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Basic aspects of methods for the determination of dioxins and PCBs in foodstuffs and human tissues
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Basic aspects of methods for the determination of dioxins and PCBs in foodstuffs and human tissues A.K. Djien Liem*
Laboratory for Organic-analytical Chemistry, National Institute of Public Health and the Environment, P.O. Box 1, NL-3720 BA Bilthoven, The Netherlands A considerable improvement can be noted in determinations of PCDDs, PCDFs and PCBs in foodstuffs and human tissues. In recent years, numerous validated schemes for extraction, clean-up and instrumental analysis have been published, enabling an accurate and precise determination of the toxicologically relevant congeners. In this article, the basic aspects of current methods and techniques are discussed. z1999 Published by Elsevier Science B.V. All rights reserved. Keywords: Foodstuffs; Human tissues; Active carbon; PCDD; PCDF; Polychlorinated biphenyls
1. Introduction Earlier methods for the determination of PCDDs and PCDFs in the study of their formation in industrial and chemical processes, and of their occurrence in products such as PCBs, chlorophenols and £ue gases from waste incineration, used packed column gas chromatography ( GC ) in combination with massspectrometric detection ( GC^MS ). It was only possible to determine the homologous groups with 4^8 Cl atoms without any speci¢cation of the levels of individual congeners. Around 1980, analytical techniques became increasingly sophisticated [ 1 ] enabling isomerspeci¢c determinations and facilitating the accurate determination of individual congeners at the low-ppt level. In addition, the introduction of the concept of toxic equivalency factors led to a shift in interest from total dioxin levels to the concentrations of the 2,3,7,8substituted tetra- to octachlorinated congeners. At present, the use of activated carbon to isolate the pla-
nar aromatic molecules such as the PCDDs and PCDFs [ 2 ], and the separation and detection of speci¢c isomers by capillary ( high resolution ) gas chromatography combined with high resolution MS ( HRGC^ HRMS ) are common features of methods for the determination of dioxins. A similar history can be noted for the PCBs. The production and use of technical mixtures of PCBs have led to their presence all over the world. Not only the low concentration level of the large number of PCB congeners, but also the simultaneous presence of other persistent contaminants ( e.g., halogenated pesticides, PCDDs, PCDFs ) pose distinct problems. In the past, the PCBs were fully chlorinated with SbCl5 to decachlorobiphenyl, with its single peak representing `total PCBs'. Since the middle of the 1980s, the determination of individual congeners is regarded as the preferred quanti¢cation technique, and legislation is based on quantifying a few selected congeners, not on reporting `total PCBs'. Several standardised schemes are used for the determination of PCB congeners 28, 52, 101, 118, 138, 153 and 180, a selection often referred to as `indicator PCBs' that is based on abundance, chromatographic resolution, response and availability as a standard, but not on toxicological considerations [ 3 ]. Indeed, considerable improvements can be noted in techniques and methods to determine the PCBs, PCDDs and PCDFs. Using appropriate instrumentation and the available knowledge of their properties during different steps of the analytical process, an experienced chemist should be able to perform accurate and precise determinations of the compounds of interest. This can however only be achieved if basic requirements are taken into account. In this article, technical aspects and critical steps for each step of the analytical process are presented. Trends and developments will be discussed in another contribution.
*Tel.: +31 (30) 274 2871; Fax: +31 (30) 274 4424. E-mail: [email protected] 0165-9936/99/$ ^ see front matter PII: S 0 1 6 5 - 9 9 3 6 ( 9 9 ) 0 0 1 1 2 - 0
ß 1999 Published by Elsevier Science B.V. All rights reserved.
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2. Basic aspects of determinations of PCDDs, PCDFs and PCBs The complexity of determinations of PCDDs, PCDFs and PCBs is mainly caused by the low levels at which these compounds occur and the pronounced differences in toxic and biological effects of the PCDD, PCDF and PCB isomers. Another complicating factor is the large number of possible interferences and matrix effects. Therefore, the following basic requirements must be met by an analytical procedure for dioxins and PCBs [ 4 ]: 1.
2.
3.
4.
High sensitivity and low limits of detection. For PCDDs and PCDFs, detectable quantities have to be in the pg range or lower and detectable concentrations in the ppt or sub-ppt range because of the extreme toxicity of some of these compounds. PCBs are known to occur at higher levels than PCDDs and PCDFs. For the most prevalent PCB congeners a sensitivity in the pg to ng range is already suf¢cient. However, for the measurement of the more toxic non-ortho-substituted congeners the same sensitivity must be reached as for the PCDDs and PCDFs. High selectivity. A distinction is required for PCDDs, PCDFs and PCBs from a multitude of other, co-extracted and possibly interfering compounds present at concentrations up to several orders of magnitude higher than those of the analytes of interest. High speci¢city. A differentiation between various isomers is desired, such as between toxic ( e.g. the seventeen 2,3,7,8-substituted PCDDs and PCDFs and the non-ortho-, mono-orthoand di-ortho-substituted PCBs ) and other isomers. High accuracy and precision. The determination should provide a valid estimate of the true concentration in a sample. Furthermore, an acceptable degree of agreement between measurements must be achieved.
For congener-speci¢c analyses of PCDDs and PCDFs, MS is ideally suited for requirements 1, 2 and 4. Especially if the mass spectrometer is operating in the selected ion recording (SIR, also referred to as selected ion monitoring, SIM ) mode, detection limits in the fg to pg range can be reached. However, MS alone cannot differentiate between most isomers ( speci¢city ) and not between different classes of
chlorinated compounds ( selectivity ). HRGC is mostly used to provide the necessary isomer speci¢city because it can be easily and directly coupled to MS. But even when appropriate GC^MS conditions are used, further ( pre )separation techniques remain necessary to prevent in£uences of interferences and matrix components. Besides the PCBs, other families of polychlorinated compounds such as the polychlorinated naphthalenes (PCNs ), diphenyl ethers (PCDPEs ), biphenylenes (PCBPs ), pyrenes (PCPYs ) and terphenyls (PCTs ) are known to interfere with determinations of PCDD / Fs [ 4 ]. Extremely high resolving powers are required in MS to distinguish TCDD from some other chlorinated compounds. At such high resolving powers, MS sensitivity will soon become insuf¢cient to identify TCDD at the required limits of detection. In the case of PCBs, GC with an electron-capture detector ( ECD ) is the instrument most commonly used in analytical practice. The ECD is suf¢ciently sensitive to detect most prominent congeners in environmental and biological samples while the required isomer speci¢city can be attained by the use of appropriate stationary phases in gas chromatography. In the case of complex GC^ECD chromatograms, subsequent GC^MS analysis may provide additional information to con¢rm the identity of the analytes. Extensive sample extraction and clean-up techniques are also required for the determination of PCBs. However, the problem of potentially interfering compounds is less severe than with dioxins as most PCB congeners are present at much higher levels than the PCDDs and PCDFs. An exception are the toxic non-ortho PCB congeners, because they are usually present at much lower concentrations than the other congeners. For the determination of these congeners in foodstuffs and human tissues, the more sensitive GC^MS is becoming the preferred technique. In the last two decades, several techniques and methods have been introduced to reach the required sensitivity and selectivity in the determination of PCDDs, PCDFs and PCBs. In the following sections, it is not intended to give an exhaustive account of available methods; instead, the methods most commonly used will be described. 2.1. Extraction procedures
The purpose of extraction is to remove the bulk of the sample matrix and to transfer the fraction containing the analytes in a suitable solvent ( mixture ).
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2.1.1. Food Extraction techniques for food are generally based on the assumption that lipophilic organic compounds such as the PCDDs, PCDFs and PCBs predominantly occur in the fat fraction of the food matrix. Sample extraction methods are therefore based on general methods for the isolation of the lipid fraction from the sample matrix [ 5 ]. Consequently, the amount to be taken as a test sample depends on the required sensitivity and the lipid content of the sample matrix. Extractions should yield at least a few grams of extractable lipids to perform reliable determinations. In duplicate diet studies, duplicate portions of all food products ( meat, fruit, vegetables, beverages, etc. ) that a volunteer consumes during a certain time period ( typically 24 h or 7 days ) are collected and subsequently mixed to one composite sample. After thorough homogenisation, these composite samples are freeze-dried to remove the water content and subsequently blended in the presence of an organic solvent mixture such as acetone^pentane or Soxhletextracted with organic solvents such as pentane or hexane to isolate the lipid fraction of the sample. Methods for the isolation of lipid fractions from individual food products depend on the type of sample. Butter, fats and oils are generally assumed to be homogeneous, and normally do not require extensive extraction procedures. Aliquots of such samples can be dissolved in n-hexane or light petroleum to the desired fat concentration. Meat and ¢sh samples are initially blended and homogenised. Next, a representative test sample is ground with anhydrous sodium sulphate, until a free £owing powder is obtained. This mixture can then be extracted using either blending techniques, a cold column extraction technique ( elution of a column packed with the powder with organic solvent or solvent mixture )[ 6 ], or a Soxhlet extraction technique [ 7 ]. Milk can either be freeze-dried or chemically dried with anhydrous sodium sulphate, followed by Soxhlet extraction with organic solvent, or subjected to a liquid^liquid extraction procedure consisting of mixing with sodium oxalate and ethanol or methanol, followed by ( repeated ) extraction steps with a combination of organic solvents such as acetone^pentane [ 8 ] or diethyl ether^light petroleum [ 9 ]. Vegetables usually have a high water content and will dehydrate prior to analysis unless extracted immediately after sampling. Vegetable matter is therefore crushed, chopped and gently dried at 40^50³C prior to
storage and analysis. Isolation procedures include grinding with coarse sea sand, blending, mixing with a more polar solvent ( acetone ) and subsequent partitioning with dichloromethane or hexane ( applicable for matrices with a high sugar content ) or mixing with a more polar solvent ( acetone ) followed by either shaking or Soxhlet extraction [ 10 ]. Saponi¢cation under alkaline conditions ( in the presence of ethanol and KOH ) followed by extraction with organic solvent is often employed for the analysis of large amounts ( up to hundreds of grams ) of fats. However, these methods are known to lead to a loss of some of the higher chlorinated PCBs [ 11 ], to degradation of PCDDs and PCDFs in proportion to their chlorine content, and, in the case of PCDFs, to production of lower chlorinated PCDFs and ethoxyPCDFs as artifacts [ 12 ]. 2.1.2. Human tissues In the analysis of human milk and adipose tissue, samples are treated in a similar way as described for milk and meat in Section 2.1.1. In the analysis of blood, methods for blood plasma or serum have been introduced. The majority of determinations use liquid^liquid partitioning techniques in a similar way as for milk samples. It should be noted that partitioning of PCBs, PCDDs and PCDFs among the cellular, serum protein and lipoprotein components of whole blood may depend on class (PCBs vs PCDD / Fs ) as well as on degree of chlorination [ 13 ]. 2.2. Sample clean-up
Additional steps are usually required to remove the bulk of co-extractants, to separate organochlorine residues into groups and to concentrate the ¢nal extract to appropriate volumes of solvent, allowing the detection of the analytes at the ultra-trace levels at which they usually occur. The choice of a particular sequence of steps will be highly dependent on the analytical system which is ¢nally used. Sample extraction, clean-up and GC method together form a delicately matched set, each of which will contribute to the ultimate speci¢city and selectivity. For PCBs, solid^liquid adsorption chromatography using Florisil, silica and alumina, gel permeation chromatography ( GPC ) and chemical methods are most commonly used [ 5 ]. For the determination of PCDDs and PCDFs, nearly all established schemes involve combinations of clean-up methods developed for the analysis of PCBs and organochlorine pesticides (OCPs ) in combination with an active carbon step to
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isolate a speci¢c fraction containing the PCDDs and PCDFs [ 2 ]. Some recent methods to isolate fractions for the determination of the non-ortho and monoortho PCB congeners frequently employ active carbon as well.
