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Anal. Chem. 1982, 54, 106-112
Capillary Gas Chromatographic Determination of Polycyclic Aromatic Compounds in Vertebrate Fish Tissue Daniel L. Vassilaros, Paul W. Stoker, Gary M. Booth, and Milton L. Lee* Departments of Chemistry and Zoology, Brigham Young University, Provo, Utah 84602
Polycycllc aromatic compound fractlons were resolved from vertebrate flsh tissue and analyzed by capillary column gas chromatography. The analytlcal procedure includes the following steps: aqueous alkaline dlgestlon, acldificatlon of the dlgestate wlth glacial acetic acld, exiractlon wlth methylene chlorlde, Ilquid-llquld partitioning wlth water and then a 10% KOH solutlon, adsorptlon chromatography on basic alumlna, uslng hexane, benzene, and chloroform sequentially, gel permeatlon chromatography on BioBeads with methylene chloride, and capillary gas chromatography and gas chromatography/mass spectrometry. Examples of chromatograms of the polycycllc aromatic compound fractions from flsh from polluted and pristlne waterways are glven. The limit of detection Is less than 0.2 ppb (ng/g wet tissue), and average recovery is 72 Yo of spiked 14C-labeled anthracene.
North American inland and coastal aquatic ecosystems have been contaminated by hydrocarbons from a variety of industrial effluents, urban stormwater runoff, oil spills, particulate matter from fossil fuel fired power plants, and aqueous effluents from coal and shale conversion processes. Increased coal utilization (direct combustion, liquefaction, and gasification) will especially add to the totalenvironmental polycyclic aromatic compound (PAC) burden. The behavior and effects of anthropogenic polycyclic aromatic hydrocarbons (PAH) in aquatic biota in chronically and acutely polluted waterways have been intensely studied for many years (1-7). Although mollusks have been shown to accumulate PAH, the question of whether the concentrations of potentially toxic and carcinogenic PAH are magnified through the food chain is not yet resolved. The analytical chemistry of PAC in tissues can provide an important part of the answer to the biomagnification question, but it must be improved by new technology and the modification of existing procedures to the point where unambiguous, detailed, and reproducible data can be obtained on a routine basis. The progress in PAC analysis of tissues is marked by the concurrent improvement in chromatographic techniques and detection systems employed. Separations of purified tissue extracts have been done with column chromatographic methods (8-14) and on TLC plates with specific wavelength UV and fluorescence detection and quantification (11-20). HPLC with UV and fluorescence detectors in series has largely supplanted TLC for sensitive analysis of PAC in complex biological matrices (21-30). The availability of selective detection (UV and fluorescence) eliminates the need for extensive cleanup of PAC fractions, but the efficiencies obtained with HPLC are not sufficient for resolving the numerous isomers present in complex PAC mixtures. Packed column and capillary column gas chromatography have been used to great advantage in identifying, profiling, and quantifying complex PAC mixtures in tissues (28-55); and capillary GC-MS has proven to be a necessary technique for confirmation of tentative GC identifications (56, 57). In a
recent review (58), Lee and Wright showed that excellent isomer resolution, separation of a wide boiling point range of compounds, increased sensitivity, and tentative identification via the PAH retention index system (59) were achieved with the use of capillary gas chromatography for the analysis of complex PAC mixtures. The only remaining obstacle for the application of capillary GC to the analysis of PAC in biological material was described by Warner et al. (28);e.g., the presence of biogenic interfering compounds in the aromatic fraction obfuscates the analysis of trace level PAC by nonselective GC methods alone. The literature contains many gas chromatograms of aromatic fractions from fish in which the PAC are minor, if resolvable, components. This problem is the major justification for the use of selective spectrophotometric detectors and selected ion mass spectrometry; but even then, the consensus drawn in several papers on hydrocarbon analysis of aquatic/marine tissues (1,2,4,28,30,37,43) is that no one analytical method in use today will provide low to sub-partper-billion sensitivity, detailed profile information, and complete resolution of a wide molecular weight range of parent and alkylated PAC from a biological matrix. Furthermore, few papers dealing with the analytical methodology for determining polycyclic aromatic sulfur heterocycles (PASH) and polycyclic aromatic nitrogen heterocycles (PANH) in fish tissues are to be found in the literature. Consideringthat the heterocyclic fractions are at least as biologically active as the PAH (60-66), it is clearly desirable that techniques be developed which will provide accurate quantitative and qualitative data on the sulfur and nitrogen heterocycles in aquatic biota. The characteristic capillary GC profiles of the sulfur and nitrogen heterocycle fractions from tissues and sediments may be helpful in tracing sources of petroleum and/or synthetic fuel pollution. The purpose of this paper is to describe the analytical methodology for the extraction, cleanup, and high-resolution gas chromatography of PAC fractions in vertebrate fish.
