Environ. Sci. Techno/. 1995, 29, 1043-1047
Determination of PCBs in Fish Tissues Usins- Supercritical Fluid Extraction
ROBERT C. HALE* AND MICHAEL 0. GAYLOR Department of Environmental Sciences, School of Marine Science, Virginia Institute of Marine Science, College of William and Mary, Gloucester Point, Virginia 23062
Methods in common use for the determination of PCBs in fish are time-consuming, expose workers to toxic solvents, and generate significant volumes of hazardous wastes. Supercritical fluid extraction (SFE), with CO2 and solid-phase cryogenic trapping, was applied to the analysis of PCBs in lyophilized fish tissues. Method evaluations were conducted using standard amended tissues and by comparison of SFE results to those obtained from conventional extraction procedures performed intra- and extramurally on fish with field-incurred PCBs. Good results for a range of PCBs were obtained after optimization of SFE conditions. Extraction required only 40 min per sample and could be automated. Extracts were collected directly in GC autosampler vials. Addition of alumina to the extraction vessel eliminated the need for offline extract purification, despite the high lipid content of the tissues. Organic solvent usage was limited to 2 mL, in contrast to conventional procedures which require several orders of magnitude more solvent.
Introduction Polychlorinated biphenyls (PCBs)are persistent environmental contaminants that may accumulate to elevated concentrations in aquatic organisms, such as fish. Consumers of these fish, includinghumans, may also be exposed and deleteriouslyaffected. As a consequence, considerable interest exists in both the scientific and the regulatory communities regarding PCB burdens in fish tissues. Although analytical approaches have improved, most studies rely on tedious, time-consuming procedures. These require large volumes of expensive and toxic solvents,much ofwhichmust eventuallybe disposed of as hazardous waste. Analysis is exacerbated by the lipids co-extracted from fish tissues. These lipids may interferewith subsequent analysis and degrade gas chromatographic (GC) columns if not removed prior to this step. Extract purification procedures in common use include liquidlliquid partitioning, acid treatment, gel permeation, and normal and reverse-phase liquid chromatography (1-3). Multiple solvent concentration steps are common, providing ample opportunity for introduction of laboratory contaminants, loss of target compounds, and worker exposure. Time and labor to perform these and other necessary tasks, such as cleaning of large amounts of glassware to remove trace levels of contaminants, are significant and result in considerable analytical costs and delays in obtaining results. Supercritical fluid extraction (SFE) offers a potential solution to some of the above-mentioned problems. SFE uses readily available materials as extracting fluids at temperatures and pressures above their critical point. The resulting supercriticalfluids possess diffusivitiesthat exceed liquids, but with solvating strengths greater than gases ( 4 ) . A commonly used extractant in SFE, COz, is a harmless gas at atmospheric pressure and temperature. It therefore presents minimal hazards to laboratory personnel and no concentration or disposal problems. This is in contrast to widely used extraction solventssuch as methylene chloride, chloroform, and benzene. Several recent papers have examined the suitability of SFE for the extraction of nonpolar pollutants from soils (5- 7). Some work has also been done on animal tissues using the selective extraction characteristics of SFE and on-line purification (8, 9). However, use of SFE for the determination of PCBs in fish has received less attention. Extraction of matices to which analytes have been added or “spiked” is inadequate to evaluate a method’s effectiveness at determining native concentrations because of the absence of typical analyte/matrixinteractionsin laboratoryamended samples (10).We report here the development of a rapid, partially automated method to analyze PCBs in fish tissues using SFE. Initially, data on the effectiveness of several SFE trapping conditions are presented using PCBamended matrices. The within laboratory comparability of Soxhlet and SFE-based methodologies for field-incurred PCBs is then examined. Finally, results from the analysis of a candidate Standard Reference Material (SRM) fish homogenate for PCBs with the SFE-based method are compared to consensus values determined by other laboratories using widely accepted methodologies. * Address correspondence to this author; Phone: (804) 642-7228; Fax: (804) 642-7186; e-mail address:
[email protected].
