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Journal of Chromatography A, 1186 (2008) 161–182

Review

Advances in the gas chromatographic determination of persistentorganic pollutants in the aquatic environment

S.P.J. van Leeuwen ∗, J. de BoerVU University, Institute for Environmental Studies (IVM), De Boelelaan 1085, 1081 HV Amsterdam, The Netherlands

Available online 21 January 2008

bstract

Environmental chemists have been challenged for over 30 years to analyse complex mixtures of halogenated organic pollutants like polychlo-inated biphenyls (PCBs), polychlorinated alkanes (PCAs), polybrominated diphenyl ethers (PBDEs) and polychlorinated dibenzo-p-dioxins andolychlorinated furans (PCDD/Fs). Gas chromatography (GC) often proved to be the method of choice because of its high resolution. The recentevelopments in the field of comprehensive two-dimensional GC (GC × GC) show that this technique can provide much more information thanonventional (single-column) GC. Large volume injection (e.g. by programmed temperature vaporiser, or on-column injection) can be employedor the injection of tens of microliters of sample extract, in that way substantially improving the detection limits. Electron-capture detectionECD) is a sensitive detection method but unambiguous identification is not possible and misidentification easily occurs. Mass spectrometric (MS)etection substantially improves the identification and the better the resolution (as with MS/MS, time-of-flight (TOF) MS and high-resolutionHR)MS), the lower the chances of misidentification are. Unfortunately, this comes only with substantially higher investments and maintenanceosts. Co-extracted lipids, sulphur and other interferences can disturb the GC separation and detection leading to unreliable results. Extraction,nd more so, sample clean-up and fractionation, are crucial steps prior to the GC analysis of these pollutants. Recent developments in samplextraction and clean-up show that selective pressurised liquid extraction (PLE) is an effective and efficient extraction and clean-up technique
hat enables processing of multiple samples in less than 1 h. Quality assurance tools such as interlaboratory studies and reference materials areery well established for PCDD/Fs and PCBs but the improvement of that infrastructure is needed for brominated flame retardants, PCAs andoxaphene. 2008 Elsevier B.V. All rights reserved.
eywords: POPs; Organohalogen compounds; GC; GC × GC; GC/MS; Selective PLE; QA

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1622. Extraction and clean-up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165

2.1. Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1652.1.1. Soxhlet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1652.1.2. Pressurised liquid extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1652.1.3. Microwave-assisted extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1652.1.4. Other extraction techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166

2.2. Clean-up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1662.3. Combined extraction and clean-up by selective pressurised liquid extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168

3. Injection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4. Gas chromatographic separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.1. Column selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4.2. Multidimensional gas chromatography . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author. Tel.: +31 20 59 89 545; fax: +31 20 59 89 553.E-mail address: [email protected] (S.P.J. van Leeuwen).

021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2008.01.044

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173

mailto:[email protected]

dx.doi.org/10.1016/j.chroma.2008.01.044

162 S.P.J. van Leeuwen, J. de Boer / J. Chromatogr. A 1186 (2008) 161–182

5. Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1756. Quality assurance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1777. Conclusions and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179

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. Introduction

Since the 1950s, persistent organic pollutants (POPs) haveeen produced in large volumes. During production, use andisposal, these POPs have entered the environment. The so-alled ‘dirty dozen’ are POPs that are toxic, bioaccumulate inatty tissues of animals and humans and do not easily degrade.hese pollutants are officially registered by the United Nationsnvironmental Programme (UNEP) under the Stockholm Con-ention [1]. They can be sub-divided as (i) eight chlorinatedesticides (dieldrin, endrin, aldrin, chlordane, heptachlor, DDT,irex and toxaphene), (ii) two industrial chemicals (hex-

chlorobenzene (HCB) and polychlorinated biphenyls (PCBs))nd (iii) two unintentionally produced compounds (polychlori-ated dibenzo-p-dioxins (PCDDs)), also abbreviated as dioxins,nd polychlorinated dibenzofurans (PCDFs), also abbreviateds furans) [2]. Although production of most POPs has ceasedor over 20 years, we are still facing considerable POP lev-ls in the environment. Apart from the aforementioned POPs,ther pollutants have been proposed as candidates for additiono the POP list, e.g. hexachlorobutadiene (HCBD), polybromi-ated diphenyl ethers (PBDE: penta, octa and deca technicalixtures), pentachlorobenzene (QCB), polychlorinated naph-

halenes (PCNs), short-chain polychlorinated alkanes (PCAs),icofol and perfluorinated octanoic acid (PFOS). Furthermore,exabromocyclododecane (HBCD) was recently recognised asbioaccumulating substance in a EU risk assessment [3] and a

uture ban on the use of HBCD is not unlikely.Dioxins and furans have never been produced intentionally

or the use in industrial or consumer products or processes.owever, they are generated in waste combustion processes.ther recorded sources are paper production, fuel burning and

s by-products in pesticide/herbicide production [4–6]. Theyave also been produced as undesired by-products in the pro-uction of technical mixtures of PCBs [6]. PCDD/Fs are veryersistent and accumulate in the lipid phase of biota or bindo the organic matter fraction of abiotic samples like sedimentnd soil [4]. Table 1 shows the theoretical number of congenersossible. The 2,3,7,8-substituted dioxins and furans are amonghe most toxic pollutants known. Apart from the PCDD/Fs, 12CBs with a non-ortho or mono-ortho chlorine substitution (so-alled dioxin-like PCBs or dl-PCBs) have a similar toxic modef action. Because of these toxic similarities, 17 PCDD/Fs and2 dl-PCBs were appointed a TCDD (tetraCDD) equivalencyactor (TEF). All 17 dioxins, furans and 12 PCBs have been
ompared to 2,3,7,8-TCDD, the most toxic congener with a TEFf 1. The other congeners are less toxic and therefore receivedTEF lower than 1. The TEFs for humans and mammals were
ecently updated by WHO [7]. Multiplying the concentration of

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congener in a sample with its respective TEF will result in aCDD equivalent (TEQ). Accumulation of all TEQs in a sample,

he sum-TEQ, is obtained. For more information on the individ-al TEFs and the TEF concept, please refer to Van den Bergt al. [7]. The high toxicity of PCDD/Fs and dl-PCBs and theow concentrations in aquatic samples (fg/g–pg/g range) callsor very sensitive, accurate, precise and selective detection tech-iques. Gas chromatography (GC) coupled to high resolutionass spectrometry (HRMS) has served as the ‘golden standard’

or this analysis since the mid-1970s [8].PCBs have been used for a number of decades, e.g. as a

ielectric in transformers and capacitors, as plasticizers and asre resistant liquid in closed systems [9]. PCBs are synthesisedith different chlorination degrees. Although theoretically 209

ongeners are possible (Table 1), the actual number of congenersound in the environment is much lower. PCBs are ubiquitouslyistributed in the (aquatic) environment [9]. The dl-PCBs areiscussed in detail together with the dioxins. The analysis ofhe other PCBs is often limited to a selection of six or sevenBs, the so-called ‘ICES-7’ or ‘indicator PCBs’. This selectiononsists of the CBs 28, 52, 101, 118, 138, 153 and 180 and coverswide range of chlorination degrees (tri- to hepta-chlorination)nd boiling points. Some specialized laboratories analyse 20–40CB congeners.

PCNs have been synthesised from melted naphthalene andhlorine in the presence of a catalyst. The application of PCNss similar to that of PCBs and includes application as dielectricsor flameproofing and insulation in various industries, additiveso rubber products, flame retardant and in lubricants [10]. PCNsre also found as impurities in PCB technical mixtures and cane formed in thermal processes (e.g. solid waste burning) [10].CNs can be potent inducers of ethoxyresorufin-O-deethylaseEROD) and the aryl hydrocarbon (Ah) receptor, and relativeotencies (REPs, relative to TCDD) were derived for some tetra-o hepta-PCN congeners [10–12]. PCNs have been found in thenvironment worldwide, mostly at concentrations lower thanhose of other POPs [13–15].

PCAs have found their application as extreme pressure addi-ives in lubricants and cutting oils, as plasticizers and flameetardants. They were also used as replacements, for exam-le for PCBs [16]. The terminology of chlorinated paraffinsCPs) is commonly used and therefore, in this paper we willse CPs rather than PCAs. Commercial CP products are classi-ed according to their carbon chain length in short-chain CPsSCCPs, C10–C13), medium-chain CPs (MCCPs, C14–C17)
nd long-chain CPs (LCCPs, >C17). CPs are produced by thehlorination of n-paraffin or paraffin wax. Their widespread useas resulted in an ubiquitous distribution in the environment17–19]. Technical CP mixtures are among the most complex

S.P.J. van Leeuwen, J. de Boer / J. Chromatogr. A 1186 (2008) 161–182 163

Table 1Pollutant groups, abbreviations and theoretical number of possible congeners (or isomers) and a selection of trade names

Name Na Production volume Typical trade names of technical mixtures Reference

Polychlorinated dibenzo-p-dioxins (PCDDs) 135 na Na [6]Polychlorinated dibenzofurans (PCDFs) 75 na Na [6]Polychlorinated biphenyls (PCBs) 209 1,000,000 tonnes (ww,

cumulative 1930–1980)Aroclor (1242, 1254 or 1260), Pyranol,Pyroclor, Phenochlor, Pyralene, Clophen,Elaol, Kanechlor, Santotherm, Fenchlor,Apirolio, Sovol

[9,54]

Polychlorinated naphthalenes (PCNs) 75 150,000 metric tonnes(ww)

Halowax (1014, 1051), Nibren wax,Seekay Wax, Clonacire wax, N-oil, N-Wax,Cerifal Matarials

[10]

Polychlorinated alkanes (PCAs, also referred to aschlorinated paraffins or CPs)

Unknown 300,000 tonnes/year (ww,currently)

SCCP: Cereclor 50LV, PCA 60, PCA 70,Witachlor149 and Witachlor 171P,Chlorawax, Chlorafin

[20]

Toxaphene (chlorinated bornanes, CHBs, PCCsb) 32,768 34,200 tonnes (USA in1974)

Alltex, Alltox, Attac 4 2, Attac 4 4, Attac 6,Attac 6 3, Attac 8, Camphechlor,Camphochlor, Camphoclor, ChempheneM5055, chlorinated camphene, Chlorocamphene, Clor chem T 590, Compound3956, Huilex, Kamfochlor, Melipax,Motox, Octachlorocamphene, Penphene,Phenacide, Phenatox, Phenphane,Polychlorocamphene, Strobane T, StrobaneT 90, Texadust, Toxakil, Toxon 63,Toxyphen, Vertac 90%

[54]

Organochlorine pesticides (OCPs) na DDT: 60,000 tonnes (wwin 1974)

DDT: Agritan, Anofex, Arkotine, Azotox,Bosan Supra, Bovidermol,Chlorophenothan, Chloropenothane,Clorophenotoxum, Citox, Clofenotane,Dedelo, Deoval, Detox, Detoxan, Dibovan,Dicophane, Didigam, Didimac, Dodat,Dykol, Estonate, Genitox, Gesafid,Gesapon, Gesarex, Gesarol, Guesapon,Gyron, Havero extra, Ivotan, Ixodex,Kopsol, Mutoxin, Neocid,Parachlorocidum, Pentachlorin, Pentech,PPzeidan, Rudseam, Santobane, Zeidane,Zerdane

[30,32–34,54]

Aldrin + dieldrin13,000 tonnes (ww in1972)

Dieldrin: Dieldrite, Dieldrix, Illoxol,Panoram D 31. ENT 16 225 (compound497), HEOD, Alvit, Octalox, OMS 18,Quintox

Endrin: 2.3–4.5 million kg(sales USA in 1962)

Endrin: Endrex, Experimental Insecticide269, Hexadrin, Nendrin, NCI-COO157,ENT17251, OMS 197, and MendrinAldrin: Aldrec, Aldrex, Aldrex 30, Aldrite,Aldrosol, Altox, Drinox, Seedrin. ENT 15949 (compound 118), HHDN, Octalene,OMS 194