EXPERIMENTAL SECTION Materials. Hexane, benzene, and methylene chloride were MCB (Omnisolve) solvents. Chloroform was Fischer Spectrograde and glacial acetic acid was Spectrum reagent grade. All solvents were used without further purification. Reagent grade KOH pellets were washed with methylene chloride/acetone and then dried and stored. “Baker Analyzed” basic alumina, Brockmann activity grade I (80-200 mesh), was used directly out of the bottle without activation/deactivation procedures. A 60-g quantity of BioBeads SX-12 (exclusion limit 400) was Soxhlet extracted with methylene chloride/acetone overnight, packed in a glass column (11 mm id.) with a Teflon stopcock, and eluted with several column volumes of methylene chloride. Initial tissue homogenation was done with a Tekmar “Tissumizer”. All glassware used in the analytical procedure was washed with soap and water, rinsed with distilled water, placed in an acid bath (41 v/v, H$04/HN03) overnight, rinsed with distilled water, and dried in an oven at 130 “C. Saponification. About 60 g of frozen composited whole fish tissue (obtained from the U.S. Fisheries and Wildlife Service, Columbia, MO) was placed in a 600-mL beaker with about 100 mL of distilled water and homogenized. After the tissue was well dispersed, 7 g of KOH pellets was added, and the mixture was
0003-2700/82/0354-0106$01.25/00 1981 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 54, NO. 1, JANUARY 1982
further homogenized until all solid matter had been finely dlispened or dissolved. The .mixture was then transferred to a 500-mL round bottom flask, several cleaned Teflon boiling chips were added, an internal standwd was spiked into the mixture, and the mixture was moderately refluxed for 4 h. Liquid-Liquid Extraction. The digestate was transferred to a 2-L separatory funnel containing 100 mL of distilled wat,er. Approximately 150 mL of glacial acetic acid was added t o the reflux mixture (to ab0u.t.pH 4). After cooling, the digestate was extracted with three separate 250-mL portions of methylene chloride. These extractri were combined, concentrated to a to'tal volume of about 250 mL by rotary evaporation, washed with distilled water, and then washed with a 10% aqueous KOH solution. If a large amount of saponifiable oils was present in the final organic portion, the hydrolysis (without refluxing) a.nd partitioning steps were repeated without suffering appreciable loss of trace organic compounds. Column Cromatogiriaphy. Several milliliters of hexane was added to the final 5-10 mL of methylene chloride solution as the more volatile solvent was being taken off by rotary evaporation; and the sample (in hex,ane)was added to the head of a 9-g basic alumina column (30 cm X 11 mm id.). 13lution with 30 mL, of hexane brought the a1:iphaticsoff the column first; the PAH/ PASH fraction was eluted next with 100 niL of benzene; and the PANH fraction was elu.ted last with 100 mL of chloroform. The two aromatic fractions were concentrated, again by rotary evaporation, and cleaned up on a BioBeads SX-12 column, using methylene chloride as the eluant. The )first 50 mL of solvent contained biogenic comlpounds (carotenoidsand steroids) and was discarded. The next 50 mL was collected, concentrated to near dryness by rotary evaporation, transferred to an acid-washed vial with a Teflon cap liner, and reduced in volume under N2gas purge to the desired volume. Capillary Gas Chraimatography and Gas Chromatography/Mass Spectrometry. The PAC fractions were chromatographed on SE-52 (film t,hicknessof 0.34 bin) fused silica capillary columns (13-20 m X 0.30 mm i.d.) mounted in a Hewlett-Packard 5880 gas chromatograplh. The temperature program was 40 to 260 "C at 4 "C/min. Hz at a linear velocity of 100 cm/s was used for the carrier gas. Tentative peak identifications were made by employing a BASIC program written for the HP 5880 which uses the PAH retention index system (59) t o identify the PAC. Dual trace chromatograms were obtained by using fused silica effluent splitters (67) int3talled in Perkin-Elmer Sigma 2 and Sigma 3 gas chromatographs. The ovens were programmed from 50 to 250 "C at 4 "C/min. Helium at a linear velocity of 40 cm/s was used for the carrier gas. All identifications were confirmed by fused silica capillary gas chromatography mass spectrometry (G'C-MS) on a HewlettPackard 5984 GC-MS-DS. The mass spectrometer end of the fused silica column wm positioned within 4 cm of the electron beam, and the direct insertion probe was placed so as to stop the hole in the DIP plug in the source body, preventing escape of GC column effluent intothe source manifold. The gas chromatograph, fitted with a Carlo Erba on-column injector,was programmed from 40 to 290 "C at 8 "C/min. GC quantification was done by coinjecting naphtho[2,3-b]thiophene as an internal standard, assuming equal response factors across the molecular weight range of the sample. Recovery Study. A radio-labeled PAH, [9-14C]anthracene, New England Nuclear, was dissolved in methylene chloride, and replicate aliquots from the stock solution were counted in an Aquasol cocktail using a Delta 300 liquid scintillation spectrometer. Three microliters (171000 disintegrations/min) of the stock solution was added to each of two bullhead catfish samples from a pristine area. The '%pike" was injected into the homogenate before commencing refluxing. Measured aliquots from known volumes of the samples were taken at every step in the analytical procedure and were counted in an Aquasol cocktail after 24 h of refrigeration.