0013-936x/95/0929-1043$09.00/0
0 1995 American Chemical Society
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with detergent and rinsed with water and acetone. Nonvolumetric glassware was then baked for 3 h at 400 O C . Prior to use, all hardware coming in contact with the samples was rinsedwith toluene, acetone, methanol, and methylene chloride. All organic solvents used were residue grade or equivalent (Burdick & Jackson). Severalteleost species were used to examine the recovery of surrogate PCB standards from tissues under different trapping conditions. Goldfish (Carassiusaurum) obtained from a Potomac River tributary (Virginia) were used to compare results between Soxhlet and SFE-based methodologies. Fish were blended and homogenized in their entirety. Additionally,carp homogenates (Cyprinus carpio, Reference Material Carp-1, National Research Council of Canada, originallycollected from SaginawBay, Lake Huron) were analyzed by the SFE methodology. All samples were lyophilized in a FTS Systems freeze-drierprior to extraction. Initial SFE method development was conducted on a PrepMaster unit (Suprex Corp.). Final phases of development were conducted on a SuprexAutoprep 44. Transition to this system was straightforward as major functional components of the instruments were identical. The restrictor systemswere of a variable-flow,heated design. Both used solid-phase, temperature-controlled traps. The AutoPrep 44 was equipped with an autosampler capable of sequential introduction of up to 44 extraction vessels and collection of corresponding extracts directly into GC autosampler vials. Aliquots of lyophilized fish tissue (ca. 1 g dry weight) were loaded into 10-mL stainless steel extraction vessels. The exit end of the extraction vessel was then filled with approximately 6 g of 150 mesh neutral alumina (Aldrich), activated at 150 "C, to retain lipids. Extraction vessels containing alumina blanks were interspaced with those with fish samples to assay reagent purity and possible cross contamination between samples. No interfering compounds were detected greater than 1%of the concentration of the target analytes. PCB surrogate congener standards 30,65, and 204 (IUPACnumbering) were added to the dried tissue and alumina blanks prior to extraction. These congeners are absent from common Aroclor formulations
Replicates (ca. 5 g) of dried goldfish tissue were also placed in fritted glass thimbles and refluxed using heating mantles with 300 mL of methylene chloride for36 h in glass Soxhlet extractors. An aliquot was then removed for gravimetric determination of extractablelipid content. Extracts required purification on an ABC Laboratories Autoprep 1002A gel permeation chromatograph (GPC)to remove the large amount of co-extracted lipids. The GPC column was equipped with a glass column packed with Bio-Beads S-X3 resin (Bio-RadLabs) (12). The column was eluted with 240 mL of a 111 (v/v) mixture of cyclohexane/methylene chloride solvent. GPC fractions containing the PCBs (100mL) were reduced in volume, and the remaining co-extractives were removed by passage over 1.0-g solid-phase florisil extraction columns (Burdick & Jackson) with 5 mL of methylene chloride. Solventwas then exchanged to hexane. Large organic solvent volumes, generated during Soxhlet extraction and GPC purifications, were reduced with a TurboVap LV or TurboVap I1 (Zymark Corp.). Solvent volumes generated for the SFE-based samples were only 2 mL. Thus, concentration in a simple heated water bath under a stream of purified nitrogen was adequate (typical final volume 0.2-0.5 mL). After addition of an internal quantitation standard (PCB 209 or pentachlorobenzene), all SFE and purified Soxhlet extracts were analyzed on aVarian 3400 gas chromatograph, equippedwith a 60 m x 0.25 mm i.d. DB-5 USCW Scientific) fused silica column (25pm film thickness) and an OIC Model 4420 electrolytic conductivity detector (ELCD). The ELCD was maintained at 850 "C. Helium carrier gas flow was 1 mllmin. Injections (1pL) were made in the splitless mode (injector split vent opened after 2 min) by a Varian 8100 autosampler. The injector was maintained at 300 "C. The column temperature was held at 90 "C for 2 min, programmed at 4 "Clmin to 320 "C, and held at that temperature for 15 min. Identification of PCBs was made using a halogen retention index (13). Quantification was accomplishedwith the use of relative response factors. These were obtained by comparison of the response of the internal standard to those of representative PCBs of varying degrees of chlorination. Standards were injected daily to verify GC system response. Corrections were not made for recovery of the surrogate PCB standards. The following PCBs were not completely resolved under the above GC conditions:
(11). A n initial 10-min static equilibration period, at 150 "C
8/5,17/18,28/31,90/101,153/132,138/158,170/190,195/ 208, and 196/203.