procedure have been introduced for the analysis of animal and human tissues [ 14^16 ]. In most methods, the carbon step is combined with one or more steps to provide additional selectivity, e.g., by means of acidor base-activated alumina column chromatography, or with GPC. The performance of several types of activated carbon for the separation of non-ortho PCBs has been studied by Kannan et al. [ 17 ]. They compared brands from six suppliers and concluded that the charcoals tested did not allow a complete separation of non-ortho PCBs from the rest of the dominant ortho-substituted PCB congeners at trace levels. Using multidimensional GC, they observed co-elution of PCB 110 with PCB 77, and PCBs 129 and 178 with PCB 126 that could lead to overestimation of the results if a single column (SE-54 ) GC determination was performed. Additional separation is obviously needed to prevent these systematic errors from occurring.
2.2.1. Carbon chromatography The basis of carbon chromatography for the selective fractionation of planar aromatic molecules such as PCDDs and PCDFs was laid by Smith and co-workers [ 2 ]. Compounds markedly retained on the carbon are certain planar multi-ring aromatic compounds, PCDDs, PCDFs, non-ortho PCBs, PCNs, PCDPEs and xanthenes [ 14 ]. The high retention is based on the coplanarity of closely situated aromatic systems and is increased by electronegative substituents ( chlorine, bromine, nitro ). The fraction containing the PCDDs and PCDFs is recovered from the carbon column by back£ush with organic solvent, e.g., toluene. A major drawback of ¢nely divided carbon as a packing material is the high back-pressure. Therefore, ¢nely divided carbon dispersed on the surface of shredded polyurethane foam, carbon dispersed on glass ¢bres and clean-up through a series of silicates ( silica, potassium silicate ) have been recommended. In this way, coloured acidic lipid materials, hydroxyPCBs and hydroxy-diphenyl ethers are retained by the silicates, allowing the carbon to be re-used [ 14 ]. The original method was used for the determination of ppt levels of PCDDs and PCDFs in environmental samples [ 2 ]. Subsequently, several modi¢cations of this
2.3. Identi¢cation and quanti¢cation
Current methods for the separation and detection of isomers of PCBs, PCDDs and PCDFs all involve GC^ ECD ( indicator, mono-ortho and di-ortho PCBs ) and, increasingly, GC^MS (PCDDs, PCDFs and non-ortho PCBs ). The identi¢cation of the target analytes is performed by comparing their relative retention times in the chromatogram of the sample extract with those in the chromatogram of a solution containing pure standards with known concentrations. To this end, each
Table 1 Separation of toxic congeners of PCDDs and PCDFs on GC columns of different polarity [ 18,29 ] Congener PCDDs 2,3,7,8 1,2,3,7,8 1,2,3,4,7,8 1,2,3,6,7,8 1,2,3,7,8,9 PCDFs 2,3,7,8 1,2,3,7,8 2,3,4,7,8 1,2,3,4,7,8 1,2,3,6,7,8 1,2,3,7,8,9 2,3,4,6,7,8
DB-1
DB-5
DB-17
DB-210
DB-225
CPS-1
SP-2331
SIL 88
Smectic
QNT QNT QTT QTT CE1
CE2 BS QTT QTT CE1
CE1 QNT BS BS BS
CE1 CE1 NBS NBS BS
QNT CE1 NBS NBS BS
CE4 BS BS BS BS
BS BS NBS NBS BS
QNT BS NBS NBS BS
BS BS QTT QTT BS
CE5 QT CE1 CE1 NBS NBS CE1
CE5 QNT CE3 CE1 QNT NBS QNT
BS CE1 BS QNT CE1 BS QNT
QNT CE2 BS CE1 CE1 CE1 BS
CE1 CE1 BS QNT CE2 BS CE1
CE1 CE1 QNT CE1 NBS BS BS
QNT CE1 BS CE1 QTT BS BS
QNT CE1 BS CE1 QTT BS BS
CE1 BS CE1 NBS NBS BS CE1
BS = no overlap with others; NBS = almost no overlap; QTT = quanti¢able, but may overlap with congener with same toxicity; QNT = quanti¢able, but may overlap with non-toxic congener; CEn = co-elution ( 100% overlap ) with n non-toxic congeners.
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compound should appear as a single peak. Quanti¢cation is carried out by comparing peak heights or areas in sample and standard solution. The use of multiple internal standards is considered essential to improve the accuracy. 2.4. Isomer identi¢cation 2.4.1. PCDDs and PCDFs The separation characteristics and elution pro¢les of GC columns with different stationary phases for the congener-speci¢c analysis of PCDDs and PCDFs have been investigated by Ryan et al. [ 18 ]. Their results are summarised in Table 1. By using apolar columns ( CP SIL 5, DB-5, HP Ultra 2 and equivalents ), a separation can be achieved between homologous groups. In addition, apolar columns can separate the 2,3,7,8-substituted congeners from each other; however, they fail to resolve these congeners from the non-toxic congeners. With polar columns ( DB-DIOXIN, CP SIL 88, SP 2331, Silar 10c and equivalents ) the resolving power improves, but incomplete separation still remains for 2,3,7,8TCDF, 1,2,3,7,8-PeCDF and 1,2,3,4,7,8-HxCDF. TCDF is most dif¢cult to resolve and requires medium polar columns ( DB-17 or equivalents ). Apolar columns are frequently used for the analysis of biological samples, including human tissues, as these samples generally do not contain congeners other than the 2,3,7,8-substituted ones. Apolar columns require shorter analysis times, have longer lifetimes and no decomposition occurs for highly chlorinated PCDD / Fs [ 19 ], a poorly understood phenomenon that occurs with the use of polar columns [ 20 ]. Polar columns are required for 2,3,7,8-isomer-speci¢c analyses in samples that may contain non-laterally substituted isomers, such as environmental samples, £y ash, ambient air and aquatic organisms and PCDD / F-contaminated products of different origins. An alternative is to use a Rtx-2330 cross-bound polar column with elution pro¢les similar to that of other polar columns, that can withstand higher temperatures ( 275³C ) enabling shorter run times, and for which no dechlorination could be observed for OCDD / F, thus avoiding re-analysis on a second column [ 19 ]. The differences in complexity of GC separations between abiotic and biotic samples are illustrated in Fig. 1.
2.4.2. PCBs The `indicator PCBs' 28, 52, 101, ( 118,) 138, 153 and 180 are routinely determined in environmental monitoring programmes and for regulatory purposes. At present, the indicator PCBs and PCBs with dioxinlike activity are frequently measured in exposure and risk assessment studies. In this regard, toxic equivalency factors (TEFs ) have been derived for a selection of non-ortho and mono-ortho PCBs to estimate their contributions to the total dioxin-like toxicity in equivalents of 2,3,7,8-TCDD [ 21 ]. The growing number of selections of relevant PCBs has inspired several analysts to tackle the problem that some of the priority PCBs are poorly resolved on the widely used SE-54 or SIL-8 type columns [ 22^24 ]. When using these stationary phases, the following separations have been reported to be critical: 28 / 31, 49 / 52, 77 / 110, 101 / 84, 118 / 149, 105 / 153 / 132, and 138 / 163. Under optimum conditions with the proper length, diameter, and ¢lm thickness, PCB pairs 28 / 31 and 118 / 149 can be separated on these columns. With more polar columns, the separation of PCBs 28 and 31 will soon become impossible, whereas the separation of PCBs 118 and 149 improves [ 25 ]. Separation problems may occur in analysing both abiotic and biotic matrices. Although PCB patterns show a general shift from lower chlorinated to higher chlorinated congeners when going from technical mixtures via soils and sediments, invertebrates and ¢sh to ¢sh-eating birds, mammals and humans, PCB patterns remain relatively complex when compared to those observed for PCDDs and PCDFs. Recently, several groups have tested narrow-bore columns for congener-speci¢c determination of PCBs in standard and technical mixtures with GC^ ECD and GC^MS using different stationary phases. The studies concentrated on possible overlap with other closely eluting PCB congeners that are potentially present in environmental and biological samples. A summary of their observations is presented in Table 2. 2.4.3. Quanti¢cation The ECD is the most widely used detector for the quanti¢cation of PCBs. The response of the ECD depends on the number and the position of the chlorine atoms in the PCB molecule. Generally, the sensitivity increases with the number of chlorine atoms. For the PCDDs, PCDFs and ( depending on their concentrations ) the non-ortho PCBs, the high resolution mass spectrometer (MS ) is currently the only technique able to provide the required sensitivity and selectivity.