RESULTS AND DISCTJSSION The majority of PA.13 analyses of aquatic tissues have been done according to the general procedure outlined in 1966 by Howard e t al. ( 1 1 ) . The elements of this procedure are (a) alkaline hydrolysis of tissues, (b) extraction of the hydrolysate
107
G i GC Id5
Figure 1. Analytical scheme for determination of PAC in tissues.
with a nonpolar solvent, (c) fractionation of the nonsaponifiable portion on an adsorption chromatographic column using an increasingly polar eluant, (d) liquid-liquid extraction, usually with hlezSO, and (e) analytical chromatography, e.g., TLC, HPLC, or GC. The results of the application of this general procedure have been useful, especially where selective detection (e.g., UV, fluorescence, and MS) has been employed. Although many variations have appeared in print, the basic theme remains essentially unchanged. In a recent interlaboratory comparison on the determination of trace level hydrocarbons in mussels (43),the laboratories which responded all used saponification, solvent extraction, adsorption chromatography, and analytical chromatography for their analyses. Only in the details of each step did the laboratories differ from each other; yet of the seven participating laboratories, only two returned extensive data on the PAH, and those data were inconsistent by a factor of 10. Careful application of the analytical procedure described herein should provide more detailed and consistent data. Figure 1diagrams the proposed analytical procedure which will be discussed below. Saponification. Grimmer and Bohnke (35)claimed that only 30% of the aromatics from a tissue sample was obtained by extraction with boiling methanol, while an additional 60% was recovered upon saponification of the tissue. They emphasized that the saponification must be complete in order to prevent the formation of stable emulsions. The preferential use of saponification as the first step is evidenced in ref 4 and 28-30. Four of the laboratories involved in the NBS mussel round-robin (43)used an alcoholic base solution for saponification, while the others used an aqueous caustic solution. Howard et al. (11-13) suggested an ethanolic KOH hydrolysis of the tissue. Dunn and others (18-20, 25-27, 39, 43, 51) employed ethanolic KOH, while Grimmer and others (10,14, 33-35, 39, 43) opted for methanolic KOH. Warner (37), Cheder et al. (29), and others (4, 23, 30, 42, 54) used an aqueous NaOH or KOH solution for the initial saponification step. The normality of the base solution ranged from 0.5 to 6 N, and the length of digestion varied from 1.5 to 24 h. Howard and Fazio (5)concluded that a base hydrolysis step was essential for the analysis of hydrocarbons in tissue but made no suggestion regarding the advantages of aqueous or alcoholic saponification steps. However, fats reacted with methanol in the presence of an acidic or basic catalyst are converted by transesterification to the fatty acid methyl esters which are then difficult to remove from the PAH fraction. Because the potassium soaps formed in the aqueous saponification process will solubilize the hydrocarbons, preventing their loss frorn the refluxing solution, the use of an alcohol in the digestate is superfluous and contributes to interference problems in the final analysis.