Bqwhmtal Sestjon All glassware and utensils used were thoroughly cleaned
and 350 atm, followed bya 30-min extraction in the dynamic or continuous flow mode at a rate of 3 mL/min (liquid flow) of unmodified supercritical COz (SFE-SFC Grade, Air Products) provided highest recoveries of surrogate and native PCBs from fish tissues during preliminary studies. Subsequent experiments were conducted at these conditions. The restrictor was heated to 100 "C. Effectiveness of methanol as a modifier for the COz was also examined. Methanol was added continuously at 10% (v/v) to the extraction stream with a Suprex MPA-1 modifier pump during modifier trials. Two types of trapping materials were examined: 100120 mesh silanized glass beads (Alltech) and 20-30 pm C18-modifiedsilica (Aldrich)mixed 1I1 (wlw) with BO/ 100 mesh Unibeads 2s (Alltech). Different trapping temperatures and elution regimes were examined. 3ptimal retention of extracted PCBs was obtained on the CIS trap at -30 "C. After completion of the extraction, PCBs were eluted from the trap with 2 mL of isooctane at 80 "C. 1044
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Results An initial 10-min staticl30-min dynamic supercritical COZ
extraction, the latter at 3 mL/min, was sufficient to isolate more than 99% of the PCBs available at 350 a m and 150 OC, as shown by sequential re-extraction of fish tissues (data not shown). This temperature and pressure regime provided the highest recoveries of native PCBs from the fish tissues examined. Addition of methanol modifier to the C02 did not increase PCB yields. SFE solid-phase trapping material as well as trap collection and elution temperatures affected the recovery and reproducibility of surrogate PCBs from both alumina and fish tissues. Table 1provides representative data for three trapping conditions (n= 10 for each condition and matrix type). Lowering the trap temperature to -30 "C and eluting with isooctane from C18 provided the best results for the recovery of three surrogate PCB standards from blank alumina. Precision of surrogate PCB recoveries from alumina,
400 7
TABLE 1
Effect of Several SFE Trapping Material/Temperature and Elution Solvenflemperature Regimes on Mean Recoveries and Precision of Three Surrogate PCB Standards from Alumina and from Fish Tissue (n = trap material trap temperature elution solvent elution temperature
PCB 30 PCB 65 PCB 204 PCB 30 PCB 65 PCB 204
n
II
Li c glass beads 20 "C benzene 40 "C mean %RSD A Iu mina 65.6 22.7 69.8 21.5 79.2 20.0 Fish 71.7 8.10 80.2 23.8 35.4 29.4
glass beads CIS -10 "C -30 "C isooctane isooctane 80 "C 80 "C mean %RSD mean %RSD 71.3 18.0 73.3 22.6 81.5 19.6
'
160
c
m
80
89.2 9.19 84.4 15.6 97.3 13.2
89.6 3.93 95.6 97.1 6.20 92.0 77.2 16.3 102
3.56 4.13 6.56
a Values are expressed as percent of PCB added to each matrix. %RSD = % relative standard deviation.
