Simultaneous extraction and clean-up of polychlorinated biphenyls and their metabolites from small tissue samples using pressurized liquid extraction

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Journal of Chromatography A, 1214 (2008) 37–46

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Journal of Chromatography A

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Simultaneous extraction and clean-up of polychlorinated biphenyls and theirmetabolites from small tissue samples using pressurized liquid extraction

Izabela Kania-Korwela, Hongxia Zhaoa,b, Karin Norstromc, Xueshu Lia,Keri C. Hornbucklec, Hans-Joachim Lehmlera,∗

a Department of Occupational and Environmental Health, Collage of Public Health, University of Iowa, Iowa City, IA 52242, USAb Key Laboratory of Industrial Ecology and Environmental Engineering, MOE, Dalian University of Technology, Dalian 116024, Chinac Department of Civil and Environmental Engineering, University of Iowa, Iowa City, IA 52242, USA

a r t i c l e i n f o

Article history:Received 16 September 2008Received in revised form 21 October 2008Accepted 24 October 2008Available online 31 October 2008

a b s t r a c t

A pressurized liquid extraction-based method for the simultaneous extraction and in situ clean-up ofpolychlorinated biphenyls (PCBs), hydroxylated (OH)-PCBs and methylsulfonyl (MeSO2)-PCBs from small(<0.5 g) tissue samples was developed and validated. Extraction of a laboratory reference material withhexane–dichloromethane–methanol (48:43:9, v/v) and Florisil as fat retainer allowed an efficient recoveryof PCBs (78–112%; RSD: 13–37%), OH-PCBs (46 ± 2%; RSD: 4%) and MeSO2-PCBs (89 ± 21%; RSD: 24%).Comparable results were obtained with an established analysis method for PCBs, OH-PCBs and MeSO2-

Keywords:Polychlorinated biphenyls (PCBs)Methylsulfonated biphenyls (MeSO2-PCBs)HP

PCBs.© 2008 Elsevier B.V. All rights reserved.

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ydroxylated biphenyls (OH-PCBs)ressurized liquid extraction (PLE)

. Introduction

Polychlorinated biphenyls (PCBs) are an important class of per-istent environmental contaminants with the general structure12HnCl10−n (n = 1–10). They were initially manufactured by batchhlorination of biphenyl. The resulting mixtures had desirablehysicochemical properties, such as high chemical and thermaltability and high dielectric constants, and were used in a largeumber of technical and consumer applications. Their productionas phased out in the United States in the 1970s due to environ-ental and public health concerns; however, the US Environmental

rotection Agency still permits the use of PCBs in enclosed sys-ems, such as transformers and capacitors. Significant quantities ofCBs have been released into the environment and transported toemote areas because of their semi-volatile character, thus result-ng in worldwide contamination [1]. PCBs can bioaccumulate and

iomagnify in the food web, which has resulted in concern aboutuman exposures and adverse human health effect [2].

PCBs can be metabolized by cytochrome P-450 enzyme sys-ems to hydroxylated PCB derivatives (polychlorinated biphenylols,

∗ Corresponding author at: Department of Occupational and Environmentalealth, Collage of Public Health, University of Iowa, 100 Oakdale Campus, #221

REH, Iowa City, IA 52242, USA. Tel.: +1 319 3354211; fax: +1 319 3354290.E-mail address: [email protected] (H.-J. Lehmler).

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021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2008.10.089

H-PCBs) [3]. This oxidation can occur either via a PCB epoxider by direct insertion of oxygen into a CAr–H bond. Alternatively,he PCB epoxide can react with glutathione, and the resulting glu-athione adduct is converted via the mercapturic acid pathway to

ethylsulfonyl derivatives (MeSO2-PCBs) [3]. These metabolitesre retained in both humans and animals, for example in blood4–7] and in liver and lung [3,8–11]. Recent studies have shownhat the levels of MeSO2-PCBs and OH-PCBs are similar or evenigher than the levels of the parent compounds [3]. These find-

ngs suggest that, in addition to PCBs themselves, their metabolitesay play an important, but frequently overlooked role in adverse

uman health effects. Therefore, laboratory as well as epidemio-ogical studies should assess tissue and/or serum levels of PCBsnd their metabolites. Unfortunately, the analysis of PCBs and theiretabolites in blood and tissue samples involves complex and time

nd solvent consuming extraction, separation and clean-up steps12].

Pressurized liquid extraction (PLE) is an established samplereparation technique for the extraction of PCBs and many otherompounds from a variety of environmental samples including sed-ments, plants and tissues [13–17]. PLE combines both elevated

emperatures and high pressures to achieve a fast, efficient andighly automated extraction of the analytes from a solid matrix.he high temperature (typically 100 ◦C) used during PLE facil-tates the distribution of the solvent into the matrix due to aecrease in solvent viscosity, assists in disrupting analyte–matrix

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8 I. Kania-Korwel et al. / J. Ch

nteractions and increases both solubility and mass transfer [18].he use of pressures between 6.9 and 17.2 MPa (1000–2500 psi)llows extractions at temperatures above the boiling point of thextraction solvent and facilitates the isolation of the analyte. Inddition to these advantages, PLE reduces the extraction time,llows the automatization of extraction procedures and requiresower solvent volumes compared to traditional Soxhlet extrac-ions.

