Similar mass spectral fragmentation patterns of
PCBs in a homologue group were previously reported for
electron impact (EI), NCI, and ion trap MS/MS mode.20,48
Having more abundant [M]þ ions than [M–Cl]þ and [M–
2Cl]þ in full-scan EI mode suggests that PCNs and PCBs
are non-dissociative.48 The impact energy of electrons onto
analyte molecules is not high enough to significantly dissoci-
ate the molecular ions. Generation of the predominant [M–
2Cl]þ ions from PCBs and PCNs in the MS/MS process sug-
gests the potential of high sensitivity and low detection limits
in CID mode for real world samples, which agrees with pre-
vious observation for PCBs.20,49
The relative abundance of [M]þ, [M–Cl]þ and [M–2Cl]þ
ions in MS/MS spectra varied with EA. For example, the
relative abundance of the parent ion (m/z 300 [M]þ) of CN 61
(PentaCN) decreased with increasing EA, and that of the two
daughter ions (m/z 263 þ 265 [M–Cl]þ and 228þ230 [M–
2Cl]þ) was enhanced with increasing EA (Fig. 2). When EA
was increased to 2.25 V, m/z 228þ230 ions became the most
sensitive daughter ions. Therefore, the parent (m/z 300) and
daughter ions (m/z 228 þ 230) were used to identify and
quantify all PentaCN congeners. All selected parent ions,
daughter ions and optimum EA values for all OCPs, PCBs
and PCNs are summarized in Table 1. For most of these OCs,
ET and IT of 5 ms and 7 ms, respectively, were found to give
good S/N ratios.
After the chromatogram had been sliced into different
retention time segments, all well-separated homologue
congeners in the same segment were identified and measured
in MS/MS mode, while 13C-PCB standards and heterologous
congeners in one segment were analyzed in multiple reaction
monitoring (MRM) mode. MRM enables MS/MS to identify
Rapid Commun. Mass Spectrom. 2005; 19: 1815–1821
1818 D. Wang et al.

196 B 264
A
231

Rel. int.
Rel. int
297
264
159 332
192 229

150 175 200 225 250 275 175 225 275 325
m/z m/z

Figure 1. MS/MS spectra of CN 47 (A) and CN 71 (B), which represent typical MS/MS spectra
of the corresponding Tetra-PCN and Hexa-PCN homologues.

300
263+265
228+230
Rel. int.

0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

Excitation voltage (V)

Figure 2. Variation of relative intensity of the parent ion (m/z 300) and two daughter ions (m/z
263 þ 265 and 228 þ 230) of CN 61 (a PentaCN congener) with increasing excitation voltage.