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Fig. 1. GC^HRMS analysis of pentachlorodibenzofurans in a sample extract of £ue gas from a municipal solid waste incinerator, and in a human milk extract. Traces are normalized molecular ion currents. GC^MS analyses were accomplished on a Rtx-2330 ( £ue gas ) and a DB-5 column ( human milk ), respectively, at a mass resolution of 5000.
The method of internal standard quanti¢cation is nowadays routinely used in GC^MS methods for the determination of PCDDs, PCDFs and non-ortho PCBs. Quanti¢cation is generally performed by using stable-isotope-labelled (13 C12 ) analogues of the compounds to be determined as internal quanti¢cation standards ( isotope dilution ). These labelled compounds ( at least one for each homologous group ) are added in known amounts to the sample prior to extraction or clean-up and are assumed to
equal the native analytes with respect to extraction, clean-up and GC properties. The labelled compounds can be easily distinguished from the native compounds on the basis of their mass spectral differences and are therefore used as guides for identi¢cation and quanti¢cation. Prior to GC^MS, other compounds can be added as internal sensitivity standards. An internal sensitivity standard is a quality control measure and can, in principle, be any compound with suitable GC^ MS properties, although the use of additional 13 C12 or
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Table 2 Separation of indicator and dioxin-like PCB congeners from other chlorobiphenyls on GC columns of different polarity [ 23,24 ] IUPACa
Structure
SIL-5
SIL-8
SIL-19
SIL-88
SIL-8 / HT-5
28 52 77 101 105 114 118 123 126 138 153 156 157 167 169 170 180 189
2,4,4P 2,2P,5,5P 3,3P,4,4P 2,2P,4,5,5P 2,3,3P,4,4P 2,3,4,4P,5 2,3P,4,4P,5 2P,3,4,4P,5 3,3P,4,4P,5 2,2P,3,4,4P,5P 2,2P,4,4P,5,5P 2,3,3P,4,4P,5 2,3,3P,4,4P,5P 2,3P,4,4P,5,5P 3,3P,4,4P,5,5P 2,2P,3,3P,4,4P,5 2,2P,3,4,4P,5,5P 2,3,3P,4,4P,5,5P
^ ^ ^ ^ 132 ^ ^ ^ 129 160+163 ^ 171 202 ^ ^ ^ ^ ^
^ ^ 110 84 132 ^ 149 149 129+178 160+163 ^ 202+171 173+200 128 ^ 190 ^ ^
31 ^ ^ ^ ^ ( 146 ) ^ 107 ^ 160+163+158 ^ ^ 180+197 ^ 203+196 190 197 ^
16 ^ ^ ( 55 ) 129 137 200+123 118+200 ^ ^ ( 110 ) ^ ^ ^ ^ ^ ( 197 ) ^
^ ^ ^ 90b ^ ^ ^ ^ 175 160a , 163a ^ 173 ^ 185a ^ ^ ^ ^
Separations on SIL-5, SIL-8, SIL-19 and SIL-88 were performed on 50 m ( HT-5: 25 m ) f.s. columns with i.d. of 0.22^0.25 mm and a stationary phase thickness of 0.10^0.26 Wm. Separations on SIL-8 / HT-5 were performed on a two-series-coupled narrow-bore fused silica WCOT columns: 25 mU0.25 mm, 0.25 Wm SIL-8 and 25 mU0.2 mm, 0.1 Wm HT-5. ^ = Well resolved CBs ( less than 10% interference from any co-elutant on the peak height ); numbers in parentheses indicate that interference is reduced to less than 10% by use of GC^MS. a Separation can be improved by optimizing temperature programme. b Congener found at insigni¢cant concentrations in technical mixtures.
other stable-isotope-labelled (13 C6 or 37 Cl4 ) analogues is preferred. GC^ECD methods for the determination of PCBs frequently use one or more PCB congeners that are known not to occur in the sample as internal quanti¢cation or sensitivity standard( s ). In GC^MS, electron impact ionisation ( EI ) is most widely used. The EI mass spectral properties of PCDFs and PCDDs have been described by Buser [ 26 ]. If the MS is operated in SIR, high sensitivity is obtained because of the abundance of the molecular ions produced. Both isomer identi¢cation and quanti¢cation can be easily done by simultaneous monitoring of the typical chlorine isotope clusters of both unlabelled and 13 C12 -labelled PCDDs and PCDFs. The measurement of 35 Cl /37 Cl ratios in analyte peaks is a reliable means for identity con¢rmation while additional structural information can be obtained by simultaneous monitoring of typical COCl losses from molecular ions [ 4 ]. Furthermore, the analyte can be positively identi¢ed if the GC retention time corresponds to that of its labelled analogue, and quanti¢cation can be performed by comparing the response of the chlorine isotopes of the molecular ion cluster with those from the labelled analogue ( cf. Fig. 2 ).
In the last few years, more 13 C-labelled reference standards have become commercially available. As a result, the isotope dilution technique is also becoming the method of choice for the analysis of PCBs, particularly for the less abundant non-ortho- and monoortho-substituted PCBs [ 3 ]. Under EI, PCBs tend to exhibit relatively high abundances of M and [ M-2Cl ] and fragmentation usually occurs with subsequent losses of chlorine atoms. The loss of Cl is favoured in the case of ortho-Cl PCBs, in which three of the four ortho positions are chlorinated. This ortho effect is also important if two of the ortho positions are chlorinated, such as 2,2P [ 27 ]. Negative chemical ionisation (NCI ) with methane as reagent gas can be used for improved molecular weight information and / or increased sensitivity. Characteristics of the NCI mass spectra of PCDDs and PCDFs have been published by Rappe et al. [ 4 ]. NCI of PCDFs will yield a base peak due to M3 , and the fragmentation produces the unusual [ M-34 ] ions, due to the uptake of Hc and the loss of Clc . Fragmentation of PCDDs in NCI mode is more conventional and results in [ M-35 ]3 due to loss of Cl [ 4,31 ]. The fragmentation and, consequently, sensitivity depend on
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3.
The signal-to-noise ratio must be 3:1 or higher. GC^MS methods:
1. 2. 3. 4. 5. 6. Fig. 2. Mass fragmentograms ( EI, MS, resolution 3000:1 ) showing traces of the most intense molecular ions of 13 C12 -labelled ( lower trace ) and native ( upper trace ) tetra-CDD ( m / z 334 and 322 ), penta-CDD ( m / z 368 and 356 ) and penta-CDF ( m / z 352 and 340 ) in a sample of cow's milk. Measured concentrations ( in pg / g fat ) are given in parentheses.
the degree of chlorination and the substitution pattern. In contrast, using the MS in the NCI mode, the determination of 2,3,7,8-TCDD appeared to be less sensitive than that for the other TCDDs and was also found to be more sensitive for PCDFs than for PCDDs. With respect to PCBs, less chlorinated PCBs have a tendency to exhibit extensive fragmentation ( e.g., Cl atoms ) and only the more chlorinated PCBs exhibit pronounced molecular ions [ 28 ]. PCBs with more than six chlorine atoms showed an increase in response of 2^3 orders of magnitude compared to GC^EI9MS. 2.4.4. Con¢rmation of positive results A number of internal quality control measures should be routinely applied for each sample, within each day or within each series of samples. These measures may help to assure that positive data reported actually refer to speci¢c PCBs, PCDDs and PCDFs. The following criteria can be used [ 4,19 ]:
GC^ECD methods: 1. 2.
Isomer speci¢city must be demonstrated initially and afterwards. The relative retention time must be within 0.1% of the one determined for standards.
Isomer speci¢city must be demonstrated initially and afterwards. The retention time must be within 1 s of that of the 13 C-labelled analogue. The signal-to-noise ratio of all peaks of the molecular ion cluster must be 3:1 or higher. The ratio between the two molecular ions monitored should be within 15% of the theoretical values. Recovery of internal quanti¢cation standards used must not exceed 120%. Fragments should con¢rm the identity of PCDDs, PCDFs and PCBs, e.g. [ M-COCl ] ions for PCDDs and PCDFs or [ M-2Cl ] ions for PCBs, with correct chlorine isotope ratio ( optional in EI^LRMS ).
2.4.5. Interferences from other co-extracted compounds Current clean-up and puri¢cation techniques are quite ef¢cient to remove the bulk of the matrix and several other classes of compounds. Nevertheless, some chlorinated compounds can still be expected in the extracts and may interfere in the analysis. Some possible interfering compounds occasionally observed in puri¢ed extracts of environmental samples are summarised in Table 3. 2.5. Method-speci¢c detection limit
In Table 4 the method detection limits (MDLs ) that can be reached for the analysis of the lipid fraction of foodstuffs and human tissues are indicated. These estimates are based on instrument detection limits as reported in the literature and are calculated for a sample size containing 1 g of lipids, and a method featuring a ¢nal extract volume of 100 Wl and a 1 Wl injection into the gas chromatograph. The ECD response of a chlorinated compound depends on both the number of chlorine atoms and their positions in the molecule. Consequently, a wide range of response factors of PCB congeners should be considered [ 30 ]. At present, detection limits in the order of 1^10 pg on column can be achieved. With respect to GC^MS, detection limits are highly dependent on the conditions and instrumentation used. When using a quadrupole instrument with EI and SIR, detection limits are about 1^10 pg for the tetra- to
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Table 3 Chlorinated compounds that may interfere in PCDD and PCDF analysis [ 4 ] Interference
PCDD / F
Solution
Class
Ion ( cluster )
Compound
Ion ( cluster )
PCBs ( non-ortho ) PCNs HxCN PCTs PCDPEs
(M-Cl2 ) (M+6 )=3 M (M+6 )=3 (M ) (M-Cl2 ) M
PCDDs PCDFs 13 C12 -TCDD PCDFs PCDFs PCDFs PCDFs
(M+2 ) M=3 M M=3 (M+2 ) (M+2 ) M
PCPYs
Fractionation on active carbon GC separation and quanti¢cation using (M+2 )=3 Other internal standard Clean-up and quanti¢cation using Quanti¢cation of PCDFs using M ions Discrimination using different chlorine isotope ratios and different GC retention times
heptachlorinated compounds and 10^50 pg for OCDD / F and DCB. Current high resolution instruments operating at a resolution of 5000^10 000 and with EI and SIR are capable of decreasing the detection limit down to 10^200 fg for the tetra- through octa-CDDs and CDFs [ 27 ]. Using low-resolution instruments in the NCI mode with methane as reagent gas, detection limits are usually in the 10^100 fg range using SIR for all PCDFs ( tetra- to octa-CDF ) as well as for the higher chlorinated PCDDs ( penta- to octaCDD ) and PCBs. This is 10^100-fold better than under EI conditions and more than 10-fold better than with GC^ECD. However, the NCI mode has a relatively poor sensitivity for 2,3,7,8-TCDD [ 31 ]. 2.6. Critical stages in the analysis procedure
Some factors that are known to be critical in methods for the determination of PCBs, PCDDs and PCDFs are presented below [ 32 ]. 2.6.1. Sample extraction Alkaline saponi¢cation may reduce recoveries of some analytes, especially for the higher chlorinated homologues [ 33 ]. At least for some food matrices such as milk and blood, non-polar solvents are not always as ef¢cient as moderately polar solvents.