PLE not only allows an efficient extraction of analytes fromvariety of matrices, but also the in situ (or in cell) clean-up

f the extract [14,19–26]. This significantly reduces the need forxhaustive post-clean-up procedures, such as column and/or gel-ermeation chromatography, and allows the automatization oflean-up steps. Especially the post-extraction removal of lipidsnd other co-extractable materials represents a challenge forCB and PCB metabolite analyses. In situ removal of these co-xtractable materials can be achieved by placing a fat retainer,uch as Florisil or silica gel, in the bottom of the PLE cell. Thisrevents the elution of co-extractable materials from the PLEell.

The present study describes the development of a PLE methodhat allows the extraction and in situ clean-up of PCBs, MeSO2-PCBsnd OH-PCBs from small tissue samples using PLE.

. Experimental

.1. Chemicals

Florisil (60–100 mesh), silica gel (70–230 mesh), absolutethanol (200 proof, 99.5%), dimethylsulfoxide (anhydrous, 99.9%),ydrochloric acid, potassium hydroxide, sodium sulfite, sulfuriccid, tetrabutylammonium sulfite and pesticide grade solventsere purchased from Fisher Scientific (Pittsburg, PA, US). Alumina

acidic, about 150 mesh) was purchased from Sigma–Aldrich (Stouis, MO, US). Diatomaceous earth (DE) was obtained from DionexSunnyvale, CA, US).

3,5-Dichlorobiphenyl (PCB 14), 2,4,6-trichlorobiphenylPCB 30), 2,3,5,6-tetrachlorobiphenyl (PCB 65), 2,3,4,5,6,4′-exachlorobiphenyl (PCB 166), 2,3,4,5,6,2′,4′,6′-octachlorobiphenylPCB 204), 2,5,2′,5′-tetrachloro-3-methanesulfonyl-biphenyl (3-

eSO2-CB 52), 2,5,2′,5′-tetrachloro-4-methanesulfonylbiphenyl4-MeSO2-CB 52), 2,3,4,2′,5′-pentachloro-4′-methanesulfonyl-iphenyl (4′-MeSO2-CB 87), 2,3,6,2′,4′-pentachloro-4-methane-ulfonyl-biphenyl (4-MeSO2-CB 91), 2,3,6,2′,4′-pentachloro-5-ethanesulfonyl-biphenyl (5-MeSO2-CB 91), 2,5,2′,3′,6′-penta-

hloro-3-methanesulfonylbiphenyl (3′-MeSO2-CB 95), 2,3,6,2′,5′-entachloro-4′-methanesulfonyl-biphenyl (4′-MeSO2-CB 95),,3,5,2′,5′-pentachloro-4′-methanesulfonyl-biphenyl (4′-MeSO2-B 101), 2,3,6,2′,3′,4′-hexachloro-4-methanesulfonyl-biphenyl4′-MeSO2-CB 132), 2,3,6,2′,3′,4′-hexachloro-5-methanesulfonyl-iphenyl (5′-MeSO2-CB 132), 2,3,6,2′,4′,5′-hexachloro-4-methane-ulfonyl-biphenyl (4-MeSO2-CB 149), 2,5,6,2′,4′,5′-hexachloro--methanesulfonyl-biphenyl (5-MeSO2-CB 149), 3,5,2′,3′,4′,5′-exachloro-biphenyl-4-ol (4′-OH-CB 159), 2,3,4,5,2′,3′,6′-eptachloro-4′-methanesulfonyl-biphenyl (4′-MeSO2-CB 174),,3,4,5,2′,3′,6′-heptachloro-5′-methanesulfonyl-biphenyl (5′-eSO2-CB 174) were purchased from Accustandard (New Haven,

T, US). 2,3,5,3′,4′-Pentachloro-4-methoxy-biphenyl (4-MeO-CB07), 2,4,5,2′,3′,4′-hexachloro-3-methoxy-biphenyl (3′-MeO-

B 138),2,3,5,2′,4′,5′-hexachloro-4-methoxy-biphenyl (4-MeO-CB46) and 2,3,5,6,2′,4′,5′-heptachloro-4-methoxy-biphenyl (4-MeO-B 187) were purchased from Wellington Laboratories (Guelph,ntario, Canada). 2,3,4,5,3′-Pentachloro-5′-methanesulfonyl-′-methyl-biphenyl (4′-Me-5′-MeSO2-CB 106) was purchased

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togr. A 1214 (2008) 37–46

rom Cambridge Isotope Laboratories (Andover, MA, US).,4,2′-Trichlorobiphenyl (PCB 28), 2,5,2′,5′-tetrachlorobiphenylPCB 52), 3,4,3′,4′-tetrachlorobiphenyl (PCB 77), 2,4,5,2′,4′,5′-exachlorobiphenyl (PCB 153), 4,5,3′,4′-tetrachloro-biphenyl-3-ol5-OH-CB 77) and 4,5,3′,4′-tetrachloro-biphenyl-2-ol (6-OH-CB7) were synthesized in our laboratory with >99% purity [27]. PCBongeners were numbered according to the corrected Ballschmiternd Zell system [28] and the metabolites are abbreviated asescribed by Maervoet et al. [29]. The chemical structure andhe concentration of the stock solutions of all compounds areummarized in Tables S1 to S3 in the Supplementary Material.