Chlordane: 9.5 million kg(USA in 1974)

Chlordane: Aspon, Belt, CD 68,Chlorindan, Chlorkil, Chlordane, Corodan,Cortilan-neu, Dowchlor, HCS3260,Kypchlor, M140, Niran, Octachlor,Octaterr, Ortho-Klor, Synklor, Tat Chlor 4,Topichlor, Toxichlor, Velsicol-1068

HCB: 10,000 tonnes/year(ww 1978–1981)

HCB: Amaticin, Anticarie, Bunt cure, Buntno more, Co op hexa, Granox, No bunt,Sanocide, Smut go, Sniecotox

Polybrominated diphenyl ethers (PBDEs) 209 Penta: 4000 tonnes (wwin 1994)

Penta-BDE: DE 60FTM, Planelon PB 501,Saytex 125, Bromkal 70 DE, Great LakesDE-60 F (85% PeBDE), Saytex 115,Tardex 50 DE 71; Bromkal 70-5 DE; FR1205/1215; Bromkal 70; Bromkal G1;Pentabromprop

[55–57]

164 S.P.J. van Leeuwen, J. de Boer / J. Chromatogr. A 1186 (2008) 161–182

Table 1 (Continued )

Name Na Production volume Typical trade names of technical mixtures Reference

Hexa-BDE: BR 33NOcta: 6000 tonnes (ww in1992)

Octa-BDE: Bromkal 79-8 DE; DE-79, FR143; Tardex 80; FR 1208; Adine 404;Saytex 111

Deca: 55,100 tonnes(sales in 2001)c

Deca-BDE: FR-300 BA; DE-83-RTM;Saytex 102; Saytex 102E; FR-1210; Adine505; AFR 1021; Berkflam B10E; BR55N;Bromkal 81; Bromkal 82-ODE; Bromkal83-10 DE; Caliban F/R-P 39P; CalibanF/R-P 44; Chemflam 011; DE 83; DP 10F;EB 10FP; EBR 700; Flame CutBR 100; FR300BA; FR P-39; FRP 53; FR-PE;FR-PE(H); Planelon DB 100; Tardex 100;NC-1085; HFO-102; Hexcel PF1;NCI-C55287

Hexabromocyclododecane (HBCD) 10 16,700 tonnes (sales in2001)c

HBCD [58,59]

Tetrabromobisphenol-A (TBBP-A) 1 130,000 tonnes (sales in2002)c

Derakane [60]

Table edited from Ref. [53].a Theoretical no. of congeners. Possible enantiomers not included. Number of congeners does not reflect the number of compounds generally encountered in

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alogenated mixtures encountered in the environment. The car-on chain length may vary (C10–C22) and isomerisation ofhe carbon chain occurs. Furthermore, the different chlorinationegree (30–70%) of the carbon chain leads to numerous possibleollutants [20]. Methods for analysis of CPs are developed forhe determination of either the SCCPs, MCCPs or the LCCPs,nd the focus in recent years has been on SCCPs mainly (proba-ly reflecting the continued production and use) [17,19,21–24].he complex nature of the technical mixtures has challengedeveral scientists trying to obtain accurate data.

Toxaphene is a very complex mixture of chlorinated bor-anes, bornenes, camphenes and dihydrocamphenes with anverage elemental composition of C10H10Cl8 [25]. It consistsf theoretically 32,768 possible congeners (Table 1). It was pro-uced in volumes estimated to be larger than those of PCBsper year) and marketed under a wide variety of trade namesTable 1) [26]. It was used as pesticide on cotton, fruits androps and for controlling ticks and mites on livestock [27]. Sev-ral nomenclature systems were developed in the past (see [26]or an overview), but the system developed by Parlar is mostlysed [28]. In environmental samples a limited number of con-eners, ca. 50–100, are found. Kimmel et al. [29] determinedhat P26, 40, 41, 44, 50 and 62 were the predominant congenersn fish oil.

Organochlorine pesticides (OCPs) are a diverse group ofhlorinated pollutants that have been used as pesticides. Well-nown examples are (see Table 1) DDT, dieldrin, endrin, aldrin,indane, HCB and chlordane. Most OCPs were very effective,
road spectrum pesticides, resulting in extensive use (Table 1).xamples of the insecticide use are on wood and structures
dieldrin, aldrin), crops (chlordane), animals (chlordane, lin-ane), seed and soil treatment (lindane) and protection of

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umans (mainly against malaria, typhus, and certain other vectororne diseases) [30–34]. Hexachlorobenzene (HCB) was useds fungicide mainly [35]. The production of OCPs was dimin-shed in North America, Europe and Japan since the (late) 1970s,ut production may have continued in other regions. The use ofDT in Africa is still supported by WHO as a cost-effective wayf reducing deaths caused by the malaria carrying mosquito [36].

Brominated flame retardants (BFRs) constitute a diverseroup of pollutants that are added to a variety of materialsn order to reduce, delay or even prevent them from catch-ng fire. A substantial part of flame retardants consists ofrominated compounds. The most frequently used BFRs areetrabromobisphenol-A (TBBP-A), hexabromocyclododecaneHBCD) and polybrominated diphenylethers (PBDEs). BFRsre used at relatively high concentrations in various materialsnd polymers, such as polyurethane and polystyrene foams, in aide range of products, such as printed circuit boards, television

ets and computers and other electronic household equipment,ars and construction materials [37]. Information on BFR usagegures (from 2003) can be found elsewhere [38]. BFRs can beeleased into the environment through production, use, and espe-ially from the disposal of the flame-retarded products. VariousFRs are present in biota [39–42] due to their lipophilicity andersistence. Although theoretically, 209 BDE congeners existTable 1), only a subset is commonly found in the environment,nd therefore analysed. This subset consists of the BDEs 28, 47,9, 100, 153, 154, 183 and 209, and maybe ca. 50 other BDEsresent in much lower concentrations. Deca-BDE is predomi-
antly found in sediments but nearly not in aquatic biota [38],lthough Eljarrat et al. [43] found levels up to 707 ng/g lipideight in fish downstream a deca-BDE discharging industrialark.

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S.P.J. van Leeuwen, J. de Boer /

Analytical chemists have been working for over 40 years toevelop a wide range of analytical methodologies for the oftenomplex mixtures of halogenated pollutants, trying to meet thengoing requests from policy makers, risk assessors, and envi-onmental scientists for accurate data on the presence of theseollutants in the environment and humans. Most halogenatedontaminants are relatively volatile, non-polar and thermallytable compounds that can perfectly be determined by GC. Cap-llary GC offers a high number of theoretical plates, resultingn a high resolution. When used in a multidimensional (MD)

ode (heart-cut MDGC or GC × GC), the resolution increasesubstantially. MS detection strongly contributes to the overallelectivity. Several excellent dedicated reviews have been pro-uced in recent years on PCDD/Fs and dl-PCBs [8,44], CPs45,46], PCBs and OCPs [47], toxaphene [26,48], BFRs [49–52]nd PCNs [48]. The aim of this work is to review recent develop-ents in GC methods for halogenated pollutants and to provide

n overview of the applicability of methods for these pollu-ants. In addition to injection, gas chromatographic separationnd detection, attention is being paid to sample pre-treatmentextraction and clean-up), as this is recognised as a critical step inhe whole analytical procedure. Finally, quality assurance issuesre discussed. This review focuses on biota (fish, shellfish andrustaceans) and sediment only, as these matrices have beenuccessfully used for several years to monitor the aquatic envi-onmental exposure to these pollutants. The analysis of theseollutants in water is not considered here, because, due to thextremely low levels of POPs in water significant errors areasily made.

. Extraction and clean-up

.1. Extraction

The determination of target pollutants typically starts withxtracting them from the sample matrix. Halogenated pollu-ants are lipophilic and stored in the body lipids in biota. Inipid-rich biota, the majority of pollutants may be stored in theepot lipids, whereas in lean biota (<1% lipids), the pollutantsre also stored in the phospholipids. By extraction, the pollutantsre liberated from the matrix and made available for further anal-sis. Several parameters influence the extraction efficiency, e.g.hoice of extraction medium (solvents), duration, temperaturef extraction medium, pressure in extraction chamber and theossibility of the solvent to penetrate the matrix. These param-ters should be optimised to exhaustively extract the pollutantsrom the matrix.

.1.1. SoxhletSoxhlet extraction is the classical method for the extrac-

ion of POPs from a variety of matrices. It has widely beensed in the past and still is an important technique disregardinghe appearance of various instrumental extraction techniques.
here are several benefits connected with Soxhlet extraction.ue to the simplicity of the method, no sophisticated (and
xpensive) equipment is needed. The method is simple toperate under routine conditions and multiple samples can be

eeo

romatogr. A 1186 (2008) 161–182 165

xtracted at the same time. The method requires long extractionimes (approximately 6–24 h), but performing the extractionsvernight can circumvent that drawback. Another benefit is thatoxhlet can be employed on a wide variety of matrices and aide range of pollutants such as PCBs, OCPs, PCDD/Fs andFRs. Extraction of lipid-rich materials (mainly triglycerides)ay be performed using a non-polar solvent only (e.g. n-hexane,

-pentane), but lean biological tissues require the use of mediumolar (binary) solvent mixtures (e.g. pentane–dichloromethaneDCM) or hexane–acetone) to extract the POPs from the phos-holipids [61]. de Boer et al. [50] evaluated several binaryolvent mixtures for the extraction of BDEs from fish tissuend sediment and concluded that mixtures of hexane–acetone1:1 or 3:1) were suitable for quantitative extraction of the tar-et analytes. Both sediments and biota need to be dried beforehey are Soxhlet extracted. This can be done by mixing themith sodium sulphate and allowing some drying time (1–2 h)r by freeze-drying (or air drying for sediment). When freeze-rying, attention should be paid to avoid cross-contaminationnd losses of volatile compounds.

.1.2. Pressurised liquid extractionPressurised liquid extraction (PLE; Dionex trade name ASE

or accelerated solvent extraction) has gained considerable inter-st over the last decade. It is a powerful technique and reducesxtraction times. Even more time is saved when extractionnd clean-up are combined in one run within the extractionell (as discussed in Section 2.3). PLE is employed for thextraction of PCBs and OCPs from biota and sediment sam-les [62]. Extraction of PCBs and OCPs from a fish samplehowed that extraction efficiencies and precision of the PLExtraction (hexane–acetone 4:1, three cycles) were similar tooxhlet extraction [62]. Josefsson et al. [63] tested the exhaus-

iveness of a 2× 5 min extraction of PCBs from sediments usinghexane–acetone mixture (1:1). Extraction efficiencies were

6–99% using this approach. They found correlations betweenxtraction efficiency, the water content and the carbon/nitrogenatio, but, surprisingly, no (significant) relation was found withotal organic carbon, soot carbon or amorphous carbon content.LE is increasingly used for the analysis of BFRs with, e.g. DCMr a DCM–hexane (1:1) mixture [64–67]. Using PLE, recoveriesere low (<60%) for the lower (mono to tri) brominated BDEs insh and sediment, but increase up to 103% for the higher bromi-ated ones [65,67]. PLE was also used for PCDD/Fs [68–70].he combination of within-cell extraction and clean-up will beiscussed later. A drawback of PLE is that the cells should beleaned thoroughly to prevent cross contamination. Because theell contains more parts than a typical Soxhlet extraction thim-le, this requires special attention. It is recommended to select aet of cells for highly contaminated samples and another set forow contaminated samples.