2.6.2. Clean-up Some adsorbents ( e.g. alumina, active carbon ) are known to show batch-to-batch differences which may alter the selectivity of the clean-up step in which they are used. This could lead to unwanted in£uences of the within-laboratory reproducibility. If the analytical process involves glassware, columns or reagents that have to be re-used, a memory effect can occur when low-level test samples are analysed after high-level test samples. A common check for possible contamination is to include one or more procedural blanks in each series of measurements. 2.6.3. GC separation GC columns are known to be susceptible to residues of matrix components ( e.g., lipids, colouring agents ) and demand high clean-up ef¢ciencies to eliminate these substances. But even if a general high performance is achieved in the clean-up process, the resolving power should be frequently checked ( see Section 2.6.2 ). 2.6.4. ECD quanti¢cation The adequate operation of the electron capture detector appears to be critical for a proper use of this measurement technique. For instance, the detector must be kept clean of carbon build-up by periodic
Table 4 Indicative method-speci¢c detection limits for the determination of PCBs, PCDDs and PCDFs in foodstuffs and human tissues with a sample size containing 1 g of ( extractable ) lipids, a sample preparation scheme resulting in a ¢nal extract volume of 100 Wl and an injection of 1 Wl of extract onto the GC Identi¢cation / quanti¢cation technique ( ionization )
Instrument detection limit ( pg )
Method detection limit ( pg / g fat )
GC^ECD GC^LRMS ( EI^SIR ) GC^HRMS ( EI^SIR ) GC^LRMS (NCI^SIR )
1^10 1^50 0.01^0.2 0.01^0.1
100^1000 100^5000 1^20 1^10
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`baking out'. The uncertainty of the response of the ECD ( non-speci¢c baseline, negative peaks, non-linearity ) is one of the most commonly reported critical factors in PCB determinations. Isotope dilution GC^ HRMS has become the method of choice because of its better overall performance compared to GC^ECD. 2.6.5. Standards Besides uncertainties associated with the EC detector, the uncertainty in the concentration of the calibration solution is another commonly reported factor introducing bias in interlaboratory performance studies. This not only concerns the original bias in the concentration of the standard as supplied ( commonly less than 10%), but also re£ects problems encountered in the preparation and storage of the solutions.
3. Conclusions Current methods and techniques provide the sensitivity, selectivity, accuracy and precision needed to investigate concentrations and £ows of the relevant congeners of the PCDDs, PCDFs and PCBs down to low-ppt levels. Important features of standard methods include the use of active carbon for effective separation of planar from non-planar compounds, the use of appropriate GC conditions to separate the different isomers present in the ¢nal extract, the MS for the detection of the PCDDs, PCDFs and non-ortho PCBs, and the ECD ( or MS ) for the detection of the more abundant mono- and di-ortho-substituted PCBs. The use of at least one 13 C-labelled analogue for each homologous group is essential to achieve the desired accuracy in determinations using GC^HRMS. A set of quality control measures should be included in each series of measurements to keep the method under control.
Acknowledgements I would like to thank Prof. Dr U.A.Th. Brinkman for his invitation to submit an essential part of my PhD thesis as a contribution to this journal, and for his valuable comments and help in preparing the manuscript.
References [ 1 ] C. Rappe, Environ. Sci. Technol. 18 ( 1984 ) 78A.
[ 2 ] L.M. Smith, D.L. Stalling, J.L. Johnson, Anal. Chem. 56 ( 1984 ) 1830. [ 3 ] P. De Voogt, D.E. Wells, L. Reutergaîrdh, U.A.Th. Brinkman, Int. J. Environ. Anal. Chem. 40 ( 1990 ) 1. [ 4 ] C. Rappe, M. Nygren and H.R. Buser, in: J.D. Rosen ( Editor ), Applications of New Mass Spectrometry Techniques in Pesticide Chemistry, Vol. 91, John Wiley and Sons, New York, 1987. [ 5 ] A.K.D. Liem, R.A. Baumann, A.P.J.M. De Jong, E.G. Van der Velde, P. Van Zoonen, J. Chromatogr. 624 ( 1992 ) 317. [ 6 ] K. Ballschmiter, H. Buëchert, S. Bihler, M. Zell, Fresenius' Z. Anal. Chem. 306 ( 1981 ) 323. [ 7 ] J. De Boer, Chemosphere 17 ( 1988 ) 1811. [ 8 ] P.A. Greve ( Editor ) Analytical Methods for Residues of Pesticides, 5th edn. Ministry of Welfare, Health and Cultural Affairs, Rijswijk, 1988. [ 9 ] AOAC ( Association of Of¢cial Analytical Chemists ), Of¢cial Methods of the Association of Of¢cial Analytical Chemists, 15th edn. AOAC, Arlington, VA, 1990. [ 10 ] D.E. Wells, Pure Appl. Chem. 60 ( 1988 ) 1437. [ 11 ] D.E. Wells, In: D. Barceloè ( Editor ), Environmental Analysis. Techniques, Applications and Quality Assurance. Elsevier Science, Amsterdam, 1993, p. 138. [ 12 ] J.J. Ryan, R. Lizotte, L.G. Panopio, B.P.-Y. Lau, Chemosphere 18 ( 1989 ) 149. [ 13 ] D.G. Patterson Jr., P. Fuërst, L.O. Henderson, S.G. Isaacs, L.R. Alexander, W.E. Turner, L.L. Needham, H. Hannon, Chemosphere 19 ( 1989 ) 135. [ 14 ] G. Lindstroëm, C. Rappe, Chemosphere 17 ( 1988 ) 921. [ 15 ] P. Fuërst, C. Fuërst, H.-A. Meemken, W. Groebel, Z. Lebensm. Unters. Forsch. 189 ( 1989 ) 338. [ 16 ] A.K.D. Liem, A.P.J.M. De Jong, J.A. Marsman, A.C. Den Boer, G.S. Groenemeijer, R.S. Den Hartog, G.A.L. De Korte, R. Hoogerbrugge, P.R. Kootstra, H.A. Van 't Klooster, Chemosphere 20 ( 1990 ) 843. [ 17 ] N. Kannan, G. Petrick, D. Schulz, J. Duinker, J. Boon, E. Van Arnhem, S. Jansen, Chemosphere 23 ( 1991 ) 1055. [ 18 ] J.J. Ryan, H.B.S. Conacher, L.G. Panopio, B.P.-Y. Lau, J.A. Hardy, Y. Masuda, J. Chromatogr. 541 ( 1991 ) 131. [ 19 ] A.P.J.M. De Jong, A.K.D. Liem, R. Hoogerbrugge, J. Chromatogr. 643 ( 1993 ) 91. [ 20 ] K. Olie, P.C. Slot, H. Wever, Chemosphere 19 ( 1989 ) 103. [ 21 ] U.G. Ahlborg, G.C. Becking, L.S. Birnbaum, A. Brouwer, H.J.G.M. Derks, M. Feeley, G. Golor, A. Hanberg, J.C. Larsen, A.K.D. Liem, S.H. Safe, C. Schlatter, F. W×rn, M. Younes, E. Yrjaënheikki, Chemosphere 28 ( 1994 ) 1049. [ 22 ] J. De Boer, Q.T. Dao, J. High Resolut. Chromatogr. 12 ( 1989 ) 755. [ 23 ] B. Larsen, S. BÖwadt, R. Tilio, Int. J. Environ. Anal. Chem. 47 ( 1992 ) 47. [ 24 ] S. BÖwadt, H. SkejÖ-Andresen, L. Montanarella, B. Larsen, Int. J. Environ. Anal. Chem. 56 ( 1994 ) 87. [ 25 ] J. De Boer, J.C. Duinker, J.A. Calder, J. Van der Meer, J. Assoc. Off. Anal. Chem. Int. 75 ( 1992 ) 1054. [ 26 ] H.R. Buser, J. Chromatogr. 114 ( 1975 ) 95. [ 27 ] D. Barceloè, Trends Anal. Chem. 10 ( 1991 ) 323. [ 28 ] H. Bagheri, P.E.G. Leonards, R.T. Ghijsen,
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U.A.Th. Brinkman, Int. J. Environ. Anal. Chem. 50 ( 1993 ) 257. [ 29 ] A.P.J.M. De Jong, A.K.D. Liem, Trends Anal. Chem. 12 ( 1993 ) 115. [ 30 ] M.D. Mullin, C.M. Pochini, S. McCrindle, M. Romkes, S.H. Safe, L.M. Safe, Environ. Sci. Technol. 18 ( 1984 ) 468.
[ 31 ] H.R. Buser, C. Rappe, P.-A. Bergqvist, Environ. Health Perspect. 60 ( 1985 ) 293. [ 32 ] W. Horwitz, R. Albert, J. Assoc. Off. Anal. Chem. 79 ( 1996 ) 589. [ 33 ] F. Van der Valk, Q.T. Dao, Chemosphere 17 ( 1988 ) 1735.