as measured by percent relative standard deviations(%RSD), were approximately 20% under both trapping conditions with glass beads alone. Values presented include not only variations associatedwith the extraction step but also those related to subsequent extract handling and GC analysis. Interestingly,mean recoveries of surrogates PCBs 30 and 65 from amended fish tissues were higher than from alumina alone under all trapping conditions (also shown in Table 1). When glass beads were used for trapping, mean recoveries of PCB 204 from fish tissue were lower than from alumina alone. Use of Cls-modified silica, lowering the collection temperature to -30 "C, and rinsingwith isooctane at an elevated temperature generally improved surrogate PCB recoveries from fish tissues. Mean recoveries under these conditions were 95.6,92.0, and 102%for PCBs 30,65, and 204, respectively. Precision of measurements also improved. %RSD dropped to 3.56, 4.13, and 6.56%, respectively. Subsequent PCB analyses were conducted with these trappinglelution conditions. Mean recoveries from Soxhlet and SFE-based methods (n = 3) for eight field-incurred PCB congeners present in fish were not significantlydifferent (paired t-test, 0.05 level). A trend was apparent in that mean recoveries for lower chlorinated biphenyls (e.g.,28/31,52, and 901101) with the SFE-based methodology appeared somewhat higher than those determined for the Soxhlet approach (Figure1).Mean concentrations of higher chlorinated biphenyls (e.g., 149, 1531105, 1381158, 180, and 1961203) appeared somewhat higher with Soxhlet. Precision of the two methods, as determined by %RSD, were both about 10%. The effectiveness of the SFE-based methodology was further evaluated by comparing results of the analysis of candidate SRM fish homogenates with those obtained during a recent multi-laboratory intercomparison exercise overseen by the National Institute of Standards and Technology or NIST (14). Mean PCB recoveries obtained with the SFE-based approach ( n = 3) were not significantly different (paired t-test, 0.05level) from the multi-laboratory consensus results (n= 16-21, as a function of analyte and after elimination of statistical outliers) or from those obtained by NIST alone (n = 3) (Table 2). PCB values are expressed on a wet weight basis. The %RSDs for the
K-I SOXHLET
0
52
28131
POI101
149
1531105 1381158
180
106RO3
PCB Congener FIGURE 1. Comparison of mean concentrations (wet weight basis) of representative field-incurred PCBs, representing trichloro- through octachlorobiphenyls, determined in homogenizedfish using Soxhlet extraction and SFE ( n = 3). TABLE 2
Comparison of Results of Determinations of PCBs in Fish SRM by SFE-Based Method (n = 3), NIST (n = 3) and Consensus Analyses (n = 16-21)a PCB 17/18 28/31 52 44 95 90/101 118 1531132 105 138 187 128 180 170/190 1951208 206 209 total
SFE mean %RSD 16.7 35.3 133 83.1 180 166 139 74.4 63.6 78.9 33.3 20.4 30.1 13.3 3.92 1.15 1.40 1070
6.41 8.10 8.67 9.69 8.52 10.3 12.2 11.0 13.3 14.3 13.7 16.3 14.6 16.2 25.4 9.41 2.06
NET mean %RSD 22.7 27.7 115 66.3 168 118 100 63.7 42.5 103 32.9 20.7 42.9 18.5 5.26 4.97 5.54 958
10.7 18.4 8.91 7.15 3.96 5.63 4.07 4.27 4.13 6.37 12.8 10.1 7.00 16.5 9.10 3.44 6.05
consensus mean %RSD 21.3 29.3 113 68.4 134 120 117 81.8 51.0 101 29.6 16.9 41.6 21.1 4.50 4.60 4.70
23.0 28.0 28.3 26.9 30.6 15.8 27.4 25.2 30.8 25.7 25.7 29.0 26.4 27.0 31.1 30.4 27.7
955
Mean values are in gglkg (wet weight basis). %RSD = % relative standard deviation. a
consensus results were higher than those obtained by the SFE-based method and NIST itself. However, these incorporate interlaboratory differences as well. Extraction methods used by the labs participating included Soxhlet, sonication, and column elution. None reported the use of SFE. Lipid contents of the goldfish and candidate SRM carp homogenates were determined by gravimetric measurements of extracts obtained from Soxhlet extraction with methylene chloride. Extractable lipids were high in these whole fish homogenates, 24% and 40%, respectively. SFE extracts obtained from these same samples contained less than 0.1% extractable lipid.