.1.3. Microwave-assisted extraction
Microwave-assisted extraction (MAE) is a very simple
xtraction technique. The requirements are the microwavequipment with vessels. This technique allows for simultane-us extraction of several (e.g. 6) samples but requires solvents

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hat can absorb the microwave radiation (due to their dielec-ric nature) such as dichlorobenzene, methanol, ethanol and, to

lesser extent, acetone, ethyl acetate and chloroform. Alter-atively, microwave transformers (e.g. Weflon discs [71]) cane used, which transform the radiation to heat, which is trans-erred to the solvents that poorly absorb microwave radiatione.g. n-hexane, dichloromethane or chloroform). The extractionolvent, temperature and time are typical conditions that requireptimisation. Care should be taken to avoid degradation of labileollutants at the elevated extraction temperatures. MAE wassed in several studies to extract PCBs and OCPs [72–76], PCNs77], PBDEs [77,78], and SCCPs [24] from biota and sediments.lthough MAE should in principle be applicable for PCDD/Fs, a

omprehensive evaluation was not found. Extraction efficienciesnd precision (<5%) were good for PCBs in cod livers [74]. Goodverage recoveries and precision were also obtained (89 ± 8% to5 ± 14%) for the extraction of BDEs 47, 99 and 100 from var-ous fish samples with 8 mL ethyl acetate–cyclohexane mixturet 115 ◦C [78]. These extraction efficiencies were only slightlyower than those of Soxhlet. Yusa et al. [77] optimised con-itions (extraction time, temperature and solvent volume) forhe extraction of PCNs, PBDEs and PBBs from spiked marineediments. At optimum conditions (24 min, 152 ◦C, 48 mL 1:1,/v, hexane–acetone mixture), recoveries were 74–93% with arecision of 4–13%, being comparable to the results of the ref-rence method (Soxhlet). Good extraction efficiencies (>90%)nd run-to-run precision (<10%) were obtained by Perera et al.,ho extracted PCBs and SCCPs from 5 g river sediment samplesing 30 mL hexane–acetone mixture (1:1, 15 min, 115 ◦C) [24].CPs were successfully isolated from oyster samples by MAE

ombined with mild saponification. At optimised conditions,esults were comparable to Soxhlet and no degradation of labileollutants was observed [79]. This shows that MAE is a viablextraction method for most of the POPs and candidate POPs.

.1.4. Other extraction techniquesMatrix solid-phase dispersion (MSPD) is an extraction

echnique in which the sample is dispersed in a solid-phaseaterial of choice (e.g. silica or C18) until a free flowing powder

s obtained. Subsequently, the dispersed material is loaded intosyringe tube. The pollutants are then eluted by, e.g. hexane,

ichloromethane or acetonitrile. MSPD has successfully beenmployed for the extraction of PCBs, OCPs and BFRs in fishamples [80–84]. The benefit of this method is the ease ofperation, low solvent consumption and no investments inexpensive) equipment are required. The application is limitedo fish samples and cannot be applied to sediments (as with, e.g.oxhlet and PLE) due to the strong adsorption of the pollutants

o the sediment, which may be a drawback for laboratoriesiming at both matrices. Recoveries of PCBs in fish samplesere 81–106% [85]. Sample intakes were as low as 0.5 g [85].are should be taken to ensure the homogeneity at such low

ample intake levels.
Supercritical fluid extraction (SFE) has been employed for
he extraction of environmental samples [86–88], but has neveround a broad application. Zougagh et al. [89] recently reviewedhe application of SFE extraction. Benefits of the technique are

olfl

matogr. A 1186 (2008) 161–182

he short extraction times (<1 h) and low solvent consumption<5 mL), but the major drawback is the labour-intensive methodevelopment. Different sample matrices require specific methodevelopment and therefore, contrary to Soxhlet, PLE and MAE,niversal methods cannot be applied.

.2. Clean-up

Clean-up is a very important and critical step in the analysisf halogenated pollutants. The extremely low concentrations ofOPs in environmental samples (e.g. sub-pg/g concentrationsor PCDD/Fs) demand a thorough clean-up of the extracts inrder to remove co-extracted substances (e.g. lipids, fatty acids,lemental sulphur) that are normally present at concentrationshat are several orders of magnitude higher than those of thearget pollutants.

Generally, the crude extract is concentrated by, e.g. rotaryvaporation or a Kuderna Danish method [90,91], in order toemove the excess solvents. We experienced that, of these twohe Kuderna Danish method is the least sensitive for cross con-amination and offers somewhat better recoveries than rotaryvaporation. It also allows more extracts to be handled at theame time with less attention [91]. The first step in the clean-upf biological extracts is the removal of bulk lipids (triglycerides),hich can be performed by destructive or non-destructiveethods. Destructive methods (sulphuric acid treatment or

aponification) efficiently remove the bulk lipids. However,ome pollutants (e.g. dieldrin and endrin) degrade under thetrong acidic conditions. Saponification can cause dechlorina-ion of higher PCBs and HCB [92]. Efficient non-destructiveemoval of lipids can be obtained by the adsorption on alu-ina [44]. Dependent on the desired fat capacity, 2–15 g glass

olumns can be used. Gel permeation chromatography (GPC)ay serve as an alternative fat separation method. The most com-only applied are polystyrene–divinylbenzene copolymeric

olumns (e.g. bio-beads SX-3) [29,77,93], although nowadaysigid PL gels appear to be more efficient [50]. GPC is not capablef removing all lipid-related substances (e.g. sterols) and there-ore, additional clean-up or repeated GPC (e.g. up to four GPColumns in series) is required. Lipids may also be removed byreezing them out the extract and subsequent filtration. This veryimple method allowed for 90% lipid removal from a mackerelxtract [94]. However, residue lipids and fatty acids that remainn the extract require additional clean-up.

For clean-up of sediment extracts, alumina columns can bepplied for the removal of non-volatile co-extractants [95]. Ele-ental sulphur is a major co-extractant from sediments. It should

e removed as it will heavily disturb the GC analysis by a broadeak somewhere half way a regular PCB chromatogram. Sulphurs not removed by alumina or silica gel column chromatographyut can be removed by several other methods, i.e. GPC, reactionith copper (curls, beads, rods, powder) (formation of CuS) ory complexation with tetrabutyl ammonium sulphite [96].

After lipid or sulphur removal, pre-fractionation is carriedut in order to separate the target pollutants from other pol-utants that may interfere during GC separation. Silica gel ororisil columns are frequently used for that purpose, sometimes

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n combination with an additional step for isolation of planarollutants such as PCDD/Fs, dl-PCBs and PCNs. Adsorptionharacteristics of the silica gel can be adjusted by heating theilica gel and subsequent addition of water. Batches preparedn the desired way need to be stored in a desiccator as SiO2 isensitive for moisture present in the air.

PCDD/Fs and dl-PCBs require (additional) clean-up byhe application of porous graphitic carbon [44,70,96,97] or 2-1-pyrenyl)ethyldimethylsilyl (PYE) column chromatography44,97–99] to separate them from the non-planar compoundse.g. bulk of PCBs). When using carbon columns, typically theon-planar pollutants are eluted by a non-polar solvent (e.g.-hexane), whereas the target pollutants are back flush elutedrom the column using toluene. Immunochromatographic LColumns may be used for the separation of OCPs and dioxin-likeompounds [100].

PCBs and OCPs. Clean-up of PCBs is often combined withhat of OCPs. After the removal of lipids or sulphur, the PCBsre separated from the more polar OCPs by silica fractiona-ion. PCBs are eluted from the column by a non-polar solvente.g. n-pentane or n-hexane), whereas elution of most OCPsequires a more polar solvent or solvent mixture (e.g. 15%iethyl ether: 85% n-pentane). Some OCPs (trans-nonachlor,is- and trans-chlordane, hexachlorobutadiene (HCBD), QCB,CB, OCS, p,p′-DDE, o,p′-DDE, pentachloroanisol and pen-

achlorothioanisol) may partially elute in the 1st fraction,ogether with the PCBs, depending on the elution volume andolarity of the solvents used [53]. Several authors applied multi-ayer silica columns, typically containing a combination of acidmpregnated, base impregnated and regular (deactivated) silicael [101,102]. These columns effectively remove potentially dis-urbing matrix constituents. More details on clean-up for PCBsnd OCPs can be found elsewhere [47,96].

PCNs. The clean-up of biota and sediment extracts can bechieved by the lipid removal by, e.g. GPC or alumina and subse-uent silica gel fractionation. Further removal of interferences ischieved using porous graphitic carbon or PYE columns becausehe molecular planarity allows to selectively separate PCNs fromnterferences (similar to PCDD/Fs) [48]. Because environmentalevels are higher compared to PCDD/Fs, a less complex clean-ups required.

CPs. The complete removal of interfering pollutants (e.g.oxaphene) is essential when using short GC columns combinedith GC–electron-capture negative ionisation (ECNI)-MS [17].lorisil can be used to effectively separate interferences (PCBs,

oxaphene, o,p′-DDT and �-HCH) from the SCCPs as demon-trated by Reth et al. [19]. Apart from pollutant class separation,ilica gel and florisil also trap polar interferences that are notemoved in earlier clean-up steps. Sometimes, a final clean-p step may be required such as treatment with sulphuric acid.hotolysis was effective for the (partial) removal of interferingollutants like PCBs, chlordanes and DDTs [103].

Toxaphene. For the analysis of toxaphene, the removal of
CBs from the extract can be achieved on silica gel but some
osses of the lower chlorinated toxaphene congeners may occurdepending on the deactivation of the silica) and care should beaken to avoid this, or correction for the losses should be made

tdmc

romatogr. A 1186 (2008) 161–182 167

104]. Krock et al. [105] found that 8 g activated silica elutedith 48 mL of n-hexane efficiently separated toxaphene fromost of the interferences (PCBs, PCNs, HCB, p,p′-DDE and

ctachlorostyrene).BFRs. The clean-up of PBDEs is similar to that used for

CBs. GPC, alumina, silica and concentrated sulphuric acidave all been successfully used for the clean-up of extracts,s showed in detail in a recent review by Covaci et al. [106].lean-up of HBCD and dimethyl-TBBP-A is partially similar

o the clean-up for PBDEs, but due to its polar character dissoci-tion should be avoided. The pKa1 and pKa2 values of TBBP-As estimated at 7.5 and 8.5, respectively [107], which meanshat in neutral environments, a substantial part of the TBBP-

is present in its dissociated state. This causes losses in thelean-up steps when a neutral environment combined with polarolvent is maintained (the polar solvent could just be a little bitf co-extracted water from the sample). Care should be takeno avoid these losses and a possible solution is to treat the rawxtract with acidified water. This results in associated TBBP-Anly, which is driven almost quantitatively towards the organichase. Concerning HBCD, care should be taken with the sil-ca elution. HBCD consists of several diastereomers (�-, �, and-HBCD are the major ones) and �-HBCD requires a largerolume of solvent for complete elution from silica columns asompared to PBDEs, me-TBBP-A and �- and �-HBCD [108]).ecause of these specific requirements it is ambitious (but fea-

ible) to combine the clean-up of extracts for PBDEs, HBCD,imethyl-TBBP-A and TBBP-A analysis.

The final step prior to GC injection is the concentration of theample extract. This is achieved by solvent evaporation (N2 blowown, Kuderna Danish or Turbovap). Care should be taken tovoid losses (of volatile) compounds during this process. Biggerosses were reported for OCPs using the Turbovap as comparedo Kuderna Danish (but nearly no losses for PCBs) [90]. Aonical Kuderna Danish receiving flask is preferred over a cylin-rical flask for reducing extract volumes to below 100 �L [91].nother way of preventing losses is the addition of iso-octaner nonane prior to the concentration step as a so-called keeper,nd these solvents are suitable for injection in the GC.