Diazomethane was synthesized from N-methyl-N-nitroso-p-oluenesulfonamide (Diazald) using an Aldrich mini Diazaldpparatus (Milwaukee, WI, US) following established procedures30]. CAUTION: Because of the highly toxic and explosive nature ofiazomethane, its preparation and use should be carried out in anfficient chemical fume hood and behind a safety shield. All glass tub-ng used to handle diazomethane solutions should have fire polishednds.

.2. Method development

.2.1. Pressurized liquid extraction of PCBs and their metabolitesA pressurized liquid extraction system ASE 200 (Dionex, Sun-

yvale, CA, US) was used for the combined extraction and in situlean-up of PCBs and their MeSO2- and OH-metabolites. In short,LE cells containing 10 g of fat retainer (Florisil or silica gel) and 2 gf diatomaceous earth were pre-extracted to avoid cell memoryffects. In case of tissue extractions, the pre-extracted diatoma-eous earth was removed from the PLE cell, thoroughly mixed withpproximately 0.3 g of liver from non-PCB exposed rats and placedack into the PLE cell. The PLE cells were then spiked with a mix-ure of standards (either PCB, OH-PCB or MeSO2-PCB standardslone or a mixture of all three standards) and extracted twice usinghe same extraction condition employed for the pre-extraction. Thextraction temperature, pressure and length of cycle were changedystematically as described under Section 3. All results are summa-ized in Figures S1 to S7 in the Supplementary Material. A heatingime of 6 min and a 60% cell volume flush were used for everyxtraction. Both parameters do not affect the extraction efficiencyf PCBs and are frequently used for PLE of environmental samples18].

.2.2. Separation of PCBs and metabolitesTissue extracts were separated into PCBs, OH-PCBs and MeSO2-

CBs using their different physicochemical properties based on therocedure described by Hovander et al. [5,31] (Fig. 1). In short, thehenolic fraction containing the OH-PCBs was separated from theeutral fraction containing PCBs and MeSO2-PCBs by partitioning

nto an aqueous-ethanolic solution of potassium hydroxide (2 ml,.5 M, 50% ethanol). The phenolic fraction was then acidified withydrochloric acid (0.5 ml of 2 M aqueous solution) and the OH-PCBsere extracted twice with hexane-MTBE (3 ml, 9:1, v/v). The OH-

CBs were derivatized with diazomethane in diethyl ether (0.5 ml,bout 5 mmol) to form MeO-PCBs. The MeSO2-PCBs were separatedrom PCBs by partitioning into anhydrous DMSO (0.5 ml, tracesf water may prevent the partitioning). Water (1 ml) was addedo the MeSO2-PCB fraction in DMSO and the metabolites were

silica gel column (1 g) and eluted with dichloromethane (15 ml).he dichloromethane was exchanged to hexane prior to GC analy-is. Traces of sulfur containing impurities were removed from theCB and MeO-PCB-containing extracts according to standard EPArotocols [32,33].

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ig. 1. Scheme of separation of PCBs, MeSO2-PCBs and OH-PCBs using PLE. Additiondotted border.

.2.3. Gas chromatographic analysis (GC-ECD)All gas chromatographic analyses were performed on an Agilent

890N gas chromatograph equipped with a 63Ni �-ECD (electronapture) detector. A SLB5MS column was used (60 m × 0.25 mmD; 0.25 �m film thickness; Supelco, Bellafonte, PA, US). Detec-or and injector temperatures were 280 ◦C and 300 ◦C respectively.CBs, MeO-PCBs (derivatized OH-PCBs) and MeSO2-PCBs werenalyzed using temperature program 1, 2 and 3, respectively (tem-erature program 1: 80 ◦C for 1 min, 25◦/min to 280 ◦C, hold for5 min; temperature program 2: 100 ◦C hold for 1 min, 25◦/min to50 ◦C, 1◦/min to 280 ◦C, hold for 20 min; temperature program 3:0 ◦C for 2 min, 10◦/min to 280 ◦C, than 1◦/min to 300 ◦C, hold formin). The internal standard (same for all three groups of com-ounds) consisting of PCB 30 and PCB 204, was added to eachample before the GC analysis [32,33]. PCB 30 and PCB 204 weresed to quantify PCBs, whereas PCB 204 was used to quantifyhe MeSO2-PCBs and MeO-PCBs. The detector was linear over thentire concentration range under investigation (Table S4, Supple-entary Material).

.3. Method validation

.3.1. Preparation of laboratory reference materialsThe PLE-based method was validated by comparison with the

stablished extraction method described by Jensen et al. [34,35]sing tissues from PCB-treated rats as laboratory reference materi-ls. The animal experiments used to generate these tissue samplesere approved by the Institutional Animal Care and Use Committee

t University of Iowa.