Discussion Recognition of the health and safety hazards of commonly used organic solvents and their increasing purchase and disposal costs have resulted in additional interest in SFE, despite the relativelylargecapital costs of these instruments. Some evaluations of the cost effectivenessof commercially available SFE instruments have been published (15,16). VOL. 29, NO. 4, 1995 / ENVIRONMENTAL SCIENCE &TECHNOLOGY
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30.00-
25.00-
E m
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a I-
,o-
s!
Ei I-
0
m m 0
aJ
a
U
0
n
cu
0 1
w
x
15.00-
10.00-
hL 15.00
20.00 25.00 30.00 Retention time in minutes
FIGURE 2. ELCD chromatogram of a candidate SRM fish tissue exti representative PCBs are identified by congener number.
Initial sample extraction typically is the least automated and the most time-consuming step in PCB analysis procedures. Setup and cleaning of apparatuses such as Soxhlets are particularly labor intensive. However, several extractions may be conducted in parallel, speeding the overall analysis of multiple samples. Until recently, commercially available SFE instruments were not highly automated. However, several are currently on the market that allow researchers to perform multiple extractions in series or parallel, including the system used here. To date, alimited number of published reports regarding the application of SFE to the isolation of PCBs from fish tissues have appeared in the literature (e.g.,refs l0,17, and 18). One problem has been the high lipid contents of these organisms, which when coextracted may block SFE restrictors. The introduction of heated, adjustable restrictors have ameliorated this problem. However, the ability to perform selective extraction or online cleanup with SFE would be highly beneficial, as extract purification steps to remove co-extracted fats represent a significant portion of the effort requiredin most PCB determinations. These steps also involve the use of additional solvents and occasionally relatively expensive equipment such as GPC or HPLC. Sample capacity of extraction vessels compatible with automated SFE systems are relatively limited. Sample size is critical for extremely low concentration analyses, e.g., for polychlorinated dioxins (19). However, lyophilization of the tissues prior to extraction conserves considerable vessel volume by eliminatingthe need for inclusion of dying 1046
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40.00
t obtained directly from the SFE with no off-line purification. Some
agents, such as sodiumsulfateor diatomaceous earth.These reagents have been used by a number of researchers to immobilize moisture in the samples and to obtain better extraction efficiencies (17, 18, 20). Water content of the fishes used in our experiments ranged from 75%to 85%of the fresh weight of the tissue. Although losses of volatile analytes are possible during lyophilization, data on concentrations of the lower molecular weight PCBs (most vulnerable to loss by this process), obtained here by SFE after freeze-drying, were comparable to results obtained by labs participating in the NIST intercomparison exercise that did not lyophilize their fish homogenates. Following freeze-drying, samples could be extracted by SFE in less than lh, with minimal glassware usage or physical sample manipulation, in contrast to most conventional methods. Organic modifiers have been required to obtain quantitative recovery of more polar contaminants, e.g., polycyclic aromatic hydrocarbons, from soils (21,221. However, the addition of methanol did not increase recoveries of PCBs from fish tissues in the current study. In agreement with Bswadt et al. (101, inclusion of a static step prior to dynamic extraction resulted in higher PCB yields from fish. Although these researchers also found higher PCB yields at elevated extraction temperature, i.e., 97 "C, attendant co-extraction of additional lipids proved problematic for their approach. The goldfish and carp homogenates contained significant amounts of methylene chloride extractable lipids,24% and 40%, respectively. As a consequence, Soxhlet extracts