Several of the aforementioned clean-up steps may be com-ined in one step. The advantage of doing so is that betweenhe various clean-up steps no concentration steps are requiredhich reduces the risk of losses due to evaporation and contam-

nation due to the use of glass ware in several steps. Also, theolume of solvent and the amount of labour are reduced in thatay. One option is the combination of several clean-up steps in a

ingle glass column (multi-layer column) loaded with, e.g. alu-ina oxide, anhydrous sodium sulphate, acidified silica, basic

ilica, neutral silica and porous graphitic carbon. The set-up ofhe method (number and type of layers) varies among the stud-es. The multi-layer clean-up was successfully applied for BFRs,CBs, OCPs, PCDD/Fs, dl-PCBs and brominated dioxins andurans [69,70,109–115]. In recent years, complete clean-up sys-
ems (e.g. PowerPrep, Fluid Management Systems, USA) wereeveloped for environmental analyses, which combine and auto-ate several clean-up steps in a modular system using disposable
olumns. After sample extraction, the extract is loaded in this

1 Chromatogr. A 1186 (2008) 161–182

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ystem and automatically processed resulting in the final extract,eady for injection. For PCDD/Fs and dl-PCBs, the used columnsre a multi-layer silica column, followed by alumina and finallyorous graphitic carbon [116]. In a parallel system, multipleamples can be processed in 1 h. Although labour reduction isonsiderable, the initial investments for such system are highnd consumables are more costly compared to a home-madeulti-layer columns. Therefore, these systems may fit perfectly

n a commercial routine laboratory for obtaining high through-ut at low labour costs, whereas flexibility may be too low foresearch laboratories. So far, they have only been applied forCDD/F analysis [117–119] and PBDEs [120,121], but appli-ation to other pollutants should also be feasible. Recently, theoncept of coupling PLE in line with the PowerPrep systemas presented [122]. This method potentially further reduces the

ample handling time, but a thorough evaluation of the systems not yet presented.

.3. Combined extraction and clean-up by selectiveressurised liquid extraction

Recently, several studies have explored the possibilities ofombined automated extraction and clean-up of environmen-al samples by means of PLE with within-cell clean-up. Suchpproach can substantially reduce the labour spent on extrac-ion and clean-up of environmental samples. This method isometimes referred to as selective PLE, or SPLE [80]. In selec-ive PLE the extraction cell is filled with sample material andhe sorbents that perform the clean-up (whereas in conventionalLE the extraction cell is only filled with sample material,ometimes mixed with anhydrous Na2SO4 for binding moisturerom the matrix). Recently, the potential of selective PLE waseviewed by Bjorklund et al. [123]. Within the DIFFERENCEroject (funded by the European Community), considerablemprovements were obtained on selective PLE for extractionnd clean-up of PCDD/Fs and dl-PCBs from feed and food (e.g.sh) [68,123–126]. Silica (or florisil in [80]) was employedor lipid-rich samples (such as herring) in order to retain theo-extracted lipids in the extraction cell (extraction with n-exane). The optimum fat-fat retainer ratio was 1:40 [124].orous graphitic carbon was applied in a specially designed cell

nlay to retain the planar compounds (Fig. 1). In the forward flushode, the lipids and non-planar compounds are extracted and

luted by n-heptane (fraction 1) and 1:1 DCM:n-heptane (frac-ion 2), whereas the PCDD/Fs and non-ortho-PCBs are retainedn the porous graphite. The latter are then backflush elutedy toluene (fraction 3). A subsequent miniaturised multi-layerlean-up of fraction 3 was sufficient for the accurate determi-ation by GC–HRMS afterwards. Results obtained by aboveethods were very well comparable (accuracy and precision)

o traditional extraction and clean-up techniques. Selective PLEas also developed for PBDEs in sediment [64,65]. For PBDE

xtraction in sediment, 1 g of sediment was mixed with alumina
nd cupper (1:2:2, w/w/w) and extracted with hexane–DCM1:1, v/v, 100 ◦C). Compared to Soxhlet, recoveries of a spikedediment were slightly lower for mono-BDEs to tri-BDEs, butomparable for tetra- to hepta-BDEs [65].
mo

CM/n-heptane (1:1, v/v, forward elution); and fraction 3: toluene (backwardlution). In backward elution mode, the cell had been turned upside down. Fromef. [123].

Extraction cells for Dionex systems are available up to00 mL. The largest cell volume is large enough to accommo-ate either a lipid-rich fish sample mixed in the proper ratiosith silica, or the fish sample and the carbon cell inlay (Fig. 1).onsidering that, especially for PCDD/F analysis, 2–6 g of lipidsre needed to obtain sufficient sensitivity, the cell volume is toomall for lean fish samples (1–5% of lipids). Therefore, largerxtraction cells are required. Splitting the sample over two orhree extraction cells can circumvent this. Method developmentf selective PLE is somewhat more laborious than conventionalLE, but the benefit is the strongly reduced sample handling

ime once the method is established. At the moment selectiveLE is one of the few techniques that offer a substantial reduc-

ion of labour time of the pre-treatment of sediments and biotaamples for POP analysis. Within the DIFFERENCE project,elective PLE was evaluated for PCDD/Fs and dl-PCBs. Theosts breakdown (Fig. 2) shows that the costs of extraction andlean-up (indicated in horizontal black lines) were similar to tra-itional extraction. Only a miniaturised additional multi-layerlean-up for the removal of residual interferences (primarilyipids) was required prior to injection [125]. It may be expectedhat due to the pressures of authorities to reduce costs of analy-es more labour-reducing methods may be developed in the nearuture.

. Injection

GC is the method of choice for the analysis of complexixtures of halogenated pollutants for its unsurpassed res-

lution offered by capillary columns. The three parts of the

S.P.J. van Leeuwen, J. de Boer / J. Ch

Fig. 2. Cost per analysis for GC–HRMS and alternative techniques (GC–ITMS/MS and GC × GC–ECD) for the analysis of dioxins and dl-PCBs (editedfrom Ref. [183]). Top: cost expressed per stage of analysis; bottom: costexpressed per item. The costs per technique are calculated on the basis oflabour (in man hours ×D 75 h−1) in each step of analysis, consumable useand costs, the costs involved with instrument investment and depreciation andinstrument maintenance costs (service costs). Costs for QA/QC (20–40%) andanalytical standards (ca. 3%) were not taken into account. *PP: PowerPrep (auto-mated extraction and clean-up, indicated in vertical grey lines (top graph only));*i

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C analysis, injection, separation and detection all need to beptimised and validated to guarantee a high-quality analysis.

Injection of POP containing extracts can be performed by var-ous automatic injection systems. The most commonly appliedystem is splitless injection [15,16,93,103,110,112,127–133],ut alternative techniques such as by programmed temperatureaporizer (PTV) and on-column injection can be applied as well134,135]. In splitless injection, 1–2 �L extract is injected in alass liner. The liner serves as the evaporation chamber wherehe liquid extract is rapidly volatilised at elevated temperatures150–200 ◦C). The liner may be open, or partially filled withplug of glass wool or other surface area increasing materi-

ls [136]. Open liners are generally preferred as glass wool orther materials can easily cause thermal degradation. This pro-ess is enhanced by active sites at elevated temperatures in the

tti

romatogr. A 1186 (2008) 161–182 169

njector. Active sites also occur in the liner due to the accumu-ation of dirt, typically after multiple injections of dirty samplextracts. Several studies reported the degradation of pollutantsue to dirty liners and the high injection temperatures, includ-ng DDT [73], toxaphene (standard mixture of 22 congeners)137,138], HBCD [51], and higher brominated BDEs (octa- toeca-BDE) [51,52,139]. Thermal degradation in the injector cane minimised by frequently replacing the liner by a clean one.urthermore, the residence time of the pollutants in the injec-

or can be minimised by the application of a pressure pulse.his pulse rapidly transfers the volatilised pollutants to the col-mn. Pressure pulse injection was applied, e.g. for PBDEs [51].nother undesired phenomenon is discrimination of pollutants.his is the fractionation of the sample in the injector whereby

he least volatile pollutants are (partly) splitted rather than beingntroduced in the GC column, resulting in a non-quantitativentroduction of these heavy compounds in the column. Thishenomenon occurs when using non optimised splitless timesnd was reported for higher chlorinated toxaphene homologousnona- and decachloro congeners) [137] and BDE 209 [139].

With (cold) on-column injection, the complete extract isntroduced directly in the first part of the GC column at roomemperature. In that way no losses can occur. The vaporisation ofhe sample extract takes place in the column at a temperature justbove the solvent boiling point. The instrumental set-up is simples well as the operation and maintenance. However, the samplextracts should be very clean to prevent the introduction of dirtrom the sample matrix. The accumulation of dirt in the firstart of the column leads to the deterioration of the GC columnnd can lead to active sites. These active sites may catalyticallyegrade labile pollutants. These phenomena can be reduced byhe application of an uncoated, deactivated retention gap. Theccumulation of dirt from the sample extract then occurs at theetention gap. However, after multiple injections system perfor-ance can decrease [114] and therefore, the retention gap should

e changed regularly. Extracts should be as clean as possible,ven when using this retention gap system. On-column injectionas successfully applied for PBDEs [51,114,139] and toxaphene

137]. On-column injection can also be used for injection of largeolumes (large volume injection, LVI) [140,141]. Bjorklund etl. [142] explored this principle for the analysis of PBDEs andnjected 50 �L into a 10 m retention gap. They evaporated theolvent through the GC column and ECD prior to GC analysis.arge quantities of solvent cannot be evaporated through an MS.

n that case, an early solvent vapour exit is required. The PTVnjector is a generic injector, some of which can be used in sev-ral modes (e.g. split–splitless injector). The more interestingpplication of PTV is that of LVI for increasing the sensitivity.sing this technique, volumes of 10–50 �L have been injected.his injector was used for PCDD/Fs and dl-PCBs [143,144],BDEs [77,145,146], and PCNs [77]. Typically, the solvent is

ntroduced in the ‘cold’ PTV injector, which is set at a temper-ture just below the solvent boiling point. Then, the solvent is

he solvent by a split flow through the opened split valve. Finally,he split valve is closed (splitless mode) and by rapidly raising thenjector temperature, the target pollutants are transferred to the

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nalytical column. Eppe et al. [143] injected 10 �L of the finalxtract (in toluene) in order to compensate for the lower sensitiv-ty of their detection method (ion-trap MS/MS). Doing so, theyrrived at an instrumental LOD (iLOD) of 200 fg/�L (S/N = 5:1).sing LVI, sample handling can be reduced by leaving out thenal extract concentration step.

. Gas chromatographic separation

.1. Column selection

The heart of the GC is the capillary column. The selec-ion of stationary phase, column dimensions and carrier gasvelocity) determines the separation characteristics. Given theomplex composition of most POP mixtures, most studiesave tried to increase and optimise the resolution of thehromatography in order to separate a maximum number ofollutants. Hydrogen and helium are generally used by carrierasses and (especially hydrogen) provide optimum resolu-ion at highest carrier gas velocities. Table 2 shows stationaryhases used in GC of halogenated pollutants. The most widelysed are non-polar to slightly polar stationary phases, suchs DB-1, DB-5, BPX-5, HT-8, CP-Sil8CB-MS or CP-Sil-9 [11,15,44,51,110,130,146–148]. The addition of MS to aolumn type name means that suppliers have minimised theleeding of the stationary phase, which is necessary when usingS detection. Column bleed may show up in the MS spectra

nd complicate the identification of the target pollutants. Filmhickness is typically 0.25 �m. Column lengths are 30–60 m,ut shorter columns are beneficial in certain cases. The col-
mn diameter is directly proportional to the resolution. Typicalolumn diameters are in the range of 0.25–0.32 mm, but nar-ow bore columns (0.10–0.15 mm) provide substantially moreheoretical plates at the same column length. These small dimen-
d[ta

able 2election of popular stationary phases used in GC analysis of halogenated pollutants

olarity scalea Stationary phase Br

100% Dimethyl polysiloxane ZB5% Phenyl-(arylene)–95% methyl polysiloxane ZB

7 50% Phenyl–50% methyl polysiloxane ZB4 75% Phenyl–25% methyl polysiloxane ZB3 50% 3-Cyanopropyl–50% phenylmethyl polysiloxane 002 Polyethylene glycol ZB8 100% 3-Cyanopropylpolysiloxane BPon-polarb 50% n-Octyl–50% dimethyl siloxane SBoderately polarb 65% Phenyl–35% methyl polysiloxane 00oderately polarb Cross-linked methyl–phenyl–polysiloxane block polymers Op

olarb Polysilphenylene phase BP

olarb 44% Methyl–28% phenyl–20% cyanopropyl polysiloxane DBBiphenylcarboxylate ester methylpolysiloxane SBDimethyl (50% liquid crystal) polysiloxane LC�-Cyclodextrin �-�-Cyclodextrin CP�-Cyclodextrin BG

a Relative polarity as determined by McReynolds and Kovats indices.b Qualitative classification. No quantitative figure on the polarity scale available.c Shape selective.d On basis of chirality.

matogr. A 1186 (2008) 161–182

ions require high gas pressures. Nowadays, GC pneumatics arequipped to accurately deliver carrier gas at these high pres-ures (up to 150 psi), which enables the use of narrow boreolumns.