and quantify multiple and coeluted analytes including Table 1. Optimized ion trap MS/MS parameters for the
isotopically labeled internal standards. After the parameters analysis of OCPs, PCBs and PCNs
had been optimized, excellent linear calibration curves were
Compounds Parent ions Daughter ions ESL EA (V)
obtained in a range of 0.5–2000 ng/mL of each OCP, 0.5–
1000 ng/mL of each PCB, and 100–5000 ng/mL of Halowax BHC 219 183 þ 183 54.1 0.30
1014, with correlation coefficients of 0.98–1.00. The limits of Heptachlor 272 235 þ 237 89.9 0.90
detection (LODs), defined as the minimum amount of target pp0 -DDE 246 176 108.4 0.90
pp0 -DDD, pp0 -DDT 235 163 þ 165 90.6 0.70
analytes producing a chromatographic peak with a S/N ratio
DiCB 222 152 73.2 1.60
of 3, were 0.5–13 pg for OCPs, PCBs and PCNs. TriCB 256 186 þ 188 112.8 1.60
13
C-PCB28 268 196 þ 198 112.8 1.60
Method performance evaluation TetraCB 292 220 þ 222 128.8 1.80
OCPs, PCBs and PCNs were well resolved and detected with PentaCB 326 254 þ 256 152.8 2.60
13
C-PCB123 338 266 þ 268 152.8 2.60
the optimized GC/MS/MS method. The average recoveries
HexaCB 360 288 þ 290 168.8 2.60
varied from 59–84% (RSDs, 3–14%) for OCPs spiked in the 13
C-PCB169 372 300 þ 302 168.8 2.60
lard samples and from 45–86% (RSDs, 2–8%) in the chicken HeptaCB 394 322 þ 324 173.9 2.60
13
heart samples (Table 2). The average recoveries were 64– C-PCB170 406 334 þ 336 173.9 2.60
125% (RSDs, 3–24%) for non-coplanar PCBs spiked in the OctaCB 430 358 þ 360 189.8 2.60
NanoCB 464 392 þ 394 204.8 2.60
lard samples and 52–123% (RSDs, 4–29%) in the chicken
DecaCB 498 426 þ 428 219.9 2.60
heart samples (Table 2). The recoveries of coplanar PCBs DiCN 196 126 97.1 1.75
(PCBs 77, 81, 126, and 169) averaged 64–137% (RSDs, 16– TriCN 230 160 þ 162 114.1 1.25
24%) from the lard samples and 67–123% (RSDs, 6–7%) TetraCN 266 194 þ 196 132.0 1.25
from the chicken heart samples. The average recoveries of PentaCN 300 228 þ 230 148.9 2.25
HexaCN 334 262 þ 264 161.8 2.25
PCNs were 36–130 % (RSDs, 7–29%) from the lard samples
HeptaCN 368 296 þ 298 182.7 2.25
and 79–152% (RSDs, 7–23%) in the chicken heart samples

Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2005; 19: 1815–1821
 GC/MS analysis of organochlorines in harbor seals 1819