2

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raction and clean-up steps required by the Jensen method are shown in boxes with

Rat liver tissue samples were prepared by administering annvironmentally relevant PCB mixture to rats. In short, six maleprague–Dawley rats, 4 weeks old, average weight 98 ± 8 g wereurchased from Harlan (Indianapolis, IN, US). The animals werellowed to acclimatize for 1 week. Five randomly selected animalseceived a single oral dose of 20 mg/kg body weight of a mixturef Aroclor 1242 and Aroclor 1254 (65:35, w/w) in corn oil (5 ml/kgody weight). The profile of this PCB mixture resembles the meanCB profile in Chicago air, as reported by Integrated Atmosphericepostition Network from 1996 to 2002 [36]. The remaining ani-al received a single oral dose of 5 ml/kg body weight of corn oil

nd was used as a sentinel. Animals were euthanized on day 7 bysphyxiation with carbon dioxide followed by cervical dislocation.iver samples were excised en bloc and stored at −20 ◦C.

.3.2. PLE of rat liver tissue (optimized method)Rat liver (0.50 ± 0.01 g) samples were mixed thoroughly with

re-extracted diatomaceous earth (as described above) and placedn the top of 10 g of pre-extracted Florisil. The samples werepiked with surrogate standards PCB 14, PCB 65, PCB 166, 4′-e-5′-MeSO2-CB 106 and 4′-OH-CB 159 and extracted with

exane–dichloromethane–methanol (48:43:9) at 100 ◦C, 1500 psind 1 static cycle of 5 min. Afterwards, extracts were concentratedo dryness, reconstituted in hexane and separated into PCBs, OH-CBs and MeSO2-PCBs as described in Section 2.2.2.

.3.3. Tissue extraction using the “Jensen method”The modified lipid extraction method described by Jensen et

l. [34,35] was scaled down and used to compare the extractionfficiency of both methods for the laboratory reference mate-

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ial. In short, liver (0.56 ± 0.05 g) samples were placed in a 10-mllass tube, weighted and spiked with the surrogate standards asescribed above. The sample was then homogenized twice in 2-ropanol (2.5 ml) and diethyl ether (1 ml) using a homogenizerIKA, Wilmington, NC, US). The organic extracts were combined andlaced over a phosphoric acid solution (5 ml, 0.1 M solution in 0.9%queous sodium chloride). After inverting for 3 min, the organichase was separated, and the aqueous phase was re-extracted withhexane–diethyl ether mixture (1 ml, 9:1, v/v). Afterwards, extractsere concentrated to dryness, reconstituted in hexane and sepa-

ated into PCBs, OH-PCBs and MeSO2-PCBs as outlined above. Sulfurmpurities were removed from the PCB and MeO-PCB fractionss described in Section 2.2.2 [32,33], followed by column chro-atography on an acidified silica gel column (0.5 g of silica gel:

oncentrated H2SO4 = 2:1 (w/w), with 0.1 g silica gel at the bot-om of the column; eluted with 10 ml of dichloromethane). The

eSO2-PCB fraction was purified using a Florisil column (1 g oflorisil, eluted with 10 ml of hexane–acetone 4:1, v/v), followed byn acidified silica gel column as described for PCBs and MeO-PCBs.he GC-ECD analysis was performed as described in Section 2.2.3,ith the exception that temperature program 4 and 5 were used to

nalyze PCBs and MeSO2-PCBs (temperature program 4: 100 ◦C formin, 1◦/min to 240 ◦C, 10◦/min to 280 ◦C, hold for 20 min; temper-ture program 5: 80 ◦C for 2 min, 10◦/min to 280 ◦C, than 1◦/min to00 ◦C, hold for 5 min). Representative chromatograms are shown

n the Supporting Material.

.4. Statistical methods

The data are presented as mean ± standard deviation (SD) ortandard error (SE), where appropriate. Differences between the

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togr. A 1214 (2008) 37–46

Jensen” and the PLE method were analyzed by two sample t-test,= 0.05. R open source statistical software (version 2.6.0, http://ww.r-project.org/index.html) was used for statistical analyses.

. Results and discussion

.1. Study outline

We herein investigate the simultaneous extraction and in situlean-up of PCBs and their metabolites using PLE to facilitate theoncurrent analysis of PCBs and their metabolites in small tissueamples. Based on our previous experience with the extractionf PCBs using PLE [33,37–40], we first investigated the effect ofLE parameters (e.g. length and number of cycles, pressure andemperature), solvents and fat retainers on the recoveries of PCBs,

eSO2-PCBs or OH-PCBs. Subsequently, different fat retainers weretudied for their effectiveness to simultaneously extract and clean-p these three groups of compounds. In this step, spiked liver tissueas subjected to PLE extraction in the presence of a fat retainer,

ollowed by the separation of PCBs, MeSO2-PCBs and OH-PCBs ashown in Fig. 1. Finally, the optimized PLE method was validatedy comparing it to the established method reported by Jensen et al.“Jensen method”) [34].