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Basic aspects of methods for the determination of dioxins and PCBs in foodstuffs and human tissues A.K. Djien Liem*
Laboratory for Organic-analytical Chemistry, National Institute of Public Health and the Environment, P.O. Box 1, NL-3720 BA Bilthoven, The Netherlands A considerable improvement can be noted in determinations of PCDDs, PCDFs and PCBs in foodstuffs and human tissues. In recent years, numerous validated schemes for extraction, clean-up and instrumental analysis have been published, enabling an accurate and precise determination of the toxicologically relevant congeners. In this article, the basic aspects of current methods and techniques are discussed. z1999 Published by Elsevier Science B.V. All rights reserved. Keywords: Foodstuffs; Human tissues; Active carbon; PCDD; PCDF; Polychlorinated biphenyls
1. Introduction Earlier methods for the determination of PCDDs and PCDFs in the study of their formation in industrial and chemical processes, and of their occurrence in products such as PCBs, chlorophenols and £ue gases from waste incineration, used packed column gas chromatography ( GC ) in combination with massspectrometric detection ( GC^MS ). It was only possible to determine the homologous groups with 4^8 Cl atoms without any speci¢cation of the levels of individual congeners. Around 1980, analytical techniques became increasingly sophisticated [ 1 ] enabling isomerspeci¢c determinations and facilitating the accurate determination of individual congeners at the low-ppt level. In addition, the introduction of the concept of toxic equivalency factors led to a shift in interest from total dioxin levels to the concentrations of the 2,3,7,8substituted tetra- to octachlorinated congeners. At present, the use of activated carbon to isolate the pla-
nar aromatic molecules such as the PCDDs and PCDFs [ 2 ], and the separation and detection of speci¢c isomers by capillary ( high resolution ) gas chromatography combined with high resolution MS ( HRGC^ HRMS ) are common features of methods for the determination of dioxins. A similar history can be noted for the PCBs. The production and use of technical mixtures of PCBs have led to their presence all over the world. Not only the low concentration level of the large number of PCB congeners, but also the simultaneous presence of other persistent contaminants ( e.g., halogenated pesticides, PCDDs, PCDFs ) pose distinct problems. In the past, the PCBs were fully chlorinated with SbCl5 to decachlorobiphenyl, with its single peak representing `total PCBs'. Since the middle of the 1980s, the determination of individual congeners is regarded as the preferred quanti¢cation technique, and legislation is based on quantifying a few selected congeners, not on reporting `total PCBs'. Several standardised schemes are used for the determination of PCB congeners 28, 52, 101, 118, 138, 153 and 180, a selection often referred to as `indicator PCBs' that is based on abundance, chromatographic resolution, response and availability as a standard, but not on toxicological considerations [ 3 ]. Indeed, considerable improvements can be noted in techniques and methods to determine the PCBs, PCDDs and PCDFs. Using appropriate instrumentation and the available knowledge of their properties during different steps of the analytical process, an experienced chemist should be able to perform accurate and precise determinations of the compounds of interest. This can however only be achieved if basic requirements are taken into account. In this article, technical aspects and critical steps for each step of the analytical process are presented. Trends and developments will be discussed in another contribution.
*Tel.: +31 (30) 274 2871; Fax: +31 (30) 274 4424. E-mail: [email protected] 0165-9936/99/$ ^ see front matter PII: S 0 1 6 5 - 9 9 3 6 ( 9 9 ) 0 0 1 1 2 - 0
ß 1999 Published by Elsevier Science B.V. All rights reserved.
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2. Basic aspects of determinations of PCDDs, PCDFs and PCBs The complexity of determinations of PCDDs, PCDFs and PCBs is mainly caused by the low levels at which these compounds occur and the pronounced differences in toxic and biological effects of the PCDD, PCDF and PCB isomers. Another complicating factor is the large number of possible interferences and matrix effects. Therefore, the following basic requirements must be met by an analytical procedure for dioxins and PCBs [ 4 ]: 1.
2.
3.
4.
High sensitivity and low limits of detection. For PCDDs and PCDFs, detectable quantities have to be in the pg range or lower and detectable concentrations in the ppt or sub-ppt range because of the extreme toxicity of some of these compounds. PCBs are known to occur at higher levels than PCDDs and PCDFs. For the most prevalent PCB congeners a sensitivity in the pg to ng range is already suf¢cient. However, for the measurement of the more toxic non-ortho-substituted congeners the same sensitivity must be reached as for the PCDDs and PCDFs. High selectivity. A distinction is required for PCDDs, PCDFs and PCBs from a multitude of other, co-extracted and possibly interfering compounds present at concentrations up to several orders of magnitude higher than those of the analytes of interest. High speci¢city. A differentiation between various isomers is desired, such as between toxic ( e.g. the seventeen 2,3,7,8-substituted PCDDs and PCDFs and the non-ortho-, mono-orthoand di-ortho-substituted PCBs ) and other isomers. High accuracy and precision. The determination should provide a valid estimate of the true concentration in a sample. Furthermore, an acceptable degree of agreement between measurements must be achieved.
For congener-speci¢c analyses of PCDDs and PCDFs, MS is ideally suited for requirements 1, 2 and 4. Especially if the mass spectrometer is operating in the selected ion recording (SIR, also referred to as selected ion monitoring, SIM ) mode, detection limits in the fg to pg range can be reached. However, MS alone cannot differentiate between most isomers ( speci¢city ) and not between different classes of
chlorinated compounds ( selectivity ). HRGC is mostly used to provide the necessary isomer speci¢city because it can be easily and directly coupled to MS. But even when appropriate GC^MS conditions are used, further ( pre )separation techniques remain necessary to prevent in£uences of interferences and matrix components. Besides the PCBs, other families of polychlorinated compounds such as the polychlorinated naphthalenes (PCNs ), diphenyl ethers (PCDPEs ), biphenylenes (PCBPs ), pyrenes (PCPYs ) and terphenyls (PCTs ) are known to interfere with determinations of PCDD / Fs [ 4 ]. Extremely high resolving powers are required in MS to distinguish TCDD from some other chlorinated compounds. At such high resolving powers, MS sensitivity will soon become insuf¢cient to identify TCDD at the required limits of detection. In the case of PCBs, GC with an electron-capture detector ( ECD ) is the instrument most commonly used in analytical practice. The ECD is suf¢ciently sensitive to detect most prominent congeners in environmental and biological samples while the required isomer speci¢city can be attained by the use of appropriate stationary phases in gas chromatography. In the case of complex GC^ECD chromatograms, subsequent GC^MS analysis may provide additional information to con¢rm the identity of the analytes. Extensive sample extraction and clean-up techniques are also required for the determination of PCBs. However, the problem of potentially interfering compounds is less severe than with dioxins as most PCB congeners are present at much higher levels than the PCDDs and PCDFs. An exception are the toxic non-ortho PCB congeners, because they are usually present at much lower concentrations than the other congeners. For the determination of these congeners in foodstuffs and human tissues, the more sensitive GC^MS is becoming the preferred technique. In the last two decades, several techniques and methods have been introduced to reach the required sensitivity and selectivity in the determination of PCDDs, PCDFs and PCBs. In the following sections, it is not intended to give an exhaustive account of available methods; instead, the methods most commonly used will be described. 2.1. Extraction procedures
The purpose of extraction is to remove the bulk of the sample matrix and to transfer the fraction containing the analytes in a suitable solvent ( mixture ).
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2.1.1. Food Extraction techniques for food are generally based on the assumption that lipophilic organic compounds such as the PCDDs, PCDFs and PCBs predominantly occur in the fat fraction of the food matrix. Sample extraction methods are therefore based on general methods for the isolation of the lipid fraction from the sample matrix [ 5 ]. Consequently, the amount to be taken as a test sample depends on the required sensitivity and the lipid content of the sample matrix. Extractions should yield at least a few grams of extractable lipids to perform reliable determinations. In duplicate diet studies, duplicate portions of all food products ( meat, fruit, vegetables, beverages, etc. ) that a volunteer consumes during a certain time period ( typically 24 h or 7 days ) are collected and subsequently mixed to one composite sample. After thorough homogenisation, these composite samples are freeze-dried to remove the water content and subsequently blended in the presence of an organic solvent mixture such as acetone^pentane or Soxhletextracted with organic solvents such as pentane or hexane to isolate the lipid fraction of the sample. Methods for the isolation of lipid fractions from individual food products depend on the type of sample. Butter, fats and oils are generally assumed to be homogeneous, and normally do not require extensive extraction procedures. Aliquots of such samples can be dissolved in n-hexane or light petroleum to the desired fat concentration. Meat and ¢sh samples are initially blended and homogenised. Next, a representative test sample is ground with anhydrous sodium sulphate, until a free £owing powder is obtained. This mixture can then be extracted using either blending techniques, a cold column extraction technique ( elution of a column packed with the powder with organic solvent or solvent mixture )[ 6 ], or a Soxhlet extraction technique [ 7 ]. Milk can either be freeze-dried or chemically dried with anhydrous sodium sulphate, followed by Soxhlet extraction with organic solvent, or subjected to a liquid^liquid extraction procedure consisting of mixing with sodium oxalate and ethanol or methanol, followed by ( repeated ) extraction steps with a combination of organic solvents such as acetone^pentane [ 8 ] or diethyl ether^light petroleum [ 9 ]. Vegetables usually have a high water content and will dehydrate prior to analysis unless extracted immediately after sampling. Vegetable matter is therefore crushed, chopped and gently dried at 40^50³C prior to
storage and analysis. Isolation procedures include grinding with coarse sea sand, blending, mixing with a more polar solvent ( acetone ) and subsequent partitioning with dichloromethane or hexane ( applicable for matrices with a high sugar content ) or mixing with a more polar solvent ( acetone ) followed by either shaking or Soxhlet extraction [ 10 ]. Saponi¢cation under alkaline conditions ( in the presence of ethanol and KOH ) followed by extraction with organic solvent is often employed for the analysis of large amounts ( up to hundreds of grams ) of fats. However, these methods are known to lead to a loss of some of the higher chlorinated PCBs [ 11 ], to degradation of PCDDs and PCDFs in proportion to their chlorine content, and, in the case of PCDFs, to production of lower chlorinated PCDFs and ethoxyPCDFs as artifacts [ 12 ]. 2.1.2. Human tissues In the analysis of human milk and adipose tissue, samples are treated in a similar way as described for milk and meat in Section 2.1.1. In the analysis of blood, methods for blood plasma or serum have been introduced. The majority of determinations use liquid^liquid partitioning techniques in a similar way as for milk samples. It should be noted that partitioning of PCBs, PCDDs and PCDFs among the cellular, serum protein and lipoprotein components of whole blood may depend on class (PCBs vs PCDD / Fs ) as well as on degree of chlorination [ 13 ]. 2.2. Sample clean-up
Additional steps are usually required to remove the bulk of co-extractants, to separate organochlorine residues into groups and to concentrate the ¢nal extract to appropriate volumes of solvent, allowing the detection of the analytes at the ultra-trace levels at which they usually occur. The choice of a particular sequence of steps will be highly dependent on the analytical system which is ¢nally used. Sample extraction, clean-up and GC method together form a delicately matched set, each of which will contribute to the ultimate speci¢city and selectivity. For PCBs, solid^liquid adsorption chromatography using Florisil, silica and alumina, gel permeation chromatography ( GPC ) and chemical methods are most commonly used [ 5 ]. For the determination of PCDDs and PCDFs, nearly all established schemes involve combinations of clean-up methods developed for the analysis of PCBs and organochlorine pesticides (OCPs ) in combination with an active carbon step to
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isolate a speci¢c fraction containing the PCDDs and PCDFs [ 2 ]. Some recent methods to isolate fractions for the determination of the non-ortho and monoortho PCB congeners frequently employ active carbon as well.