Table 2. Mean recoveries of PCBs and OCPs from spiked Table 3. Mean recoveries of PCNs from spiked lard (n ¼ 4)
lard (n ¼ 4) and chicken heart samples (n ¼ 7) and limits of and chicken heart samples (n ¼ 7) and limits of detection
detection (LODs) of PCBs and OCPs in chicken heart (LODs) of PCNs in chicken heart samples (n ¼ 7)
samples (n ¼ 7) Lard Chicken heart
Larda Chicken heartb
Spiked Spiked
Recovery RSD Recovery RSD LOD level Recovery RSD level Recovery RSD LOD
Compound (%) (%) (%) (%) (pg/g ) Compounds (ng/g) (%) (%) (ng/g) (%) (%) (pg/g)
13
a-BHC 67 5 45 5 0.8 C-PCB169 2.5 99 20 1.0 81 13
b-BHC 69 3 64 8 1.9 CN19 0.3 36 7 0.06 116 19 0.5
g-BHC 63 3 54 4 0.8 CN24/14 2.9 37 9 0.59 128 15 4.3
d-BHC 84 8 80 2 0.7 CN23 1.3 40 9 0.27 134 14 1.8
Heptachlor 59 9 54 6 1.2 CN42 0.2 45 10 0.03 131 14 0.6
pp0 -DDE 71 10 66 7 1.7 CN33/34/37 1.7 42 12 0.33 136 12 2.0
pp0 -DDD 95 4 86 5 1.7 CN47 0.6 48 10 0.13 133 14 0.9
pp0 -DDT 65 14 63 5 1.1 CN28/43 0.6 44 12 0.13 143 10 0.7
Mean for OCPs 72 7 64 5 CN35 0.7 45 11 0.15 138 12 0.9
PCB8 87 3 101 4 1.6 CN38 1.9 49 11 0.38 138 9 1.8
PCB18 86 5 101 6 2.3 CN46 1.0 48 13 0.19 145 16 1.7
PCB28 83 9 100 11 4.5 CN52/60 1.8 51 10 0.36 142 16 3.0
13
C-PCB28 68 13 104 19 7.9 CN61 1.9 52 8 0.38 125 9 1.6
PCB52 103 8 104 6 2.5 CN50 0.5 73 29 0.10 125 17 0.8
PCB44 119 7 102 4 1.5 CN57 2.8 76 13 0.56 124 15 3.8
PCB66 103 5 88 7 2.9 CN62 3.2 79 10 0.65 125 20 6.0
PCB81 64 18 88 7 2.3 CN53 2.4 68 8 0.48 128 11 2.5
PCB77 65 17 111 7 2.8 CN59 4.2 67 12 0.84 114 7 2.6
PCB101 105 8 88 4 1.6 CN66/67 0.5 80 10 0.09 114 23 0.9
PCB123 87 7 119 16 7.5 CN64/68 1.5 99 10 0.30 152 15 2.6
13
C-PCB123 74 10 92 18 6.7 CN69 3.5 130 17 0.71 79 22 4.6
PCB118 99 7 123 19 8.9 CN71/72 6.7 62 8 1.33 92 22 10.1
PCB114 101 3 101 5 2.2 CN63 1.7 100 23 0.33 127 17 2.7
PCB105 110 5 92 10 3.4 CN65 2.4 86 8 0.49 104 7 1.3
PCB153 96 7 101 5 2.2 CN73/74 2.4 119 7 0.48 90 18 2.9
PCB138 100 9 92 7 2.3 Mean 67 12 123 15
PCB126 137 16 123 6 2.9
PCB128 96 6 88 6 2.5
PCB167 102 15 76 10 2.5 background interferences from this tissue. The MDLs of
PCB156 108 3 77 6 4.6
OCPs, PCBs and PCNs were 0.7–1.9, 1.5–8.9 and 0.5–
PCB157 89 12 55 29 6.3
PCB169 108 24 67 6 1.6
10 pg/g dry weight (dw), respectively (Tables 2 and 3).
PCB187 87 9 100 5 2.1 Although only 13C-PCB surrogates were used to quantify
PCB180 97 6 79 16 4.8 all selected OCs in this study, the precision and accuracy of
PCB170 125 10 76 10 2.6 this method have met the requirements of environmental
13
C-PCB170 79 16 75 17 4.8 trace analysis.23
PCB189 111 11 57 21 4.6
PCB195 101 9 52 19 3.5
PCB206 125 9 68 17 4.3
PCB209 102 5 80 13 3.7 Analysis of harbor seal samples
Mean for PCBs 97 9.4 90 11 This method has been used for the analysis of OCs in harbor
a
The fortification level was 10 ng/g dw. seal tissues collected from subsistence hunters in Alaska. The
b
The fortification level was 5 ng/g dw. samples were processed according to the extraction and
clean-up procedures described above. GC/MS/MS chroma-
tograms of PCBs, OCPs and PCNs in the two fractions col-
(Table 3). In general, the mean recoveries and RSDs of PCB lected from activated carbon/silica gel columns show that
analysis were better than those of OCPs in spiked lard and all OCPs, PCBs and PCNs were sliced into appropriate seg-
chicken heart samples. Good recoveries of some PCNs in ments (Fig. 3). Individual OCs in each segment were well
spiked chicken heart samples were also found. A general separated and could be accurately detected. This method is
trend of lower and higher recoveries of OCPs (64–72%) and validated as a reliable and valuable technique for monitoring
PCNs (67–123%), respectively, relative to those of PCBs (90– of OCs in harbor seals. Table 4 shows lipid contents and the
97%) may be related to the use of 13C-PCB surrogate stan- total concentrations (S) of different classes of OCs detected in
dards only. The 13C-PCB surrogates may behave differently the harbor seal tissues. The lipid contents were 12–28% in
from OCPs and PCNs in the analytical process, which may kidney and liver tissues, and 77–99% in blubber samples.
cause errors for OCP and PCN recovery quantitation. High Such high lipid contents extracted and detected in blubber
recoveries of PCNs (123%) from chicken heart samples may tissues suggest that the ASE method in this study can effec-
also relate to their low fortification levels (0.03–1.33 ng/g of tively extract lipids from the tissues. In all the tissues, PCBs,
each PCN) as compared to those of PCBs (5–10 ng/g) and and pp0 -DDT, pp0 -DDE and pp0 -DDD were the predominant

Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2005; 19: 1815–1821
1820 D. Wang et al.
pp’-DDE PCB52
pp’-DDT

Rel. int

Rel int
pp’-DDD
α-BHC β-BHC heptachlor PCB28
PCB66
γ-BHC PCB8 PCB44
PCB18

20.0 22.5 25.0 27.5 30.0 32.5 35.0 20.0 22.5 25 0 27 5 30.0
PCB107 PCB180
PCB101

Rel.int
PCB114

PCB118
Rel. int

PCB105
PCB170

30.0 32.5 35.0 37.5 40.0 35 36 37 38 39 40 41

PCB153 CN24/14

Rel. int
PCB138 CN23
Rel. int

PCB128
PCB156/157
PCB167 CN19

32 33 34 35 36 37 38 39 25 26 27 28 29
CN38
CN33/34/3 PCB77
Rel. int

CN46
Rel. int

CN28

CN 47

CN42

29 30 31 32 33 29 30 31 32 33 34
CN61
PCB126
CN59 CN53
Rel. int
Rel. int

CN52/60 CN57

39.5 40.0 40.5 41.0 41.5 42.0 34 35 36 37 38 39

RT (min) RT (min)

Figure 3. GC/MS/MS chromatograms of OCPs, PCBs and PCNs in the extract of a harbor seal liver
(Sample ID: 18855) (Rel. int, relative intensity; RT, retention time).

Table 4. Concentrations (ng/g dry weight) of OCs in harbor seal tissues.

Total concentration (S) of different classes of OCsa
Sample
type ID Lipid % PCBs DDTsb BHCsc PCNs Heptachlor
d
Liver 18855 16 114 54 6.4 0.25 0.15
18901d 16 18 24 0.66 0.28 0.085
18902d 17 64 23 1.2 0.12 0.096
Kidney 18907e 28 19 13 3.9 0.34 0.16
18908d 12 9.9 5.3 0.74 0.18 0.065
47021e 18 13 6.2 1.6 0.17 0.054
Blubber 47026e 99 90 88 6.6 1.9 0.63
47027e 77 98 173 7.5 1.2 0.38
47033f 99  0.3 220  62 220  54 4.6  2.7 2.5  0.80 0.78  0.37
a
Specific OCs detected are shown in Table 2.
b
DDTs include pp,-DDT, pp0 -DDE and pp0 -DDD.
c
BHCs include a-BHC, b-BHC, g-BHC and
-BHC isomers.
d
Average values of two replicates due to limited amount of samples available.
e
Values of single analysis due to limited amount of samples available.
f
Average values of three replicates  standard deviations.

Copyright # 2005 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2005; 19: 1815–1821

pollutant groups, while BHCs, heptachlor and PCNs were
the minor pollutants.
CONCLUSIONS
The ASE procedure offers good recoveries of the OCPs, PCBs
and PCNs. The clean-up and fractionation procedures allow
the clean-up and concentration of the sample extracts to low
volumes to enhance MDLs for the analysis of lower concen-
trations of the PCNs and coplanar PCBs. The GC/MS/MS
method resolved all the analytes selectively and sensitively
using isotopic dilution techniques. The precision and accu-
racy of the described procedure met the requirements speci-
fied by the US EPA for environmental trace analysis.23 The
analysis of the harbor seal blubber, liver and kidney samples
shows the applicability of the described method for the ana-
lysis of OCs in free-ranging mammals that are hunted for sub-
sistence use.
Acknowledgements
The harbor seal tissue samples were collected with the help
of the Alaska Native Harbor Seal Commission Biosampling
Program. This study was supported by the Alaska SeaLife
Center Harbor Seal Research Program, with funds from the
National Marine Fisheries Service and Exxon Valdez Oil Spill
Trustee Council (grant to SA).
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