Co-elution of pollutants with other pollutants of interest orith interferences is a common problem in GC separation. No

ingle analytical column is able to separate all PCBs [147],BDEs [149], HBCDs [42] or even the 17 WHO PCDD/Fs112,127,143]. The identity of co-eluting pollutants can be deter-ined by the elution over a secondary column with a different

tationary phase (either or not in a dual-column system). Polarhases like CP-Sil 88 have been employed for that reason. Fur-hermore, liquid crystalline columns show distinct separationharacteristics based on molecular structure rather than on boil-ng point [150]. Unfortunately, these columns suffer from higholumn bleed [8,112].

PCDD/Fs and dl-PCBs. Column manufacturers have devel-ped dedicated columns for a number of applications toesolve critical pollutants, e.g. for dioxin analysis (e.g. DB-ioxin, BPX-DXN, RTX-Dioxin2). These columns have aore polar stationary phase (e.g. DB-Dioxin: 44% methyl–28%

henyl–20% cyanopropyl polysiloxane) and enable the separa-ion and quantification of critical pairs (e.g. 2,3,7,8-TCDD beingeparated from 2,3,4,7,8-pentaCDF, 2,3,4,6,7,8-hexaCDF).owever, incomplete separation of all 17 WHO PCDD/F con-eners remains. Details on co-elution of PCDD/Fs can be foundlsewhere [151].

PCBs and OCPs. PCBs and OCPs are typically sepa-ated on non-polar (e.g. BPX-5, HP-5MS, DB-5MS, VF-5MS)r slightly polar (e.g. CP-Sil 8CB) stationary phases with
imensions of 30–60 m × 0.25 mm I.D., 0.25 �m film thickness72,83,110,130,152,153]. These columns are not able to separatehe complete set of 209 PCBs, but the indicator-PCBs can nearlyll be separated from other PCBs. Well-known co-elutions on
and and type names

-1(ms), CP-Sil5CB, DB-1, HP-1(ms), PE-1, RTX-1, BP(x)-1, Ultra-1-5(ms), CP-Sil8CB, DB-5(ms), HP-5(ms), PE-5, RTX-5(ms), BPX-5, Ultra-2-50, DB17(ms), HP-50+, HP-17, PE-17, RTx-50, BPX-50, OV-17, Optima 17-50, CP Sil 24 CB

7-225, CP-Sil 43 CB, AT-225, BP-225-WAX, ZB-WAXplus, DB-WAX, CP-Wax 52 CBX70, CP-Sil 88 CB, DB23, HP23, PE-23, RTX-2330, VF-23MS-octyl 507-65HTtima delta-3X-DXN, RTX-Dioxin2, SP-2331, 007-23, RTX-2332, DB-Dioxin

-Dioxin-smectic-50

DEX 120-Chirasil-Dex CB, �-DEX 120, Cyclodex-B, HP-Chiral- �, Rt-�DEXB-176SE, BGB-172, Rt-�DEX

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on-polar phases are PCB 28 and 31 and 138 and 163 [47,95].he OCP fraction may contain many interferences that prefer-bly are removed by clean-up of the extract as they may leado inaccurate results, especially when using non-selective ECDetection. In these cases, a column length of 50–60 m is rec-mmended for maximum separation. Furthermore, confirmationay be required by analysis on a second column with different

polar) stationary phase. de Boer et al. [154] investigated the sep-ration of PCBs on several narrow bore columns (0.15 mm I.D.)nd although the resolution further improves using these smallerimensions, narrow bore columns have not found a wide appli-ation. Separation based on chirality will be discussed below.

PCNs. Jarnberg et al. [155] determined the retentionehaviour of a PCN standard mixture on six capillary columns:ltra 1 and Ultra 2; HT-5 (5% phenyl–dimethylpolysiloxane on

arborane); CP-Sil-88, SB-octyl 50 and SB-smectic. None ofhe columns was able to resolve all congeners, but Ultra 1 andltra 2 were able to separate 44 out of 75 possible congeners. On

hese columns, the different homologue groups eluted as distinctlusters, whereas on CP-Sil 88 an overlap between clusters wasound because of a higher resolution within each homologueroup [155]. The SB-octyl 50 and SB-smectic columns wereble to resolve specific pairs, although resolution was highlyemperature-dependent. Specific hexa–CN pairs can be sepa-ated on alpha-cyclodextrin and beta-cyclodextrin (�-DEX 120,-DEX 120, Supelco) columns [132].

CPs. The technical mixtures of SCCPs are so complex thaturrent state-of-the-art capillary GC does not provide a solutionor the separation of all congeners. CPs are generally separatedn non-polar columns (DB-5MS, HP-1) with a length of approx-mately 15–30 m [16,19]. Complete separation has to date noteen feasible and is not likely to be achieved in the near future.omplete separation may sometimes even not be a desirableoal, as such separations would generate extensive amounts ofata that are not easy to handle, and not very informative foruthorities. Instead, it is desirable to focus on the determinationf a representative selection of compounds (as with toxaphene)r on the toxicological relevant isomers (similar to PCDD/Fsnd dl-PCBs). It should be noted that such selection of ‘relevant’somers has not yet been proposed.

Toxaphene. In the case of toxaphene, not all congeners inechnical mixtures are present in environmental samples andnly a small selection is typically observed (i.e. P26, 39, 40,1, 44, 50, 62). These congeners can be separated chromato-raphically. Oehme and Baycan-Keller [138] have reviewed theC separation of toxaphene on capillary columns. Non-polar

tationary phases like DB 5, CP-Sil 8, HP 5, Ultra 2 are com-only used and allowed for the separation of P26, 50 and 62

26,93,138,156]. The congeners P39-44 are generally difficult toesolve, but medium polar columns like Optima delta-3 and HT-successfully resolved these congeners (in a standard mixture

f 23 congeners) [138]. Polar columns should be used with cau-ion, as considerable toxaphene degradation may take place (see
138] for details). Baycan-Keller and Oehme [157] conductedemperature programming experiments and found the best res-lution with 10 ◦C/min temperature ramping as compared to◦C/min.
fi[1

romatogr. A 1186 (2008) 161–182 171

BFRs. Korytar et al. [149] created an extensive PBDEetention-time database for 126 PBDEs, HBCD, TBBP-A andBBs on 7 GC columns (17–30 m), i.e. DB-1, DB-5, HT-5, HT-, DB-17, DB-XLB and CP-Sil 19. None of the columns wasble to separate all major PBDEs, but the most abundant BDEs47, 99 and 100) were baseline separated on the DB-1, DB-5,B-XLB, HT-8 columns. BB 153 and me-TBBP-A co-elute withDE 154 on a DB-1 and DB-5 column [149]. This could result in

naccuracies because BB 153 and me-TBBP-A can be found innvironmental samples at significant concentrations [51]. Tech-ical HBCD consists predominantly of three diastereomers (�,and �) and each of those has two enantiomers [58,158,159].

hese cannot be separated by GC. Furthermore, at tempera-ures >160 ◦C, the diastereomer composition changes [51] andonsidering the different response factors of the diastereomers40], this may result in a response that does not represent thectual concentrations in the extract. HBCD and TBBP-A can beetermined by HPLC–electrospray ionisation (ESI)-MS(/MS)s well [108]. The benefit of this technique is the chromato-raphic separation of the individual diastereomers and thus,iastereomer profile information can be obtained. Furthermore,C–ESI-MS/MS does not suffer from thermal degradation in the

njection system and the isomerisation of the diastereomers inhe column. Considerable differences, up to a factor 5, are some-imes observed between GC- and HPLC-generated results [40],hereas Goemans et al. [160] found a smaller difference (<2)etween GC and LC results. This calls for further explorationf the underlying reasons, but the application of LC–ESI-S/MS using clean extracts and 13C12-labelled standards (for

ll three diastereomers) appears to be the best road to accurateesults.

Chiral compounds can be separated using columns witheta-cyclodextrin stationary phases specifically developed forhat purpose. Chiral PCBs were separated on Chirasil-Dex,GB-176SE and BGB-172 [129,161] columns and Bordajanit al. were able to separate nine out of the nineteen enan-iomeric PCBs (PCB 84, 91, 95, 132, 135, 136, 149, 174, and76) on the Chirasil-Dex phase. Cyclodextrin stationary phasesave also been employed for enantioselective separation of �-CH, chlordanes, and DDTs [162–165]. Vetter and Luckas

166] studied the enantioselective separation of toxapheneongeners on a tert-butyldimethylsilylated beta-cyclodextrinolumn (�-BSCD; 30 m × 0.25 mm I.D. × 0.25 �m film) and onpermethylated �-cyclodextrin column (�-PMCD; dimensionsre not reported). They separated several enantiomeric pairs andoing so, they were able to determine that selective enantiomericnrichment took place at high throphic level biota.

The use of shorter columns (5–15 m) for the analysis ofalogenated pollutants has not (yet) found a wide application.owever, short columns enable rapid analysis of compounds andithout sacrificing resolution, provided small internal diameters

re being used. Faster analysis also means shorter residue timen the column at elevated oven temperatures, which is beneficial
or thermo-labile compounds such as BDE 209 for which min-mised column residence times are crucial [49,50]. Binelli et al.114] determined the response of BDE 209, BB 209 and BDE83 and found a 50-fold response increase for BDE 209 when

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hortening the column from 16 to 6 m (RTX-5 MS, 0.25 mm.D., 0.25 �m film), and optimisation of the carrier gas flow.he authors concluded that interactions with active sites in theolumn were the cause for poor chromatography on the longerolumn lengths they tested. Bjorklund et al. [139] tested dis-rimination on analytical columns (15 m × 0.25 mm I.D.) andound that severe discrimination occurred for the higher bromi-ated BDEs (BDE 203, 209) on DB-XLB, HP-1 and RTX-500tationary phases. They also found that a small film thickness of.1 �m (instead of the commonly used 0.25 �m) was beneficial
or the yield of the hepta to deca-BDEs. Finally, they deter-ined that for the temperature program a final oven temperature
f 300 ◦C was a good compromise between degradation and

awa

ig. 3. GC × GC–�ECD overlay plot of various pollutant groups on a DB-1 × 007-6CDEs, ( ) PBDEs, ( ) PCDTs, (�) PCNs, (�) PCDD/Fs, ( ) OCPs, ( ) indiAroclors 5442 + 5460), and toxaphene technical mixture; (C) ( ) PCDTs, and (�)

matogr. A 1186 (2008) 161–182

and broadening [139]. Stejnarova et al. [17] used a very shortolumn without stationary phase (1.3 m, 0.15 mm I.D. quartzolumn) coupled to ECNI-MS for the determination of SCCPs17]. Coelhan et al. [167] used even a shorter column of 0.65 mnly. Short columns provide condensed chromatograms and nar-ow peaks (few seconds only), resulting in increased sensitivity167], but care should be taken in operating the detector at suf-ciently high frequency to record 10–12 datapoints over thehole peak (for modern ECD and MS detectors, that should

mount of sample that can be loaded on the column decreases,hich counteracts the sensitivity improvement discussed

bove.