procedure have been introduced for the analysis of animal and human tissues [ 14^16 ]. In most methods, the carbon step is combined with one or more steps to provide additional selectivity, e.g., by means of acidor base-activated alumina column chromatography, or with GPC. The performance of several types of activated carbon for the separation of non-ortho PCBs has been studied by Kannan et al. [ 17 ]. They compared brands from six suppliers and concluded that the charcoals tested did not allow a complete separation of non-ortho PCBs from the rest of the dominant ortho-substituted PCB congeners at trace levels. Using multidimensional GC, they observed co-elution of PCB 110 with PCB 77, and PCBs 129 and 178 with PCB 126 that could lead to overestimation of the results if a single column (SE-54 ) GC determination was performed. Additional separation is obviously needed to prevent these systematic errors from occurring.
2.2.1. Carbon chromatography The basis of carbon chromatography for the selective fractionation of planar aromatic molecules such as PCDDs and PCDFs was laid by Smith and co-workers [ 2 ]. Compounds markedly retained on the carbon are certain planar multi-ring aromatic compounds, PCDDs, PCDFs, non-ortho PCBs, PCNs, PCDPEs and xanthenes [ 14 ]. The high retention is based on the coplanarity of closely situated aromatic systems and is increased by electronegative substituents ( chlorine, bromine, nitro ). The fraction containing the PCDDs and PCDFs is recovered from the carbon column by back£ush with organic solvent, e.g., toluene. A major drawback of ¢nely divided carbon as a packing material is the high back-pressure. Therefore, ¢nely divided carbon dispersed on the surface of shredded polyurethane foam, carbon dispersed on glass ¢bres and clean-up through a series of silicates ( silica, potassium silicate ) have been recommended. In this way, coloured acidic lipid materials, hydroxyPCBs and hydroxy-diphenyl ethers are retained by the silicates, allowing the carbon to be re-used [ 14 ]. The original method was used for the determination of ppt levels of PCDDs and PCDFs in environmental samples [ 2 ]. Subsequently, several modi¢cations of this
2.3. Identi¢cation and quanti¢cation
Current methods for the separation and detection of isomers of PCBs, PCDDs and PCDFs all involve GC^ ECD ( indicator, mono-ortho and di-ortho PCBs ) and, increasingly, GC^MS (PCDDs, PCDFs and non-ortho PCBs ). The identi¢cation of the target analytes is performed by comparing their relative retention times in the chromatogram of the sample extract with those in the chromatogram of a solution containing pure standards with known concentrations. To this end, each
Table 1 Separation of toxic congeners of PCDDs and PCDFs on GC columns of different polarity [ 18,29 ] Congener PCDDs 2,3,7,8 1,2,3,7,8 1,2,3,4,7,8 1,2,3,6,7,8 1,2,3,7,8,9 PCDFs 2,3,7,8 1,2,3,7,8 2,3,4,7,8 1,2,3,4,7,8 1,2,3,6,7,8 1,2,3,7,8,9 2,3,4,6,7,8
DB-1
DB-5
DB-17
DB-210
DB-225
CPS-1
SP-2331
SIL 88
Smectic
QNT QNT QTT QTT CE1
CE2 BS QTT QTT CE1
CE1 QNT BS BS BS
CE1 CE1 NBS NBS BS
QNT CE1 NBS NBS BS
CE4 BS BS BS BS
BS BS NBS NBS BS
QNT BS NBS NBS BS
BS BS QTT QTT BS
CE5 QT CE1 CE1 NBS NBS CE1
CE5 QNT CE3 CE1 QNT NBS QNT
BS CE1 BS QNT CE1 BS QNT
QNT CE2 BS CE1 CE1 CE1 BS
CE1 CE1 BS QNT CE2 BS CE1
CE1 CE1 QNT CE1 NBS BS BS
QNT CE1 BS CE1 QTT BS BS
QNT CE1 BS CE1 QTT BS BS
CE1 BS CE1 NBS NBS BS CE1
BS = no overlap with others; NBS = almost no overlap; QTT = quanti¢able, but may overlap with congener with same toxicity; QNT = quanti¢able, but may overlap with non-toxic congener; CEn = co-elution ( 100% overlap ) with n non-toxic congeners.
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compound should appear as a single peak. Quanti¢cation is carried out by comparing peak heights or areas in sample and standard solution. The use of multiple internal standards is considered essential to improve the accuracy. 2.4. Isomer identi¢cation 2.4.1. PCDDs and PCDFs The separation characteristics and elution pro¢les of GC columns with different stationary phases for the congener-speci¢c analysis of PCDDs and PCDFs have been investigated by Ryan et al. [ 18 ]. Their results are summarised in Table 1. By using apolar columns ( CP SIL 5, DB-5, HP Ultra 2 and equivalents ), a separation can be achieved between homologous groups. In addition, apolar columns can separate the 2,3,7,8-substituted congeners from each other; however, they fail to resolve these congeners from the non-toxic congeners. With polar columns ( DB-DIOXIN, CP SIL 88, SP 2331, Silar 10c and equivalents ) the resolving power improves, but incomplete separation still remains for 2,3,7,8TCDF, 1,2,3,7,8-PeCDF and 1,2,3,4,7,8-HxCDF. TCDF is most dif¢cult to resolve and requires medium polar columns ( DB-17 or equivalents ). Apolar columns are frequently used for the analysis of biological samples, including human tissues, as these samples generally do not contain congeners other than the 2,3,7,8-substituted ones. Apolar columns require shorter analysis times, have longer lifetimes and no decomposition occurs for highly chlorinated PCDD / Fs [ 19 ], a poorly understood phenomenon that occurs with the use of polar columns [ 20 ]. Polar columns are required for 2,3,7,8-isomer-speci¢c analyses in samples that may contain non-laterally substituted isomers, such as environmental samples, £y ash, ambient air and aquatic organisms and PCDD / F-contaminated products of different origins. An alternative is to use a Rtx-2330 cross-bound polar column with elution pro¢les similar to that of other polar columns, that can withstand higher temperatures ( 275³C ) enabling shorter run times, and for which no dechlorination could be observed for OCDD / F, thus avoiding re-analysis on a second column [ 19 ]. The differences in complexity of GC separations between abiotic and biotic samples are illustrated in Fig. 1.
2.4.2. PCBs The `indicator PCBs' 28, 52, 101, ( 118,) 138, 153 and 180 are routinely determined in environmental monitoring programmes and for regulatory purposes. At present, the indicator PCBs and PCBs with dioxinlike activity are frequently measured in exposure and risk assessment studies. In this regard, toxic equivalency factors (TEFs ) have been derived for a selection of non-ortho and mono-ortho PCBs to estimate their contributions to the total dioxin-like toxicity in equivalents of 2,3,7,8-TCDD [ 21 ]. The growing number of selections of relevant PCBs has inspired several analysts to tackle the problem that some of the priority PCBs are poorly resolved on the widely used SE-54 or SIL-8 type columns [ 22^24 ]. When using these stationary phases, the following separations have been reported to be critical: 28 / 31, 49 / 52, 77 / 110, 101 / 84, 118 / 149, 105 / 153 / 132, and 138 / 163. Under optimum conditions with the proper length, diameter, and ¢lm thickness, PCB pairs 28 / 31 and 118 / 149 can be separated on these columns. With more polar columns, the separation of PCBs 28 and 31 will soon become impossible, whereas the separation of PCBs 118 and 149 improves [ 25 ]. Separation problems may occur in analysing both abiotic and biotic matrices. Although PCB patterns show a general shift from lower chlorinated to higher chlorinated congeners when going from technical mixtures via soils and sediments, invertebrates and ¢sh to ¢sh-eating birds, mammals and humans, PCB patterns remain relatively complex when compared to those observed for PCDDs and PCDFs. Recently, several groups have tested narrow-bore columns for congener-speci¢c determination of PCBs in standard and technical mixtures with GC^ ECD and GC^MS using different stationary phases. The studies concentrated on possible overlap with other closely eluting PCB congeners that are potentially present in environmental and biological samples. A summary of their observations is presented in Table 2. 2.4.3. Quanti¢cation The ECD is the most widely used detector for the quanti¢cation of PCBs. The response of the ECD depends on the number and the position of the chlorine atoms in the PCB molecule. Generally, the sensitivity increases with the number of chlorine atoms. For the PCDDs, PCDFs and ( depending on their concentrations ) the non-ortho PCBs, the high resolution mass spectrometer (MS ) is currently the only technique able to provide the required sensitivity and selectivity.