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ANALYTICAL CHEMISTRY, VOL. 54, NO. 1, JANUARY 1982
Liquid-Liquid Extraction. Isooctane (10, 13, 25, 39), cyclohexane (14,36,43),hexane (23,26,42,43),pentane (29, 31, 33, 34,53), ethyl ether (30, 37, 54), benzene (33),and a mixed ethyl ether/methylene chloride solvent (43) have all been used for the extraction of the nonpolar, nonsaponifiable materials from the caustic digestate. Methylene chloride is an excellent solvent for PAC; but ethyl ether will extract a wide polarity range of compounds from the digestate which will have to be eliminated in subsequent separations. This partitioning step is usually accompanied by the formation of a stable emulsion (especially with the first extraction) which can decrease the efficiency of the partitioning mechanism. Even partial elimination of the emulsifying soaps formed in the base hydrolysis step significantly improves the quantitative extraction of nonpolar compounds from the aqueous hydrolysate. Acidifying the digestate with glacial acetic acid (mineral acids are implicated in the formation of esters and are to be avoided) frees a sufficient quantity of fatty acids from their potassium salts such that the emulsions are practically eliminated from the partitioning steps. Although the free long-chain fatty acids are extracted into the organic phase, they are readily removed in subsequent treatments of the organic extract. Extraction with methylene chloride removes steroids, carotenoids, free fatty acids, phthalate esters, hydrocarbons, and some acetic acid from the digestate. The acetic acid and other aqueous-soluble species can be extracted from the methylene chloride by a single water wash. If the two phases do not separate cleanly, adding a small amount of clean NaCl will break up the emulsion. The yellow carotene color is retained in the organic phase through this step, but partitions into the aqueous layer upon addition of an approximately equal volume of aqueous 10% KOH solution. As the layers separate after vigorous shaking, the upper layer takes on a distinct yellow tinge, leaving the lower phase fairly colorless. The light, persistent emulsion which usually forms a t the interface a t this point can be eliminated by the judicious use of small amounts of NaCl and MeQH. GC and GC-MS analyses show that the PAH, PASH, and PANH are retained in the organic phase, as well as large amounts of biogenic hydrocarbons such as cholesterol and some carotenoids. Adsorption Chromatography. Florisil(11,13,19,25,27, 55), alumina (11, 1 4 , 1 6 , 3 9 , 5 1 ) silica , gel (14,30,33,35-37, 43,54,55),silicic acid (32,52),and combinations of alumina and silica gel (26,31,33,34,40,43,53)have been employed for fractionating the hydrocarbon extract into aliphatic and aromatic portions. Solvents similar to those used in the liquid-liquid extraction step are commonly the eluants. Work performed in this laboratory on the class separation of a synthetic fuel on neutral alumina was recently published (68). This procedure gives a clean separation between aliphatic, aromatic, and nitrogen heterocyclic hydrocarbons in one chromatographic step. Further cleanup is required for the fish extract subfractions, however, because high levels of biogenic compounds elute with the PAH and P A " from the alumina column. For example, up to l/z g of lycopersene (mol wt. 546, C40H66),a viscous, colorless carotenoid, can elute in the PAH alumina cut, causing severe interference problems with the capillary GC analysis of the fraction. Cholesterol saturates the PANH fraction and must be completely removed before reasonable capillary GC analysis can be performed. MezSO Extraction/Gel Permeation Chromatography. Several authors reported an extensive MezSO partitioning scheme to clean biogenic materials from hydrocarbon fractions (11,13,19,25-27,39).Dunn and Armour (25) suggested that MezSO extraction and gel chromatography on LH-20/2propanol gave complementary separations. Perusal of the
Natusch and Tomkins article (69) reveals that the MezSO partitioning procedure will not separate the PAH from the phthalates, high molecular weight n-aliphatic acids, and other neutral aromatic species. Experience has shown that the long-chain fatty acids, phthalate esters, and other neutral compounds (carotenoids, steroids) interfere with the determination of high molecular weight PAH. Adsorption chromatography on Sephadex LH-20 (57) has been employed by a few authors in lieu of the MezSQ extraction (28, 52, 54). The size difference between the PAC and the biogenic compounds can be exploited for sample cleanup by gel permeation chromatography. The BioBeads column used in this procedure was calibrated with a mixture of carotenes and PAH; the carotenoids and steroids eluted