5HT column combination. Pollutant groups: (A) ( ) PCBs, ( ) PBBs, ( )vidual toxaphene standards, and PCAs (PCA-60); (B) PCAs (PCA-60), PCTsPCDD/Fs; (D) ( ) PCDEs and ( ) PBDEs. From Ref. [111].

J. Ch

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.2. Multidimensional gas chromatography

Heart-cut multidimensional GC was used in the 1990s fornalysis of, e.g. toxaphene and (chiral) PCBs making use of theeans switch for transfer of the heart-cut to the 2nd dimension

olumn [129,161,168,169]. With the introduction of modula-ors such as the sweeper [170,171], and later, the cryogenic

odulators, the field of GC × GC has made a breakthroughn the analysis of halogenated pollutants in recent years. AC × GC system consists of two GC columns connected byconnector (e.g. press fit). The first column is often a tradi-

ional 30–50 m column with a non-polar phase, which separateshe pollutants of interest based on their boiling points. The sec-nd column is a very short (0.5–1.5 m) column with a differenttationary phase (e.g. polar or shape-selective). The pollutantsluting from the 1st column are trapped (often cryogenically)or a short period of time (modulator time) and subsequentlyeleased by heating for the separation on the second column (aisualisation of this process can be found elsewhere [172]). Theodulation causes a focussing of the peaks, which improves the

ensitivity of the system. Where traditionally a two-dimensionallot is obtained (retention time and response), here a three-imensional plot is obtained. The 1st dimension (x-axis) isimilar to a conventional chromatogram (with retention timesf typically 40–90 min) and the 2nd dimension with very shortetention times (typically 6–9 s) are plotted on the y-axis. Theeak response rises from this two-dimensional plane (z-axis).or a graphic explanation of the resulting chromatogram, pleaseefer to Adahchour et al. [173]. Fig. 3 shows examples ofeveral GC × GC chromatograms. More details on the princi-les of this technique can be found elsewhere [172–174]. Thenterpretation of the chromatograms requires specific GC × GCmaging software and several GC × GC suppliers provide soft-are with their instruments (e.g. Thermo Finnigan and Leco).eneric GC × GC software is available from Zoex (TX, USA).three-dimensional peak is composed of several individual 2nd

imension chromatograms in which the compound of interestlutes. The quantification of peaks is thus based on summaris-ng the peak areas of the individual 2nd dimension peaks. Theptimisation of the GC × GC separation is more laborious thanraditional GC separation and involves a proper selection of theolumn combination, temperature programming of one or twovens, carrier gas velocity and modulating time.

GC × GC provides a very strong separation method, and haseen used for the separation of complex mixtures of PCDD/s and (dl-)PCBs [111,112,129,175–178], BFRs [111,179],

oxaphene [111,169,180], CPs [111,181], OCPs [111,182] andCNs [111]. The DIFFERENCE and DIAC projects [183,184]ave given the development of GC × GC for the analysis ofCDD/Fs and dl-PCBs a considerable push forward by evalu-ting crucial parameters such as column selection, modulatorype and detection method [111,112,175,177,185,186]. Korytart al. [112] investigated a range of column combinations and
btained complete separation of all 29 WHO dioxins and dl-CBs on a DB-XLB column (1st dimension) combined with007-65HT, VF-23MS or LC-50 columns (2nd dimension).
iquid crystalline phases (e.g. LC-50) in combination with a

tccu

romatogr. A 1186 (2008) 161–182 173

on-polar column also allowed for the separation of the 29 pollu-ants from matrix constituents. Unfortunately, the LC-50 columns not widely available due to column bleed of this column type.owever, as long as it is used as the 2nd dimension column

he bleeding does not play a role because of the short lengthnd thin film thickness. Focant et al. [144] achieved separationf all 12 PCDD/Fs and 4 dl-PCBs on a RTX-500 × BPX-500ombination. Modulators using CO2 as cryogenic coolant arereferred over other types (e.g. thermal modulation (sweeper)nd liquid nitrogen cooled jets) for producing narrow peaksnd a broad application range [187]. In the framework of theIFFERENCE project, an extensive validation took place of

he DR-CALUX bioassay, GC–ITMS/MS and GC × GC–ECDs. GC–HRMS for the detection of PCDD/Fs and dl-PCBs inood and feed samples [186]. Three datasets on GC × GC–ECDere obtained and these showed that performance compared toC–HRMS was comparable for a cleaned fish extract, a fishil, a spiked vegetable oil and a herring sample [183]. Someverestimation that was found could easily be explained by theomewhat higher detection levels of the GC × GC–ECD systemhat resulted in higher numbers when applying the upper-boundpproach [183]. The results complied with the EU method per-ormance requirements on screening techniques for the detectionf PCDD/Fs and dl-PCBs in food and feed [188], which showshat GC × GC–ECD is a viable method.

Harju et al. [176] and Focant et al. [178] studied the sep-ration of all 209 CBs in a standard mixture on a DB-XLB,B-1, HT-8 (1st dimension, 30–60 m) combined with a HT-8,PX-50, BPX-70, SP-2340 or LC-50 column (2nd dimension,.9–2.3 m). The DB-XLB × BPX-70 combination provided theest resolution with only 15 co-elutions but at the cost of a40 min runtime, whereas in approximately 144 min, nearlyimilar results were obtained when using BPX-50 as the 2ndimension column (see [174] for an overview table). With theB-XLB × SP-2340, HT-8 × BPX-50 and HP-1 × HT-8 combi-ations, group separation information was obtained [176–178].pplication of GC × GC using column combinations likehirasil-Dex/SUPELCOWAX-10 and Chirasil-Dex/VF-23 msrovided enantiomeric separation as well as separation fromhe non-enantiomeric PCBs and matrix pollutants [129]. Kory-ar et al. [179] evaluated column combinations for GC × GC of25 PBDEs, some BBs, HBCD and (me-)TBBP-A. On a DB-(1st dimension) × 007-65HT (2nd dimension) combination,

hey resolved 90 out of 125 PBDEs, including the environ-entally relevant BDEs (i.e. BDE 28, 47, 99, 100, 153, 209).

n addition, the 2nd dimension column was able to separateeTBBP-A, TBBP-A, BB 169 and two metabolites of BDE

7 which interfere in the 1st dimension [179]. The potential ofC × GC was also investigated for technical toxaphene [180].t optimised conditions (30 m × 0.25 mm × 0.25 �m HP-1 1stimension and 1 m × 0.1 mm × 0.1 �m HT-8 2nd dimensionolumn, GC × GC–�ECD) over 1000 individual toxapheneongeners could separately be determined in the technical mix-
ure. In the same study, a standard containing 23 individualongeners was analysed (GC × GC–TOF-MS, 1st dimensionolumn 10 m × 0.25 mm × 0.25 �m DB-1; 2nd dimension col-mn: 1 m × 0.1 mm × 0.1 �m HT-8). Nearly all congeners were

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aseline separated and group separation of the chlorinated bor-anes and camphenes was obtained based on the number ofhlorine substitutions). Using this method, they were able toonfirm that the technical mixture consists primarily (97%) ofexa- to nona-chlorinated compounds. Korytar et al. [181] evalu-ted GC × GC for CPs. They evaluated six column combinationsnd found that DB-1 × 007-65HT provided most informationn group separation (homologue groups). No complete sep-ration of congeners was obtained, but the technique provedo be a strong additional tool for profiling CPs in environ-

ental samples. Finally, Korytar et al. [111] challenged theC × GC separation by trying to achieve group separation of

everal contaminant classes in a single column combination.he DB-1 (1st dimension) × LC-50 (2nd dimension) column setrovides group separation based on planarity and planar com-ounds such as PCDD/Fs, polychlorinated dibenzothiophenesPCDTs) and PCNs are more retained on the 2nd dimen-ion LC-50 column than non-planar analytes. The DB-1 (1stimension) × 007-65HT (2nd dimension) column set effectivelyeparates PCAs and PBDEs from all other compound classesig. 3), and provides a good separation of brominated and chlo-inated analogue classes from each other [111]. This column setas the most efficient one for within-class separation of OCPs

nd PCNs.Comprehensive GC × GC has proven to be a strong technique

or the separating complex mixtures and provides considerablyore information on the pollutant profile when compared to

raditional GC. GC × GC is excellent for the identification ofnknown compounds appearing (or interfering) in the chro-atogram. For example, unknown PCBs in a sample can be

dentified based on the number of chlorines [177] or based onhe number of ortho-substituted chlorines [176]. Similar char-
cterisations can be achieved for toxaphene [111] and PCAs181]. The use of selective detectors such as TOF-MS furtherncreases the identification possibilities. A current drawback ishe interpretation of the complex chromatograms, in particular
Iot

able 3ualitative scoring of the various methods for the analysis of trace levels of halogenat

±) intermediate; (+) good; (++) excellent choice)

Sample extraction and clean-up Injection method

Soxhlet MAE PLE PowerPrep Split–splitless On

ase of methoddevelopment

++ ++ + + + +

obustness ++ ++ ++ + ++ +ensitivity n.a. n.a. n.a. n.a. + +

electivity n.a. n.a. (+)a ++abourc + + + (++)a ++ ++ ++peed/throughput ± + + (++)a ++ + +osts: investment ++ + ± ± ++ ++osts, otherd ++ + + + ++ +

.a., not applicable.a Between parentheses: combined extraction and clean-up.b Between parentheses: when used in LVI mode.c (−) very laborious; (±) intermediate; (+) not very laborious; (++) not laborious ad Other costs: consumables and maintenance.

matogr. A 1186 (2008) 161–182

hen quantitative analyses are needed of low concentrations.his is currently a very labour intensive task as software isot yet capable of automatic accurate identification and inte-ration of peaks close to the limit of quantification (LOQ).nstrument suppliers put much effort in software developmentnd it is therefore expected that this is only a temporary problem.urthermore, at very low concentrations, as with PCDD/Fs inelected fish and sediment samples, more effort should be put inlean-up. It should be noted that this is less of a problem whenollutant concentrations in the samples are higher. Finally, theC × GC optimisation and maintenance is less straightforward

han the traditional GC set-up. Presumably these issues have pre-ented up-to-date a wide acceptance of GC × GC as a routinenstrument.

When comparing costs per analysis (in the framework of theIFFERENCE project, Fig. 2), the costs for a GC × GC–ECDetermination of PCDD/Fs and dl-PCBs (ca. 900 Euro) are muchigher than for GC–HRMS (275–500 Euro). This is mainlyaused by the low contaminant concentrations combined withhe considerable data treatment as discussed above. Further-

ore, more emphasis was put on clean-up for the removal ofnterferences from extracts. Investment costs per sample are,s expected, much lower (see also Table 3). It is believed thathe price difference will become less in the future when soft-are developments allow for rapid data treatment. It shoulde noted that in total, the cost data of five GC–HRMS lab-ratories were obtained, whereas the cost estimation for thether techniques is based on a lower number of laboratories.herefore, for GC–HRMS the range of costs among labo-

atories can be determined whereas this is not feasible forC × GC–ECD for which only the data of one laboratory werebtained.

Fast GC [189] is basically a variety of multidimensional GC.t has, for example been applied at airports for the fast detectionf explosives. Until now, it has hardly found its application inhe analysis of the pollutants discussed here.

ed contaminants in aquatic sediment and biota (valuing: (−) not recommended;

GC method Detection

-column PTV GC GC × GC ECD LRMS MS/MS HRMS

± ++ ± ++ + + ±

+ ++ ± ++ ++ + ++ (++)b + ++ ++ ECNI:

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t all.