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Fig. 1. GC^HRMS analysis of pentachlorodibenzofurans in a sample extract of £ue gas from a municipal solid waste incinerator, and in a human milk extract. Traces are normalized molecular ion currents. GC^MS analyses were accomplished on a Rtx-2330 ( £ue gas ) and a DB-5 column ( human milk ), respectively, at a mass resolution of 5000.
The method of internal standard quanti¢cation is nowadays routinely used in GC^MS methods for the determination of PCDDs, PCDFs and non-ortho PCBs. Quanti¢cation is generally performed by using stable-isotope-labelled (13 C12 ) analogues of the compounds to be determined as internal quanti¢cation standards ( isotope dilution ). These labelled compounds ( at least one for each homologous group ) are added in known amounts to the sample prior to extraction or clean-up and are assumed to
equal the native analytes with respect to extraction, clean-up and GC properties. The labelled compounds can be easily distinguished from the native compounds on the basis of their mass spectral differences and are therefore used as guides for identi¢cation and quanti¢cation. Prior to GC^MS, other compounds can be added as internal sensitivity standards. An internal sensitivity standard is a quality control measure and can, in principle, be any compound with suitable GC^ MS properties, although the use of additional 13 C12 or
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Table 2 Separation of indicator and dioxin-like PCB congeners from other chlorobiphenyls on GC columns of different polarity [ 23,24 ] IUPACa
Structure
SIL-5
SIL-8
SIL-19
SIL-88
SIL-8 / HT-5
28 52 77 101 105 114 118 123 126 138 153 156 157 167 169 170 180 189
2,4,4P 2,2P,5,5P 3,3P,4,4P 2,2P,4,5,5P 2,3,3P,4,4P 2,3,4,4P,5 2,3P,4,4P,5 2P,3,4,4P,5 3,3P,4,4P,5 2,2P,3,4,4P,5P 2,2P,4,4P,5,5P 2,3,3P,4,4P,5 2,3,3P,4,4P,5P 2,3P,4,4P,5,5P 3,3P,4,4P,5,5P 2,2P,3,3P,4,4P,5 2,2P,3,4,4P,5,5P 2,3,3P,4,4P,5,5P
^ ^ ^ ^ 132 ^ ^ ^ 129 160+163 ^ 171 202 ^ ^ ^ ^ ^
^ ^ 110 84 132 ^ 149 149 129+178 160+163 ^ 202+171 173+200 128 ^ 190 ^ ^
31 ^ ^ ^ ^ ( 146 ) ^ 107 ^ 160+163+158 ^ ^ 180+197 ^ 203+196 190 197 ^
16 ^ ^ ( 55 ) 129 137 200+123 118+200 ^ ^ ( 110 ) ^ ^ ^ ^ ^ ( 197 ) ^
^ ^ ^ 90b ^ ^ ^ ^ 175 160a , 163a ^ 173 ^ 185a ^ ^ ^ ^
Separations on SIL-5, SIL-8, SIL-19 and SIL-88 were performed on 50 m ( HT-5: 25 m ) f.s. columns with i.d. of 0.22^0.25 mm and a stationary phase thickness of 0.10^0.26 Wm. Separations on SIL-8 / HT-5 were performed on a two-series-coupled narrow-bore fused silica WCOT columns: 25 mU0.25 mm, 0.25 Wm SIL-8 and 25 mU0.2 mm, 0.1 Wm HT-5. ^ = Well resolved CBs ( less than 10% interference from any co-elutant on the peak height ); numbers in parentheses indicate that interference is reduced to less than 10% by use of GC^MS. a Separation can be improved by optimizing temperature programme. b Congener found at insigni¢cant concentrations in technical mixtures.
other stable-isotope-labelled (13 C6 or 37 Cl4 ) analogues is preferred. GC^ECD methods for the determination of PCBs frequently use one or more PCB congeners that are known not to occur in the sample as internal quanti¢cation or sensitivity standard( s ). In GC^MS, electron impact ionisation ( EI ) is most widely used. The EI mass spectral properties of PCDFs and PCDDs have been described by Buser [ 26 ]. If the MS is operated in SIR, high sensitivity is obtained because of the abundance of the molecular ions produced. Both isomer identi¢cation and quanti¢cation can be easily done by simultaneous monitoring of the typical chlorine isotope clusters of both unlabelled and 13 C12 -labelled PCDDs and PCDFs. The measurement of 35 Cl /37 Cl ratios in analyte peaks is a reliable means for identity con¢rmation while additional structural information can be obtained by simultaneous monitoring of typical COCl losses from molecular ions [ 4 ]. Furthermore, the analyte can be positively identi¢ed if the GC retention time corresponds to that of its labelled analogue, and quanti¢cation can be performed by comparing the response of the chlorine isotopes of the molecular ion cluster with those from the labelled analogue ( cf. Fig. 2 ).
In the last few years, more 13 C-labelled reference standards have become commercially available. As a result, the isotope dilution technique is also becoming the method of choice for the analysis of PCBs, particularly for the less abundant non-ortho- and monoortho-substituted PCBs [ 3 ]. Under EI, PCBs tend to exhibit relatively high abundances of M and [ M-2Cl ] and fragmentation usually occurs with subsequent losses of chlorine atoms. The loss of Cl is favoured in the case of ortho-Cl PCBs, in which three of the four ortho positions are chlorinated. This ortho effect is also important if two of the ortho positions are chlorinated, such as 2,2P [ 27 ]. Negative chemical ionisation (NCI ) with methane as reagent gas can be used for improved molecular weight information and / or increased sensitivity. Characteristics of the NCI mass spectra of PCDDs and PCDFs have been published by Rappe et al. [ 4 ]. NCI of PCDFs will yield a base peak due to M3 , and the fragmentation produces the unusual [ M-34 ] ions, due to the uptake of Hc and the loss of Clc . Fragmentation of PCDDs in NCI mode is more conventional and results in [ M-35 ]3 due to loss of Cl [ 4,31 ]. The fragmentation and, consequently, sensitivity depend on
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3.
The signal-to-noise ratio must be 3:1 or higher. GC^MS methods:
1. 2. 3. 4. 5. 6. Fig. 2. Mass fragmentograms ( EI, MS, resolution 3000:1 ) showing traces of the most intense molecular ions of 13 C12 -labelled ( lower trace ) and native ( upper trace ) tetra-CDD ( m / z 334 and 322 ), penta-CDD ( m / z 368 and 356 ) and penta-CDF ( m / z 352 and 340 ) in a sample of cow's milk. Measured concentrations ( in pg / g fat ) are given in parentheses.
the degree of chlorination and the substitution pattern. In contrast, using the MS in the NCI mode, the determination of 2,3,7,8-TCDD appeared to be less sensitive than that for the other TCDDs and was also found to be more sensitive for PCDFs than for PCDDs. With respect to PCBs, less chlorinated PCBs have a tendency to exhibit extensive fragmentation ( e.g., Cl atoms ) and only the more chlorinated PCBs exhibit pronounced molecular ions [ 28 ]. PCBs with more than six chlorine atoms showed an increase in response of 2^3 orders of magnitude compared to GC^EI9MS. 2.4.4. Con¢rmation of positive results A number of internal quality control measures should be routinely applied for each sample, within each day or within each series of samples. These measures may help to assure that positive data reported actually refer to speci¢c PCBs, PCDDs and PCDFs. The following criteria can be used [ 4,19 ]:
GC^ECD methods: 1. 2.
Isomer speci¢city must be demonstrated initially and afterwards. The relative retention time must be within 0.1% of the one determined for standards.
Isomer speci¢city must be demonstrated initially and afterwards. The retention time must be within 1 s of that of the 13 C-labelled analogue. The signal-to-noise ratio of all peaks of the molecular ion cluster must be 3:1 or higher. The ratio between the two molecular ions monitored should be within 15% of the theoretical values. Recovery of internal quanti¢cation standards used must not exceed 120%. Fragments should con¢rm the identity of PCDDs, PCDFs and PCBs, e.g. [ M-COCl ] ions for PCDDs and PCDFs or [ M-2Cl ] ions for PCBs, with correct chlorine isotope ratio ( optional in EI^LRMS ).
2.4.5. Interferences from other co-extracted compounds Current clean-up and puri¢cation techniques are quite ef¢cient to remove the bulk of the matrix and several other classes of compounds. Nevertheless, some chlorinated compounds can still be expected in the extracts and may interfere in the analysis. Some possible interfering compounds occasionally observed in puri¢ed extracts of environmental samples are summarised in Table 3. 2.5. Method-speci¢c detection limit
In Table 4 the method detection limits (MDLs ) that can be reached for the analysis of the lipid fraction of foodstuffs and human tissues are indicated. These estimates are based on instrument detection limits as reported in the literature and are calculated for a sample size containing 1 g of lipids, and a method featuring a ¢nal extract volume of 100 Wl and a 1 Wl injection into the gas chromatograph. The ECD response of a chlorinated compound depends on both the number of chlorine atoms and their positions in the molecule. Consequently, a wide range of response factors of PCB congeners should be considered [ 30 ]. At present, detection limits in the order of 1^10 pg on column can be achieved. With respect to GC^MS, detection limits are highly dependent on the conditions and instrumentation used. When using a quadrupole instrument with EI and SIR, detection limits are about 1^10 pg for the tetra- to
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Table 3 Chlorinated compounds that may interfere in PCDD and PCDF analysis [ 4 ] Interference
PCDD / F
Solution
Class
Ion ( cluster )
Compound
Ion ( cluster )
PCBs ( non-ortho ) PCNs HxCN PCTs PCDPEs
(M-Cl2 ) (M+6 )=3 M (M+6 )=3 (M ) (M-Cl2 ) M
PCDDs PCDFs 13 C12 -TCDD PCDFs PCDFs PCDFs PCDFs
(M+2 ) M=3 M M=3 (M+2 ) (M+2 ) M
PCPYs
Fractionation on active carbon GC separation and quanti¢cation using (M+2 )=3 Other internal standard Clean-up and quanti¢cation using Quanti¢cation of PCDFs using M ions Discrimination using different chlorine isotope ratios and different GC retention times
heptachlorinated compounds and 10^50 pg for OCDD / F and DCB. Current high resolution instruments operating at a resolution of 5000^10 000 and with EI and SIR are capable of decreasing the detection limit down to 10^200 fg for the tetra- through octa-CDDs and CDFs [ 27 ]. Using low-resolution instruments in the NCI mode with methane as reagent gas, detection limits are usually in the 10^100 fg range using SIR for all PCDFs ( tetra- to octa-CDF ) as well as for the higher chlorinated PCDDs ( penta- to octaCDD ) and PCBs. This is 10^100-fold better than under EI conditions and more than 10-fold better than with GC^ECD. However, the NCI mode has a relatively poor sensitivity for 2,3,7,8-TCDD [ 31 ]. 2.6. Critical stages in the analysis procedure
Some factors that are known to be critical in methods for the determination of PCBs, PCDDs and PCDFs are presented below [ 32 ]. 2.6.1. Sample extraction Alkaline saponi¢cation may reduce recoveries of some analytes, especially for the higher chlorinated homologues [ 33 ]. At least for some food matrices such as milk and blood, non-polar solvents are not always as ef¢cient as moderately polar solvents.