of these homogenates required extensive purification, i.e., GPC and florisil column treatment, prior to GC analysis to remove these co-extracted lipids. Some form of pcistextraction purification is essential to most organic solventbased procedures. In contrast, inclusion of alumina in the SFE extractionvessel eliminatedthe need for any additional off-line purification. The combination of selective SFE extraction and alumina retained more than 99%of the lipids. Previously, France et al. used alumina to retain co-extracted fats during SFE of chlorinated pesticides added to poultry fat (8).Johansen et al. (18) recently reported the analysis of biota for laboratory-amended PCBs with coupled SFEl GC, incorporating on-line retention of fats with alumina. During our initial method development, a 5-mL extraction vessel was used, and only 1-2 g of alumina was added to the 1-g fish samples. This reduced the amount of coextracted lipids to the point where only a single florisil solidphase extraction column chromatographystep was required prior to GC injection. Similarly, Bowadt et al. (10) used a single acid silica cleanup step in conjunction with milder SFE extraction conditions prior to on-column GC injection. These simple steps still represent a s i m c a n t improvement over post-extraction purification required following the Soxhlet approach. However, use of the 10-mLvessel and additional alumina eliminated the need for any supplemental off-line post-extraction cleanup. Although inclusion of alumina for on-line retention of fats reduces sample capacity, advantages of this step outweigh this drawback. Method quantitation limits using the ELCD varied from about 0.5 to 0.1 pglkg (with a final extract volume of 0.2 mL), as a function of analyte degree of chlorination. Substitution of electron capture detection (ECD)would reduce this limit, although ECD is less specific than the ELCD. Thus, current sensitivity of the method appears adequate without major modification for most regulatory and scientific studies of PCB burdens in fish. Studies of specific low concentration coplanar PCBs may require some additional modifications. A number of researchers have investigated the use of different trap sorbents such as silica, florisil, and CISphases (23-25). In the current study, Cle-modified silica in combination with low trap temperatures provided excellent recoveries of amended PCBs from fish tissue (Table 1). Johansen et al. (18)also found that a Cl8-basedtrap at -30 "C gave good results for PCBs extracted from fish. A small volume of isooctane, 2 mL, was the only organic solvent required for the optimized SFE-basedprocedure. This volume is several orders of magnitude less than the total required to complete Soxhlet and other conventional approaches. Evaluation of a PCB analysis method should include an examination of its ability to produce accurate and precise measurements of native PCBs in representative matrices. PCB values determined in fish using SFE were similar to those obtained by the widely used Soxhlet approach. Further evaluation was conducted by the analysis of a candidate SRM fish sample. Results of the SFE-based method compared favorably to those obtained by experienced external laboratories using widely validated methods (Table 2). Somewhat lower concentrations of higher molecular weight PCBs were observed with the SFE methodology, as was seen during the Soxhlet trials. Concentrations higher than consensus values were measured by the SFE-based method for some intermediate weight PCBs (e.g., PCB 101). This may be due to contributions by co-eluting pesticides or other halogenated com-
pounds. A supplemental post-extraction column chromatography step to separate PCBs from pesticides was not included in this SFE-based method. A sample ELCD chromatogramof a SRM extract generatedby SFE extraction is provided in Figure 2. In conclusion, SFE was shown to be effective in isolating native PCBs from lipid-rich fish tissues without the need for an intermediate off-linelipid removal step. SFE extracts generated could be collected in autosampler vials and injected directly onto a gas chromatograph. Solid-phase trapping onto C1e-modified silica at -30 "C was effective at retaining a wide range of PCBs. The method was less laborious and required far less organic solvent than conventional procedures, such as Soxhlet extraction, but produced comparable results.