.1.1. Selection of model compoundsSeveral representative PCBs, MeSO2-PCBs and OH-PCBs were

elected for the method development based on their use as analyti-al standards or their environmental and/or toxicological relevance.CB 14, PCB 65 and PCB 166 were included in our study becausehey are commonly used as surrogate standard for PCB determina-ions [32,33], whereas PCB 28, PCB 52 and PCB 153 were selected as

ecoveries of model compounds (n = 5). Recoveries of (A) PCBs, (B) MeSO2-PCBs and

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mportant components of commercial and environmental PCB mix-ures [32]. 4′-MeSO2-PCB 87 and 4′-MeSO2-PCB 101 were chosens representative MeSO2-PCB metabolites that have been detectedn human tissue samples [4,8,41–43], whereas 3-MeSO2-CB 52nd 4-MeSO2-PCB 52 are metabolites of PCB 52 [44]. 4′-Me-5′-eSO2-CB 106 was included in this study because of its use as a

urrogate standard for the analysis of methylsulfonated compounds8,42,43]. Finally, 5-OH-CB 77 and 6-OH-CB 77 were included in ourtudy because they are hydroxylated metabolites of PCB 77 [45], aCB congener that has been extensively studied in our laboratory40,46].

.1.2. Selection of tissueThe PLE method development outlined herein focused on the

iver. The liver is not only the major site of PCB metabolism, butlso a storage site for PCBs and MeSO2-PCBs, both in laboratorynimal models [10,11] and in wildlife [3]. Although OH-PCBs areostly retained in blood, they have also been found in the liver [9].

.2. Effect of solvent and PLE parameters on recoveries frompiked PLE cells

.2.1. Effect of solventIn general, the choice of solvent is important for the extraction

f environmental contaminants from the matrix of interest. Theevelopment of PLE methods is not different in this regard [13].

he major challenge in the present study was the selection of aolvent system that allows the simultaneous extraction and clean-p of PCBs, MeSO2-PCBs and OH-PCBs. Hexane–acetone (1:1, v/v)47–51], dichloromethane–acetone (1:1) [18,52], dichloromethane48,49,53,54] and hexane–dichloromethane (1:1, v/v) [55,56] were

d(oCm

5, v/v) on recovery of model compounds in PLE (120 ◦C, 10.3 MPa, 1 static cycle of

elected because these solvent systems have been frequentlysed to extract PCBs from abiotic (e.g., sediments) and bioticamples (e.g., fish and mussels) using PLE. The hexane–acetoneixture was also successfully used for the extraction of PCBs

rom tissue samples in our laboratory [33,40]. In addition,ther combinations of the above mentioned solvents, such asexane–dichloromethane–methanol (50:45:5, v/v) were initially

nvestigated. Based on an earlier investigation [57], methanol wasf particular interest as a polar modifier to facilitate the extractionf polar OH-PCBs from fat retainers, such as Florisil.

PLE with all five solvent combinations resulted in comparablyood recoveries of all PCBs from spiked PLE cells (Fig. 2A), with noCBs detectable in the second, independent extraction step. Sim-larly, recoveries of all MeSO2-PCBs were close to 100% for mostolvent combinations investigated (Fig. 2B). The only exceptionas hexane–dichloromethane (1:1, v/v), which had recoveries ofnly 30–50% in the first extraction step. Additional 9–13% of theespective MeSO2-PCBs were extracted during the second, inde-endent extraction step (Fig. S1C and D, Supplementary Material),hich suggests that the extraction of MeSO2-PCBs was not com-lete under these PLE conditions.

The recoveries of the two model OH-PCBs were signifi-antly lower compared to PCBs and MeSO2-PCBs (Fig. 2C)ecause the extraction was incomplete with hexane–acetone1:1, v/v), dichloromethane, hexane–dichloromethane (1:1,/v) and dichloromethane–acetone (1:1, v/v). Only hexane–

ichloromethane–methanol (50:45:5, v/v) gave higher recoveriesi.e., 30% for 5-OH-CB77 and 58% for 6-OH-CB77). Independentf the extraction solvent, recoveries followed the order 5-OH-B77 < 6-OH-CB77, which suggests that the recoveries of OH-PCBsay depend on their chemical structure.

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42 I. Kania-Korwel et al. / J. Chromatogr. A 1214 (2008) 37–46

Fig. 4. The effect of extraction temperature, pressure and duration of extraction cycle on the recoveries of PCBs, MeSO2-PCBs and OH-PCBs. Effect of extraction temperatureo on thet All exa

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n the recoveries of (A) PCBs, (B) MeSO2-PCBs and (C) OH-PCBs. Effect of pressurehe extraction cycle on the recoveries of (G) PCBs, (H) MeSO2-PCBs and (I) OH-PCBs.t 120 ◦C and 10.3 MPa using 1 static cycle of 5 min (n ≥ 2).