2.6.2. Clean-up Some adsorbents ( e.g. alumina, active carbon ) are known to show batch-to-batch differences which may alter the selectivity of the clean-up step in which they are used. This could lead to unwanted in£uences of the within-laboratory reproducibility. If the analytical process involves glassware, columns or reagents that have to be re-used, a memory effect can occur when low-level test samples are analysed after high-level test samples. A common check for possible contamination is to include one or more procedural blanks in each series of measurements. 2.6.3. GC separation GC columns are known to be susceptible to residues of matrix components ( e.g., lipids, colouring agents ) and demand high clean-up ef¢ciencies to eliminate these substances. But even if a general high performance is achieved in the clean-up process, the resolving power should be frequently checked ( see Section 2.6.2 ). 2.6.4. ECD quanti¢cation The adequate operation of the electron capture detector appears to be critical for a proper use of this measurement technique. For instance, the detector must be kept clean of carbon build-up by periodic
Table 4 Indicative method-speci¢c detection limits for the determination of PCBs, PCDDs and PCDFs in foodstuffs and human tissues with a sample size containing 1 g of ( extractable ) lipids, a sample preparation scheme resulting in a ¢nal extract volume of 100 Wl and an injection of 1 Wl of extract onto the GC Identi¢cation / quanti¢cation technique ( ionization )
Instrument detection limit ( pg )
Method detection limit ( pg / g fat )
GC^ECD GC^LRMS ( EI^SIR ) GC^HRMS ( EI^SIR ) GC^LRMS (NCI^SIR )
1^10 1^50 0.01^0.2 0.01^0.1
100^1000 100^5000 1^20 1^10
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`baking out'. The uncertainty of the response of the ECD ( non-speci¢c baseline, negative peaks, non-linearity ) is one of the most commonly reported critical factors in PCB determinations. Isotope dilution GC^ HRMS has become the method of choice because of its better overall performance compared to GC^ECD. 2.6.5. Standards Besides uncertainties associated with the EC detector, the uncertainty in the concentration of the calibration solution is another commonly reported factor introducing bias in interlaboratory performance studies. This not only concerns the original bias in the concentration of the standard as supplied ( commonly less than 10%), but also re£ects problems encountered in the preparation and storage of the solutions.
3. Conclusions Current methods and techniques provide the sensitivity, selectivity, accuracy and precision needed to investigate concentrations and £ows of the relevant congeners of the PCDDs, PCDFs and PCBs down to low-ppt levels. Important features of standard methods include the use of active carbon for effective separation of planar from non-planar compounds, the use of appropriate GC conditions to separate the different isomers present in the ¢nal extract, the MS for the detection of the PCDDs, PCDFs and non-ortho PCBs, and the ECD ( or MS ) for the detection of the more abundant mono- and di-ortho-substituted PCBs. The use of at least one 13 C-labelled analogue for each homologous group is essential to achieve the desired accuracy in determinations using GC^HRMS. A set of quality control measures should be included in each series of measurements to keep the method under control.
Acknowledgements I would like to thank Prof. Dr U.A.Th. Brinkman for his invitation to submit an essential part of my PhD thesis as a contribution to this journal, and for his valuable comments and help in preparing the manuscript.
References [ 1 ] C. Rappe, Environ. Sci. Technol. 18 ( 1984 ) 78A.
[ 2 ] L.M. Smith, D.L. Stalling, J.L. Johnson, Anal. Chem. 56 ( 1984 ) 1830. [ 3 ] P. De Voogt, D.E. Wells, L. Reutergaîrdh, U.A.Th. Brinkman, Int. J. Environ. Anal. Chem. 40 ( 1990 ) 1. [ 4 ] C. Rappe, M. Nygren and H.R. Buser, in: J.D. Rosen ( Editor ), Applications of New Mass Spectrometry Techniques in Pesticide Chemistry, Vol. 91, John Wiley and Sons, New York, 1987. [ 5 ] A.K.D. Liem, R.A. Baumann, A.P.J.M. De Jong, E.G. Van der Velde, P. Van Zoonen, J. Chromatogr. 624 ( 1992 ) 317. [ 6 ] K. Ballschmiter, H. Buëchert, S. Bihler, M. Zell, Fresenius' Z. Anal. Chem. 306 ( 1981 ) 323. [ 7 ] J. De Boer, Chemosphere 17 ( 1988 ) 1811. [ 8 ] P.A. Greve ( Editor ) Analytical Methods for Residues of Pesticides, 5th edn. Ministry of Welfare, Health and Cultural Affairs, Rijswijk, 1988. [ 9 ] AOAC ( Association of Of¢cial Analytical Chemists ), Of¢cial Methods of the Association of Of¢cial Analytical Chemists, 15th edn. AOAC, Arlington, VA, 1990. [ 10 ] D.E. Wells, Pure Appl. Chem. 60 ( 1988 ) 1437. [ 11 ] D.E. Wells, In: D. Barceloè ( Editor ), Environmental Analysis. Techniques, Applications and Quality Assurance. Elsevier Science, Amsterdam, 1993, p. 138. [ 12 ] J.J. Ryan, R. Lizotte, L.G. Panopio, B.P.-Y. Lau, Chemosphere 18 ( 1989 ) 149. [ 13 ] D.G. Patterson Jr., P. Fuërst, L.O. Henderson, S.G. Isaacs, L.R. Alexander, W.E. Turner, L.L. Needham, H. Hannon, Chemosphere 19 ( 1989 ) 135. [ 14 ] G. Lindstroëm, C. Rappe, Chemosphere 17 ( 1988 ) 921. [ 15 ] P. Fuërst, C. Fuërst, H.-A. Meemken, W. Groebel, Z. Lebensm. Unters. Forsch. 189 ( 1989 ) 338. [ 16 ] A.K.D. Liem, A.P.J.M. De Jong, J.A. Marsman, A.C. Den Boer, G.S. Groenemeijer, R.S. Den Hartog, G.A.L. De Korte, R. Hoogerbrugge, P.R. Kootstra, H.A. Van 't Klooster, Chemosphere 20 ( 1990 ) 843. [ 17 ] N. Kannan, G. Petrick, D. Schulz, J. Duinker, J. Boon, E. Van Arnhem, S. Jansen, Chemosphere 23 ( 1991 ) 1055. [ 18 ] J.J. Ryan, H.B.S. Conacher, L.G. Panopio, B.P.-Y. Lau, J.A. Hardy, Y. Masuda, J. Chromatogr. 541 ( 1991 ) 131. [ 19 ] A.P.J.M. De Jong, A.K.D. Liem, R. Hoogerbrugge, J. Chromatogr. 643 ( 1993 ) 91. [ 20 ] K. Olie, P.C. Slot, H. Wever, Chemosphere 19 ( 1989 ) 103. [ 21 ] U.G. Ahlborg, G.C. Becking, L.S. Birnbaum, A. Brouwer, H.J.G.M. Derks, M. Feeley, G. Golor, A. Hanberg, J.C. Larsen, A.K.D. Liem, S.H. Safe, C. Schlatter, F. W×rn, M. Younes, E. Yrjaënheikki, Chemosphere 28 ( 1994 ) 1049. [ 22 ] J. De Boer, Q.T. Dao, J. High Resolut. Chromatogr. 12 ( 1989 ) 755. [ 23 ] B. Larsen, S. BÖwadt, R. Tilio, Int. J. Environ. Anal. Chem. 47 ( 1992 ) 47. [ 24 ] S. BÖwadt, H. SkejÖ-Andresen, L. Montanarella, B. Larsen, Int. J. Environ. Anal. Chem. 56 ( 1994 ) 87. [ 25 ] J. De Boer, J.C. Duinker, J.A. Calder, J. Van der Meer, J. Assoc. Off. Anal. Chem. Int. 75 ( 1992 ) 1054. [ 26 ] H.R. Buser, J. Chromatogr. 114 ( 1975 ) 95. [ 27 ] D. Barceloè, Trends Anal. Chem. 10 ( 1991 ) 323. [ 28 ] H. Bagheri, P.E.G. Leonards, R.T. Ghijsen,
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U.A.Th. Brinkman, Int. J. Environ. Anal. Chem. 50 ( 1993 ) 257. [ 29 ] A.P.J.M. De Jong, A.K.D. Liem, Trends Anal. Chem. 12 ( 1993 ) 115. [ 30 ] M.D. Mullin, C.M. Pochini, S. McCrindle, M. Romkes, S.H. Safe, L.M. Safe, Environ. Sci. Technol. 18 ( 1984 ) 468.
[ 31 ] H.R. Buser, C. Rappe, P.-A. Bergqvist, Environ. Health Perspect. 60 ( 1985 ) 293. [ 32 ] W. Horwitz, R. Albert, J. Assoc. Off. Anal. Chem. 79 ( 1996 ) 589. [ 33 ] F. Van der Valk, Q.T. Dao, Chemosphere 17 ( 1988 ) 1735.
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