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. Detection

The third step in the GC analysis of halogenated pol-utants is the detection. The predominantly used detectorsre ECD, LRMS, ITMS/MS, TOF-MS and HRMS. Tripleuadrupole MS/MS has not found a wide application [190].S techniques can be used either with electron impact (EI)

r ECNI ionisation. Table 3 shows a qualitative evaluation ofhe pros and cons of the various detectors. ECD has foundts application mainly in the analysis of PCBs and OCPs105,191–197], toxaphene [29,105,198] PBDEs [149] and asdetector for GC × GC applications on PCDD/Fs and dl-PCBsnd PBDEs [175,179,187]. The application of ECD detections straightforward, it is sensitive and provides fairly simple andasy-to-interpret chromatograms. However, ECD detection isensitive to electronegative interferences. 13C12-labelled stan-ards cannot be used and co-elutions can cause biased results.hen not successfully resolved chromatographically, such com-

ounds can complicate the interpretation of the chromatogramsnd may result in inaccurate quantification. Therefore, muchffort should be put in the GC separation and in the removalf interferences by clean-up of the extract, as discussed ear-ier. Compared to normal ECD, �ECD is equipped with amall volume detection cell (e.g. 150 �L), which is essentialor maintaining narrow peaks after GC separation, especiallyith applications that produce narrow peaks (e.g. GC × GC andarrow bore short column separations). The benefit of MS tech-iques is the improved identification compared to ECD. In theS, compounds are being ionised and subsequently separated

ased on their mass-to-charge ratio (m/z). There are two ioni-ation techniques: EI and ECNI. The separation of ions takeslace in an electromagnetic field induced by a quadrupole, aagnetic field or based on the time it takes the ions to arrive at

he detector in an electromagnetic field (time-of-flight, TOF).ith low-resolution instruments, the mass resolution is unitass generally, whereas with high-resolution instruments mass

esolution of over 10,000 are achieved. LRMS detection (EI orCNI combined with single quadrupole separation of the result-

ng ions) has been used in a variety of studies for its sensitivity,electivity and the fact that this type of instrument is widelyvailable and fairly easy to operate and optimise. Applicationsf LRMS include analysis of BFRs [50,51,106], CPs, toxaphene199] and PCNs [11,13,14,132]. Ion trap-MS/MS (ITMS/MS)as been used in several studies on toxaphene [93,156,200–201]nd on PCDD/Fs [119,130,202] and CPs [203,204]. The benefitf ITMS/MS is its higher selectivity and sensitivity when used inhe MS/MS mode and the confirmation possibilities by recordingull scan spectra of product ions [190]. TOF-MS is a very strong

S technique that is increasingly used in environmental anal-sis. It provides excellent resolution and mass accuracy [190].ull scans are being generated continuously (i.e. throughout thehromatogram), which allows for unambiguous identification.hromatographically unresolved and interferences may be sep-
rated using the deconvolution software, a feature that is onlyvailable for TOF analysers. Prices of TOF-MS instrumentsre considerably higher than those of LRMS. This limits theroad application of the technique (Table 3). Finally, HRMS
[sdt

romatogr. A 1186 (2008) 161–182 175

as widely been applied for the analysis of PCDD/Fs and dl-CBs [119,205], toxaphene [29,206] and PBDEs [121]. The

echnique provides excellent sensitivity (down to 100 fg for,3,7,8-TCDD) and mass resolution [190]. Unfortunately, thenvestment and maintenance costs are high (Table 3 and Fig. 2),hich also has limited its broad application. Please refer to the

eview by Santos and Galceran for MS techniques applied innvironmental analysis [190].

Because of the narrow peaks provided by GC × GC100–600 ms at the baseline), high data-acquisition rates areequired in order to obtain sufficient data points accuratelyescribing the eluting peak [174,207]. Its high operating speedup to 500 spectra/s) makes TOF-MS the ideal detector for thearrow peaks from GC × GC [116,144]. In spite of its rela-ively low frequency, LRMS (ECNI mode) has been successfullypplied as detector for GC × GC [185]. Other detectors used inC × GC studies, including �ECD [112,175] and ITMS/MS

144].PCDD/Fs and dl-PCBs. In the detection of PCDD/Fs and

l-PCBs, EI–HRMS is currently the golden standard. Alter-ative techniques are ECD (for mono-ortho CBs, but witho-elution risks [151]), ECNI–LRMS (for non-ortho CBs)44,96], ITMS/MS (see below) and GC × GC–TOF-MS [144].emmochi et al. [208] optimised collision characteristics in

TMS/MS and thereby improved the mass resolution. As aesult, the iLOD for 2,3,7,8-TCDD decreased from 100 to0 fg. Within the framework of the DIFFERENCE project183], GC × GC–�ECD, GC–ITMS/MS were developed andubjected to an extensive validation against the GC–HRMSechnique. For real samples, accuracy, precision and LOQsere in the same range (fish oil, fish), or slightly less (milk,ork) compared to GC–HRMS results [119,144,183,186], con-rming the potential of these alternative techniques. However,

t should be noted that although the GC × GC–�ECD andC–ITMS/MS techniques require lower investments, the sam-les may require more labour due to additional clean-up,ore frequent maintenance of the instrument (GC–ITMS/MS)

r more data treatment time to evaluate the complex chro-atograms (GC × GC–�ECD) [144]. The overall costs for

he ITMS/MS analysis of PCDD/Fs and dl-PCBs (Fig. 2)re comparable to the lower range of GC–HRMS and muchower than for those of GC × GC–�ECD. For GC × GC anal-sis, ECNI–LRMS is a suitable detector for most PCDD/FsiLOD = 10–110 fg injected), except for the important 2,3,7,8-CDD congener and OCDD, for which ECNI provided notnough sensitivity (430–710 fg injected) to compete with HRMS185].

PCBs and OCPs. ECD and MS may both be used forhe detection of PCBs and OCPs. ECD detectors are attrac-ive because of their low costs and high sensitivity. However,heir selectivity is limited. 13C12-labelled standards cannot besed and co-elutions and other interferences can cause biasedesults, as is experienced in PCB–OCP interlaboratory studies
209]. �ECDs are even more sensitive (5–10-fold) due to themaller cell volume. MS techniques are preferred for accurateetermination, because of the unambiguous identification andhe possibility to use 13C12-labelled standards [209], although

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t should be noted that for PCB homologues the spectra aredentical (which limits the selectivity gain compared to ECD).I–LRMS is less sensitive (low pg) than ECNI–LRMS (low fg).he latter technique is especially sensitive to higher chlorinatedompounds [47]. Gomara et al. [110] evaluated EI–ITMS/MS.hey isolated the [M+2]•+ and [M+4]•+ as precursor ions and

he resulting daughter ions were obtained through loss of twohlorine ions ([M−235Cl]+ and [M−35Cl37Cl]+) [110]. Veren-tch et al. [130] selected slightly different precursor and daughterons. The iLOQ in both studies was approximately 0.1–1.2 pgnjected, being somewhat higher than that of EI–HRMS (but this

ay be compensated by using LVI). Depending on the PCB ofnterest, sensitivity was only slightly better or worse compared toECD. EI–ITMS/MS and EI–HRMS results were comparable

or marine biota extracts [130]. EI–HRMS was also used in sometudies and provides excellent selectivity [110,130]. The mostbundant isotope ions monitored are M+, [M+2]+ and [M+4]+

130]. In GC × GC analysis, TOF-MS [210] and �ECD [111]ere used for the detection of OCPs and �ECD [112,129,177],I–LRMS [211] and TOF-MS [111,176] were used as detectors

or PCBs.PCNs. ECD and MS may both be used for the detection of

CNs, although the latter technique is more selective. Mostlypplied are EI–LRMS and ECNI–LRMS [11,14,148,212]. SIMs used for quantification of the individual congeners and homo-ogue groups. Ion trap-MS was used by Wiedman et al. [213],sing molar responses for the quantification. Wang et al. reportedhe use of ITMS/MS detection [15], but without reporting the

S/MS transitions used. ITMS/MS provides good sensitivitynd improved selectivity compared to single MS techniques.I–HRMS has also been used for the detection by several labsroviding excellent sensitivity and selectivity [13,148,214]. ForC × GC detection of PCNs, �ECD has been used [111].CPs. The problem of CP analysis is the extreme complexity

f the technical mixture and of the patterns in the environmen-al samples. When using ECD the chromatogram shows oneuge hump, which can of course be quantified, but which at theame time lacks any accuracy because of differences betweenhe technical standards and the samples [103]. In ECNI-MS, theain ions produced are [M−Cl]−, [M−HCl]− and [M+Cl]−.CNI response factors vary with chlorination degree: three to

our chlorines are not detected whereas congeners with sevenr more chlorine atoms are overestimated [19]. ECNI–LRMSuffers from some mass interferences from ions with five car-on atoms less and two chlorine atoms more [16]. Many of suchpairs’ exist (e.g. C10H14Cl8 and C15H26Cl6, see [16] for anverview table) when both SCCPs and MCCPs are present innvironmental samples. The determination of isotope ratios cane used for tracking possible interferences. Identification is oftenerformed by summarising the possible isomer ions per numberf carbons (e.g. C11Cl15 to C11Cl19) [17,19]. This results in aotal response per chain length, which can be compared with theotal response per chain length obtained from a selected tech-
ical mixture. Another means to determine the carbon chainength profile is by carbon skeleton reaction GC, in which thePs are dechlorinated in the injector with a palladium catalyst
215], but this method has to our knowledge not been applied to

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matogr. A 1186 (2008) 161–182

nvironmental samples. For GC × GC detection of CPs, �ECDas been used [111].

Toxaphene. ECD has yet been used for the detection ofoxaphene in several studies [29,198]. ECD response factorsor the predominant congeners vary from 0.6 to 2.0 [29]. ECDs much less sensitive for toxaphene than, for example PCBs,ue to the aliphatic character of toxaphene. Again, ECD lackspecificity and OCPs in particular can interfere. A selectivelean-up can minimise but not omit these interferences (foretails see the sample clean-up section). Therefore, ECD resultsn environmental samples tend to be higher than results obtainedy MS detection. With the introduction of ECNI-MS, moreeports on toxaphene in the European environment becamevailable. The higher selectivity of MS provides, informa-ion on homologue groups (hexa- through decachlorobornanesnd -bornenes). EI is less sensitive than ECNI, except forhe lower chlorinated congeners where EI provides best sen-itivity [26]. With ECNI, the [M]− and [M−Cl]− ions cane monitored [26,104,131,137,198]. ECNI–HRMS used at aesolution of 10,000 is a very selective method of detection,irtually free of interferences [29,131]. Gouteux et al. [93] eval-ated EI–ITMS/MS for the detection of individual congeners.he EI mass spectra are rich of ions that can be chosen asarent ion in MS/MS experiments. They tested several transi-ions and concluded that for P26, 40, 41, 44 transition of m/z25 → 89 was most sensitive, whereas for P50 it was 279 → 243nd 305 → 267 for P62. Their detection limits were 0.08 and.37 ng/g ww. A similar EI–ITMS/MS method was used byernardo et al. [156]. For additional information, one shouldonsult the comprehensive reviews available [26,48,104]. ForC × GC detection of toxaphene, �ECD and ECNI-TOF-MSave been used [111,180].

BFRs. EI–HRMS and ECNI–LRMS are the detection tech-iques most commonly applied [50,51]. Other techniques usedre EI–LRMS and EI–ITMS/MS [146]. ECD [114] has beensed as well. ECNI-MS provides a good sensitivity and selec-ivity for the detection of BFRs. The most commonly monitoredons are m/z 79 and 81, representing the two bromine isotopes50]. These ions are not very specific, but the molecular ions arenly produced at low yields, resulting in insufficient sensitivity.or BDE 209, the m/z 484.7 and 486.7 can be monitored as wellnd for HBCD m/z 561 can be used as qualifier ion (but due to theow yield, its not suitable for quantitation when aiming for lowevel samples). In EI-MS, the most commonly monitored ionsre [M−Br2]+ and [M]+. They provide a good selectivity, butlower sensitivity, especially for the higher brominated PBDE

ongeners (hepta- to deca-BDE) [51]. This can be overcomey LVI of larger volumes (e.g. 20 �L [216]). EI-MS enables these of 13C12-labelled standards, which is important for a reliableuantification. HRMS provides good sensitivity and selectivity,ut at higher instrumentation investment and maintenance costs.n EI–ITMS/MS the molecular BDE ion fragmented (using CID)n the [M−Br2]+ or [M−COBr]+ ion. The instrumental LOD
as 0.1–1.3 pg/�L (4 �L injected) [146]. TOF-MS may be used
or the detection of PBDEs at a sensitivity comparable to otherS techniques [182,217]. Due to the limited linear range of

he instrument, samples with large variation in concentration of

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BDEs often require re-analysis [217], which hampers a broadpplication of the instrument. An overview of detection tech-iques, benefits, drawbacks and ions monitored in MS detectionan be found elsewhere [51,106]. For GC × GC detection ofFRs, TOF-MS [179,210] and �ECD [111,179] have been used.