Because the hexane–dichloromethane–methanol solvent sys-em gave the best OH-PCB recoveries in all experimentsnvestigating solvents, PLE parameters and fat retainers (see below),

e revisited the role of different amounts of methanol on PCB,eSO2-PCB and OH-PCB recoveries. An increase in methanol con-

ent from 0 to 30% had no effect on the recoveries of PCBsFig. 3A). A methanol content of 5% improved the recovery of

eSO2-PCBs to approximately 100%; however, residue extractionrom the PLE cell was observed with a methanol content of 10%Fig. 3B), followed by decreasing recoveries (80–100%) at methanoloncentrations of 20–30%. Also, an improvement of the recover-es of both model OH-PCBs was observed (Fig. 3C), from 33 to4% at 5% methanol to 48–58% at 30% methanol. Similar recov-ries of OH-PCBs from different animal tissues were reportedreviously by Saito et al. in their PLE method for extraction of

CBs and their metabolites [58]. Ultimately, a methanol con-ent of 10% (hexane–dichloromethane–methanol = 48:43:9, v/v)as selected for the method validation experiments because itrovided optimum recoveries for PCBs, MeSO2-PCBs and OH-CBs.

Pm[la

recoveries of (D) PCBs, (E) MeSO2-PCBs and (F) OH-PCBs. Effect of the duration oftractions were performed with hexane–dichloromethane–methanol (50:45:5, v/v)

.2.2. Effect of PLE parametersIn addition to different solvent systems, several PLE parame-

ers were studied in order to optimize the recovery of selectedarget analytes (Fig. 4). Specifically, temperature (60–120 ◦C),ressure (6.9–13.8 MPa, 1000–2000 psi) and the length ofhe static cycle (1–9 min) were investigated using hexane–ichloromethane–methanol (50:45:5, v/v) as extraction solvent. Inddition, the number of extraction steps required to quantitativelyecover each model compounds was assessed by extracting eachLE cell twice using the same extraction condition.

The extraction temperature is a critical parameter for PLE extrac-ions because it greatly improves the extraction efficiency byncreasing the solubility of the analyte in the solvent, improv-ng mass transfer from the matrix to the solvent and disruptingnalyte–matrix interactions [18]. Several studies have shown that

LE recoveries for PCBs are typically very good. However, the opti-al extraction temperature depends on the nature of the matrix

20,52,59,60]. In the present study, recoveries of PCBs were excel-ent (85–122%) over the entire temperature range investigated andppeared to be independent of the extraction temperature (Fig. 4A).

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ig. 5. The effect of fat retainer on recoveries of model compounds after extracti0.3 MPa, 1 static cycle of 5 min) with and without liver tissue present (n ≥ 2). RecovD) MeSO2-PCBs, with tissue; (E) OH-PCBs, standards only; (F) OH-PCBs, with tissue

CB recoveries above 100% are probably due to minor, co-elutingmpurities present in some sample extracts.

In contrast, the recoveries of MeSO2-PCBs reached a maximumt temperatures ranging from 90 to 110 ◦C (Fig. 4B) and decreasedoth at lower (70–80 ◦C) and higher temperatures (120 ◦C). Thereere some differences in the recoveries of OH-PCBs with increasing

emperature, but there was no obvious trend between 60 and 100 ◦CFig. 4C); however, the recoveries of OH-PCBs decreased slightly at20 ◦C. Overall, an extraction temperature of 100 ◦C appears to beptimal for the extraction of PCBs and their metabolites.

There was no effect of pressure on recoveries of PCBs (Fig. 4D),hich is also in agreement with earlier studies [60,61]. This isot surprising because the primary function of the high pres-ure is to keep the solvent in the liquid state in the PLE cell12,13,61]. However, the lowest (6.9 MPa, 1000 psi) and highest

P[

Pi

th hexane–dichloromethane–methanol mixture (50:45:5, v/v) using PLE (120 ◦C,of (A) PCBs, standards only; (B) PCBs, with tissue; (C) MeSO2-PCBs, standards only;

13.8 MPa, 2000 psi) pressure investigated had a negative effect onhe recoveries of MeSO2-PCBs (Fig. 4E). In both cases, an additional0–20% of the model compounds were recovered with the secondxtraction (i.e., the extraction was incomplete at the lowest andighest pressure investigated). The biggest effect of pressure wasbserved for OH-PCBs (Fig. 4F), with no model compounds recov-red at 6.9 MPa (1000 psi) and 13.8 MPa (2000 psi). Together, theseesults suggest that a pressure of 10.3 MPa (1500 psi) is optimal forhe extraction of PCBs and their metabolites from tissue samples.imilarly, other studies have used pressures ≥1500 psi to extract

CBs and their metabolites from complex environmental matrices18,25,48,49,52,54,55].

Extraction times up to 7 min had no effect on the recoveries ofCBs, MeSO2-PCBs or OH-PCBs (Fig. 4G–I); however, at 9 min anncrease of residue extraction from the PLE cell was observed for

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44 I. Kania-Korwel et al. / J. Chromatogr. A 1214 (2008) 37–46

Table 1Precision and accuracy of PLE method compared to “Jensen method” [34].