Acknowledgments Field samplesused in the trap evaluations and Soxhlet comparison exercises were collected by theVirginia Department of Environmental Quality. Suprex Corp. loaned a Prepmaster SFE system during initial method development. Dr. Craig L. Smith and Ellen Harvey are acknowledged for their excellent technical support. VIMS Contribution No. 1921.
literature Cited (1) Maack, L.; Sonzogni,W. C.Arch.Environ. Contam.Toxicol. 1988, 17, 711. (2) Schmitt, C. J.: Zajicek, J. L.; Peterman, P. H. Arch. Environ. Contam. Toxicol. 1990, 19, 748. (3) Hale, R. C.; Greaves, J. J. Chromatogr. 1992, 580, 257. (4) Camel, V.; Tambute, A.; Caude, M. 1. Chromatogr. 1993, 642, 263. (5) Hawthorne, S. B.; Miller, D. J. J. Chromatogr. 1987, 403, 63. (6) Langenfeld, J. J.; Hawthorne, S. B.; Miller, D. J.; Pawliszyn, J. Anal. Chem. 1993, 65, 338. (7) Lee, H.-B.;Peart, T. E.; Hong-You,R. L.; Gere, D. R.J. Chromatogr. 1993, 653, 83. (8) France, J. E.; King, J. W.; Snyder, J. M. 1.Agric. Food Chem. 1991, 39, 1871. (9) Snyder, J. M.; King, J. W.; Rowe, L. D.; Woerner, J.A. J. AOAClnt. 1993, 76, 888. (10) Bswadt, S.; Johansson, B.; Fruekilde, P.; Hansen, M.; Zilli, D.; Larsen, B.; de Boer, J. J. Chromatogr.1994. 675, 189. (11) Schulz, D. E.; Petrick, G.; Duinker, J. C. Environ. Sci. Technol. 1989, 23, 852. (12) Hale, R. C.; Bush, E.; Gallagher,K.; Gundersen, J. L.; Mothershead, R. F., I1 J. Chromatogr. 1991, 539, 149. (13) Mothershead, R. F., 11; Hale, R. C.; Greaves, J. Environ. Toxicol. Chem. 1991, 10, 1341. (14) NISTlNOAA NS&T/EPA E M . Intercomparison Exercise Program for Organic Contaminants in the Marine Environment: Fish Homogenate I QA93FSHl. Draft results from the NOM NS&T quality assurance workshop Dec 9, 1993, Miami, FL. (15) Lopez-Avila, V.; Dodhiwala. J. Chromatogr. Sci. 1990, 28, 468. (16) King, J. W.; Snyder, J. M.; Taylor, S. L.; Johnson, J. H.; Rowe, L. D. J. Chromatogr.Sci. 1993, 31, 1. (17) Nam, K. S.; Kapila, S.; Yanders, A. F, Puri, R. K. Chemosphere 1990, 20, 873. (18) Johansen, H. R.; Becher, G.; Greibrokk,T. Freseniml.Anal. Chem. 1992, 344, 486. (19) Larsen, B.; Facchetti, S. FreseniusJ.Anal. Chem. 1994,348, 159. (20) Hopper, M. L.; King,J. W.J.Assoc. OR Anal. Chem. 1991, 74,661. (21) Hawthorne, S. B. Anal. Chem. 1990, 62, 633A. (22) Levy, J. M.; Donata, L.; R a y , R. M.; Storozynsky,E.; Holowczak, K. A. 1.High Resolut. Chromatogr. 1993, 16, 368. (23) King, J. W. J. Chromatogr.Sci. 1989, 27, 355. (24) Mulcahey, L. J.; Hedrick, J. L.; Taylor, L. T.Ana1. Chem. 1991,63, 2225. (25) Bswadt, S.; Johansson, B. Anal. Chem. 1994, 66, 667.
Received for review July 28, 1994. Revised manuscript received December 2, 1994. Accepted December 19, 1994.@ ES9404734 @
Abstract published in Advance ACS Abstracts, February 1, 1995.
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