. Quality assurance

The analysis of organic pollutants is laborious and complexnd involves many steps. Errors are easily made in extraction,lean-up, GC determination and quantification, as discussed ear-ier. Accurate analysis of halogenated pollutants is important forcientists and policy makers who rely on the data produced innvironmental laboratories. To minimise the chance of errors,teps should be taken to improve the analysis and quality con-rol systems should be established and routinely applied (e.g.ccording to ISO-17025). This includes the use of high-qualitytandards and internal standards, blank tests, replicate analysis,ecovery experiments, plotting quality control charts, partici-ation in interlaboratory studies and the analysis of certifiedr standard reference materials (CRM, SRM) and laboratoryeference materials (LRMs) [218].

High-quality standards are commercially available from var-ous suppliers. Internal standards should preferably be massabelled and used in combination with MS detection. Mass-abelled standards are available, e.g. for PCDD/Fs, PCBs, OCPsnd BFRs. Most compounds can be quantified individually, butn case of total-toxaphene and SCCPs, quantification is based onvailable technical mixtures. Because of that, large inaccuraciesesult as will be discussed below.

Participation in interlaboratory studies and analysis of CRMsnd SRMs on a regular basis provides a performance testompared with external sources. An overview of frequentlyrganised interlaboratory studies can be found in Table 4. Unfor-unately, no frequent interlaboratory studies are available forCCPs and PCNs. Fish and sediment CRMs and SRMs arevailable for PCBs, OCPs, PCDD/Fs, PBDEs and toxaphene219–223]. Wet, sterilised matrix-type CRMs produced by theommunity Bureau of Reference of the European Commis-

ion (BCR) are favourite over non-matrix-type CRMs for theirery close matrix resemblance [224]. These CRMs are availablehrough the Institute for Reference Materials and Measurements,
eel, Belgium. Feasibility studies showed that a successful cer-
ification of low level CRMs is possible for BFRs, PCDD/Fs,CBs and OCPs, but unfortunately, these materials have notecome available [223]. Again, no RMs are available for PCNs

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CDD/Fs and dl-PCBs Fish, shellfish, sedimentCBs, OCPs Fish, shellfish, sedimentCNs naoxaphene Fish, shellfishPs naFRs Fish, shellfish, sediment

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romatogr. A 1186 (2008) 161–182 177

nd SCCPs. This is surprising as SCCPs are produced by farore than other compounds (Table 1) and the especially theCCP analysis is vulnerable for large inaccuracies.

PCDD/Fs and dl-PCBs. Because of the low concentrationevels, laboratories should take care of cross contaminationetween high and low contaminated samples. Specialised lab-ratories often use separate glassware and clean rooms forow-level samples. Laboratories are often accredited for this

ethod and although the analysis is very laborious and com-licated, the results within one laboratory can be very accuratewith repeatability as low as 5% for individual congeners),ainly because of the application of mass-labelled internal stan-

ards. The agreement between data on individual congeners,xpressed as relative standard deviation, ranged from 21 to00%, with the ‘difficult’ congener, OCDD, showing the leastccuracy [225]. Interlaboratory studies (ILS) and RMs are avail-ble.

PCBs and OCPs. High-quality standards (individual OCPsnd PCB congeners) are widely available and several CRMs forCBs and OCPs in biota (fish oils, whale blubber, mussel tissuesnd fish tissues) and sediment are available. ILS are availableTable 4) and they show that laboratories have generally moreifficulties in producing good quality data for OCPs than forCBs. ECD is a commonly used detector, but inaccuracies due

o interferences often occur. At recent dedicated QUASIMEMEorkshop it was concluded that MS detection is preferred overCD [209]. This also allows the use of mass-labelled standards,

urther improving the accuracy.PCNs. Several PCN congeners are commercially available

s standards [226]. Wiedmann and Ballschmiter [227] devel-ped a GC–MS quantification method using molar responses oflectron-impact ionisation. They were able to quantify all con-eners on the basis of a small set of PCNs. Due to a lack oftandards, response factors for homologue groups have repeat-dly been used [148]. In a PCN ILS, nine laboratories quantifiedomologue groups and individual congeners in test solutionserived from Halowax 1014. The variability in homologueuantification was slightly better (11–43% RSD) than for thendividual congeners (18–51%, excl. CN-29) [148]. The resultsf the announced 2nd phase ILS (including real environmentalamples) are not reported yet. To our knowledge no CRM isvailable.

CPs. Quantification of CPs is mainly done by the calibrationith technical mixtures. Recently, individual congeners haveeen produced and are commercially available [181]. Coelhant al. [22] have quantified SCCPs in fish samples using C10, C11,

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12 and C13 CPs with different chlorination degrees (47–68%)s well as quantification against Cerechlor SCCP technical mix-ure (63% chlorination). Differences were as high as 1100%hen fish samples were quantified against a low chlorinatedr a highly chlorinated standard. The authors recommendedhe use of single-chain length standards for quantification inrder to meet the specific profiles found in the fish samples22]. The use of technical mixtures that do not match the pat-ern as observed in the sample decrease the accuracy of dataeported [22]. The quality of reported data is also decreased bynterference of other chlorinated pollutants in the extract (e.g.CPs, toxaphene) when using total ion current-MS. More spe-

ific information on individual formula and homologue groups isbtained by the detection with high-resolution (HR)-MS [228].urther details on MS detection and quantification can be foundlsewhere [46]. Tomy et al. [229] organised an interlaboratorytudy on the quantification of SCCPs. The data from the sevenarticipating laboratories showed that the true value of standardolutions was overestimated up to 150%. The coefficient of vari-tion for the fish extracts was 27 and 47%, which is reasonablyood taking into account the lack of reliable standards. Givenhe lack of accuracy, it is highly surprising that many studieseport data with high level of suggested accuracy (e.g. reportingn several decimals). The reporting should be adjusted so as toeally represent the level of accuracy of the methods applied. NoRMs are available for CPs and although CPs were detected in

tandard reference materials (SRMs) from the National Instituteor Standards and Technology (NIST) [228], these SRMs areot certified for CPs. There is a clear need for further methodevelopment and ILS for CPs also because they are included inhe target contaminants list of the European Water Frameworkrogramme.

Toxaphene. For the quantification of toxaphene often tech-ical mixtures are used as standards and levels are reported asotal-toxaphene resulting in a mismatch between the congenerrofile present in the sample and the technical mixture [104,206].

change in the composition of technical toxaphene may forxample occur in split/splitless injection [137], leading to biasedesults when quantifying against technical toxaphene mixtures.lthough there is a lack of standards for individual congeners,limited number is commercially available (e.g. Parlar nos. 26,2, 40, 41, 44, 50 and 62) [104]. Currently, no CRM is available219]. The NIST SRM 1588a and 1945 have indicative valuesor toxaphene [104,230]. ILS is available from QUASIMEMETable 4).

BFRs. Methods for PBDE analysis have been developedy a vast number of laboratories worldwide in the last 5ears. Recently, over 170 PBDE congeners, individual HBCDiastereomers, (me-)TBBP-A and others became commerciallyvailable. For many of those, isotope-labelled and fluorinatednternal standards are available. A number of ILS have beenrganised for sediment and biota samples. Improvement waseen over the years for most PBDEs, although BDE 183 and
09 remain problematic [49]. de Boer and Wells [49] providedeveral analytical solutions for these problems. Recently, SRMsor sediment and fish were analysed (and certified for fish only)or PBDEs [231]. Blank tests are important, because of the pres-
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matogr. A 1186 (2008) 161–182

nce of organohalogen pollutants in dust, electric equipment anduilding materials [232–235] present in laboratories. de Boernd Wells [49] presented an overview of blank problems in theFR analysis. Special care should be taken to avoid contami-ation of samples and extracts by BDE 209 from dust and air.urthermore, it is essential to use separate sets of glasswarend extraction and clean-up equipment for high contaminatedamples and low contaminated samples.

. Conclusions and outlook

Extraction, and more so, clean-up and fractionation, are cru-ial steps prior to the GC analysis of halogenated pollutantsecause co-extracted compounds such as lipids and sulphur havemajor negative effect on their delectability at the trace lev-

ls at which they normally occur in the environment. SelectiveLE provides an effective and efficient extraction and clean-p technique that enables processing of multiple samples in ahort time (less than 1 h). Developments in injection have beenomewhat limited over the recent years. Large volume injectione.g. by PTV, or cold on-column) is interesting for obtainingetter LOQs. Septumless injection has been introduced to avoideptum particles to enter the column. A wide choice of autosam-lers is now available, both for on-column and splitless injection.C × GC is a strong technique for unravelling complex mix-

ures. By selecting the right column combinations, structuralnformation can be obtained. The narrow peaks offer a betterensitivity compared to single-column GC that even enableshe determination of low (pg/g) dioxin concentrations. Masspectrometry in various set-ups is the preferred detection tech-ique. QA tools such as interlaboratory studies, use of LRMsnd CRMs are very well established for PCDD/Fs, OCPs andCBs but improvement of that infrastructure is needed for BFRs,Ps, PCNs and toxaphene.

Future developments in the analysis of halogenated con-aminants will focus on a reduction of sample handling time.he QuEChERS approach (quick, easy, cheap, effective, ruggednd safe (QuEChERS) fit in that development. Basically, theomogenised sample is dispersed with adsorption material (e.g.18, active carbon) and a buffered medium polar solvent (e.g.cetonitrile) [236]), resulting in a ready-to-inject extract. So far,he method was only applied to polar pesticides in agriculturalroducts [237,238], but adoption of this dispersive method toon-polar contaminants should be feasible. As selective PLEffers a substantial gain in extraction and clean-up time, it willost likely see a further development in the near future.Pressure on fast analyses may also stimulate developments

n GC and MS techniques. Direct analysis real time (DART)s a non-contact analysis technique for MS at atmosphericressure [239]. The sample is placed in a warm gas jet andn electrode generates electrons. Ionised compounds are thenransferred into the MS for direct characterisation. DART cannalyse gases, liquids, solids and materials on surfaces. It was
lready applied for drugs, metabolites, explosives, forensics,hemical weapon agents, synthetic organics, organometallicompounds, pesticides and toxic industrial materials [239,240].ther new techniques are desorption electrospray ionisation

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DESI)-MS and desorption atmospheric pressure chemical ion-sation (DAPCI)-MS, in which the (more polar) compoundsf interest are directly desorbed into the MS without chro-atographic separation. Applications have been developed for

xplosives, mycotoxins and pharmaceuticals in various sampleatrices [241,242].Bio-specific analysis relies on an interaction of the pollu-

ant with an antibody or other type of receptor. With minimalample pre-treatment only, contaminant concentrations can beetermined. When binding of a pollutant to a specific antibodys obtained, detection can be achieved by, e.g. surface plasmonesonance (SPR) technology [243].

Finally, the increasing number of halogenated (and non-alogenated) industrial contaminants that are being found inur environment may stimulate non-target approaches such asioassay analysis. Effect-directed analysis (EDA), in which tar-et analysis is only carried out to identify a contaminant whenspecific effect is found after a series of fractionations, will

ombine the advanced analytical methods described above andoxicological information [244,245].

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