Compound PLE method “Jensen method”

Recovery ± SD (%) RSD (%) Recovery ± SD (%) RSD (%)

A. Surrogate standards for PCBs (n = 6)PCB 14 84 ± 31 37 83 ± 18 22PCB 65 112 ± 37 33 119 ± 29 22PCB 166 78 ± 10 13 68 ± 9 13

Compound PLE method “Jensen method”Mean tissue concentration (ng/g) Mean tissue concentration (ng/g)

B. PCB concentrations in liver tissue (n = 5)PCB 28 151 ± 24 148 ± 49PCB 52 24 ± 14 12 ± 3PCB 77 (+PCB 110)a ND NDPCB 91 ND NDPCB 95 254 ± 77 250 ± 92PCB 132 (+105)a ND NDPCB 136 ND NDPCB 149 (+PCB 123)a 26 ± 12 23 ± 11PCB 153 61 ± 24 66 ± 28PCB 174 ND NDPCB 176 (+PCB 137 + PCB130)a 13 ± 5 17 ± 6PCB 183 2.8 ± 1.0 3.2 ± 0.8

Compound PLE method “Jensen method”

Recovery ± SD (%) RSD (%) Recovery ± SD (%) RSD (%)

C. Surrogate standard for OH-PCBs (n = 6)4′-OH-CB159 46 ± 2* 4 52 ± 5 10

Compound PLE method “Jensen method”Mean tissue concentration (ng/g) Mean tissue concentration (ng/g)

D. OH-PCB concentrations in liver tissue (n = 5)4-MeO-CB107 3.70 ± 1.40 4.90 ± 2.103′-MeO-CB138 0.14 ± 0.06** 0.28 ± 0.104-MeO-CB146 ND ND4-MeO-CB187 ND ND

Compound PLE method “Jensen method”

Recovery ± SD (%) RSD (%) Recovery ± SD (%) RSD (%)

E. Surrogate standard for MeSO2-PCB (n = 6)4′-Me-5′-MeSO2-CB 106 89 ± 21 24 103 ± 30 30

Compound PLE method “Jensen method”Mean tissue concentration (ng/g) Mean tissue concentration (ng/g)

F. MeSO2-PCB concentrations in liver tissueb

3-MeSO2-CB 52 ND ND4-MeSO2-CB 52 1.4 0.574′-MeSO2-CB 87 ND ND4-MeSO2-CB91 ND ND5-MeSO2-CB91 4.0 1.23′-MeSO2-CB95 ND ND4′-MeSO2-CB95 ND ND4′-MeSO2-CB 101 ND ND4′-MeSO2-CB132 ND ND5′-MeSO2-CB132 ND ND4-MeSO2-CB149 ND ND5-MeSO2-CB149 0.8 ND4′-MeSO2-CB174 ND ND5′-MeSO2-CB174 ND ND

a The target PCB congeners co-eluted with congeners in parentheses as described in [32].

-test, ˛

-test, ˛

Ptrgi

b Pooled samples.* Significantly different between the PLE and the “Jensen” method. Two sample t

** Significantly different between the PLE and the “Jensen” method. Two sample t

CBs and MeSO2 (recoveries >100%). Overall, a 5-min extractionime appeared to be optimal because it provides good recoveries in aeasonable length of time [13]. Finally, 1 static cycle was found to beenerally sufficient for the extraction of PCBs and their metabolitesn this and other studies [51].

3a

c

= 0.05, P < 0.01.= 0.05, P < 0.05.

.3. Effect of fat retainers on PLE recoveries in the presence andbsence of clean tissue samples

PLE offers to advantage that the extraction of the analytes can beombined with an in situ clean-up step by placing the sample on top

Page 9

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f a suitable fat retainer [19–26]. This approach typically requires aat-to-fat retainer ratio of 0.025 [19,25] and is, therefore, suitable forhe simultaneous extraction and clean-up of small tissue samples<0.5 gram) using 33 ml PLE cells. Here we investigated the effectf Florisil, silica gel, silica gel acidified with concentrated H2SO41:0.66, w/w) and alumina as fat retainers on the recovery of targetnalytes in the presence and absence of rat liver tissue spiked withCBs, MeSO2-PCBs and OH-PCBs. Florisil has been successfully useds fat retainer for the simultaneous extraction and clean-up of PCBsrom animal tissues by PLE in our [33,40] and other laboratories20]. Silica gel, especially acidified silica gel, has been found to ben excellent fat retainer for the clean-up of fish and other foodstuffssing PLE [19,25].

In preliminary experiments, acidified silica gel (but not sil-ca gel alone) caused the degradation of the extraction solventi.e., hexane–dichloromethane–methanol 50:45:5, v/v). Further-

ore, extraction of alumina with the same solvent mixture resultedn yellowish extracts. Similar observations have been reported byjorklund et al. for PLE extractions with alumina [19]. Therefore,nly Florisil and silica gel were systematically investigated as fatetainers.

Recoveries of PCBs in the presence of Florisil and silica gel werexcellent using hexane–dichloromethane–methanol (50:45:5, v/v)s extraction solvent, with no notable difference between bothat retainers (Fig. 5A). Furthermore, PCB recoveries were compa-able when the PLE extraction was performed in the presence orbsence of rat liver tissue (Fig. 5A and B). Similarly, recoveries ofeSO2-PCBs extracted in the presence of Florisil and silica gel were

xcellent and independent of the fat retainer employed (Fig. 5C);owever, recoveries in the presence of rat liver samples duringxtraction were lower for both fat retainers compared to the extrac-ion of MeSO2-PCB standards alone (Fig. 5D).

In contrast to PCBs and MeSO2-PCBs, recoveries of OH-PCB stan-ards were systematically lower (30–45%) during PLE extraction,egardless of the fat retainer employed (Fig. 5E and F). Further-ore, recoveries of OH-PCBs extracted from empty PLE cells or in

he presence of diatomaceous earth, sodium sulfate or sand wereomparably low. These findings suggest that the PLE extraction ofH-PCBs generally gives poor-to-moderate recoveries [31,58,62],robably due to the chemical instability of OH-PCBs.

.4. Method validation

The optimized PLE conditions (i.e., hexane–dichloromethane–ethanol (48:43:9, v/v), 100 ◦C, 1500 psi with a 6-min heating

ime, 1 static cycle of 5 min and a 60% cell volume flush) weremployed to validate the PLE extraction method by verifying itsinearity and repeatability. In addition, the PLE method was com-ared with the “Jensen method” [34], an established method forhe analysis of PCBs and their metabolites in tissue samples. Ashown in Fig. 1, the Jensen method requires more extraction andlean-up steps than the PLE method described herein. Specifically,he Jensen method involves three labor-intensive extraction stepsith different solvent combinations of increasing polarity, the PLEethod only requires a single, automated PLE extraction step that,

s discussed above, combines an extraction and an in situ clean-p step. In addition, the Jensen method requires additional columnlean-up steps for both PCBs and MeSO2-PCBs.

.4.1. Method linearity and repeatability

The linearity of the method was studied over a range of con-

entrations (from 10 to 1000 ng) for selected target analytes, withnd without liver tissue present in the samples. The method wasinear, with correlation coefficients >0.999 for most model com-ounds (Table S5, Supplementary Material). At the same time, the

ogr. A 1214 (2008) 37–46 45

epeatability of recoveries of surrogate standards was assessed withnd without liver tissue present in the PLE cell. The RSD was ≤16%or PCBs (PCB 14, 65 and 166) and 4′-Me-5′-MeSO2-CB 106. Thenly exception was the extraction of 4′-Me-5′-MeSO2-CB 106 inhe absence of liver tissues (RSD = 34%).

.4.2. Comparison with the “Jensen method”Since no Standard Reference Material is certified for PCBs

nd their hydroxylated and methylsulfonyl metabolites, a well-stablished reference method was used to verify the performancef the method. In an initial experiment, rat liver tissue from PCBreated animals was extracted in parallel with the optimized PLE

ethod and the “Jensen method” [34]. Seven PCB congeners (outf 12 congeners analyzed) were detected in the livers of PCBreated rats, with mean tissue concentrations ranging from 2.9 to54 ng/g liver tissue (Table 1). The major OH-PCB detected in the

iver was 4-MeO-CB107. Similarly, Bergman et al. have reportedhat 4-MeO-CB107 is the major OH-PCBs formed in rats treatedith Aroclor 1254 [6]. Small quantities of 3′-MeO-CB138 were

lso detected. The tissue concentrations of all 14 MeSO2-PCBsnalyzed were below the respective detection limit in all sam-les (Table S4 in the Supplementary Material), independent of theethod employed. However, 4-MeSO2-CB 52, 5-MeSO2-CB91 and

-MeSO2-CB149 were detected in pooled extracts from the PLEethod, and 4-MeSO2-CB 52 and 5-MeSO2-CB91 were detected in

xtracts from the “Jensen method”. These metabolites were alsoeported in livers of rats exposed to Clophen A50 [63]. However,n this earlier study the 4-substituted methylsulfonyl metabo-ites of PCB 132 and PCB 149 were preferentially formed over the-substituted metabolites, and the ratio changed with time of expo-ure.

Comparison of the recoveries of surrogates standards as wells selected PCBs revealed no differences between the two methodstwo sample t-test, ˛ = 0.05, P value <0.05; Table 1). This observations not surprising because the recovery of PCBs is typically excellent,ndependent of the extraction and clean-up methods employed.ecovery of the OH-PCB surrogate standard, 4′-OH-PCB159, andhe two OH-PCBs detected in liver samples tended to be slightlyower with the PLE method compared to the “Jensen method”. Thisifference was statistically significant for surrogate standard and′-MeO-CB138. In contrast, mean tissue concentrations of MeSO2-CBs (determined in pooled samples) were higher with the PLEethod compared to the “Jensen method”.

cknowledgments

We thank Dr. Wei Xie, Dr. Sanjay Telu and Mr. Bartlomiejilanowski for their help with the animal studies. The research was

upported by grants ES05605, ES013661 and ES012475 from theational Institute of Environmental Health Sciences, NIH, and Majoresearch Instrumentation grant BES-0420378 from the Nationalcience Foundation.

ppendix A. Supplementary data

Supplementary data associated with this article can be found,n the online version, at doi:10.1016/j.chroma.2008.10.089.

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