in the first 50 mIJ of methylene chloride, and the aromatics (both PAH and PA") eluted in the next 50 mL, The total time required to prepare one fish sample for GC analysis is 6-8 h; multiple samples can be prepared at the same time. Analytical Chromatography. Benzo[a]pyrene can be separated from its structural isomers by TLC on celluloseacetate and then quantified by fluorometric techniques (18). TLC was used in the early stages of PAH analysis but has been largely replaced by HPLC and GC methods. HPLC on reversed-phase columns will not separate certain PAH isomer pairs (25),but the selective and sensitive detection afforded by UV and fluorescence detectors in series can give data on quantities at the picogram level. However, the unique response of the fluorescence detector at different wavelengths to each PAH requires an extensive calibration procedure in order to properly perform quantitative analysis. Furthermore, the resolution and profile information generated by HPLC (e.g., ref 25), although useful in certain situations, are not always comparable with other high-resolution techniques. PAH fractions from aquatic tissues have been analyzed by GC on packed columns (35-37,39,42,51)and on capillary columns (4, 28, 30, 43, 50, 52, 54, 55). Capillary gas chromatography is considered an excellent method for the analysis of complex PAC samples from petroleum, coal liquids, and environmental samples (58). With the development of the PAH retention index system (59),software incorporating the retention index system has been written which calculates retention indices for the peaks in the run, compares them with a data bank of standard retention indices and corresponding compounds, outputs all the calculated indices, and names those peaks whose indices fall within the tolerance windows established in the program. The softwave provides the means of rapidly and inexpensively screening PAC fractions for individual components and their amounts prior to confirmatory GC-MS. Figure 2 shows a capillary gas chromatogram of the PAH/PASH fraction of a brown bullhead catfish (Zctalurus nebulosus) from the Black River in Ohio. (The Black River flows through a heavily industrialized region which includes at least one coking plant.) A recent study (70) showed that this particular fish had several cholangiomas (bile duct tumors). Table I lists the peak numbers, compound names, retention indices, and amounts of the peaks numbered in Figures 2-5. Peak amounts are given in parts per billion (ng/g wet tissue), and have been corrected assuming an average 72% recovery. The fraction consisted primarily of PAH ranging from 2-methylnaphthalene to benzo[ghi]perylene, with fairly high levels of PASH. The major components are acenaphthylene, dibenzofuran, fluorene, phenanthrene, fluoranthene, and pyrene. The lower detection limit for this sample was about 0.5 ppb. Total sample volume was 50 pL, and the injection volume was 0.3 bL. The high efficiency of the SE-52 capillary column used in this analysis is demonstrated in the
ANALYTICAL CHEMISTRY, VOL. 54, NO. 1, JANUARY 1982
109
\?
Flgure 2. Capillary gas chromatogram of PAHIPASH fraction from Black River bullhead catfish. See Table for chromatographic conditiions.
I for peak identifications and text
11
I
2
1 0
1 0 TIME (rnn i)h-
0
10
0
2
do
0
Flgure 3. Capillary chromistogram of PAH/PASH fraction from Buckeye Lake bullhead catfish. See Table I for peak identifications and text for chromatographic conditions.
TEMPITI
7
40 5 IM E(m1n)
10
2
1 0
1 0
7 - I 20
JO
20
2 0
20
Flgure 4. Capillary gas chromatogram of PAI-VPASH fraction from Potomac River striped bass. See Table I for peak identifications and text for chromatographic condlitlons.
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ANALYTICAL CHEMISTRY, VOL. 54, NO. 1, JANUARY 1982
Table I. PAH and PASH Determined in Three Fish Samples
peak no. 1 2 3 4 5 6 7 8 9 10 11
12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
amount, ppb Buckeye Black River Lake 6
5
14 100
17
270 39
Potomac River
retention index
compound name
200.000 220.400 223.240 236.237
naphthalene 2-methylnaphthalene 1-methylnaphthalene biphenyl C,-naphthalenes acenaphthylene acenaphthene dibenzofuran C,-naphthalenes fluorene methylbiphenyls or methylacenaphthenes methyldibenzofurans C,-biphenyls methylfluorenes dibenzothiophene phenanthrene anthracene naphtho [ 2,3-b]thiophene methyldibenzothiophenes 1-phenylnaphthalene methylphenanthrenes 4H-cyclopenta[de flphenanthrane C,-dibenzothiophenes 2-phenylnaphthalene C,-phenanthrenes fluoranthene acephenanthrylene phenanthro[ 4,5-bcd ]thiophene pyrene methylfluoranthenes and methylpyrenes benzo [alfluorene benzo[ b ]fluorene benzo[ b]naphtho[ 2,ldlthiophene benzo [ghilfluoranthene benzo[c]phenanthrene benzo[ b ]naphtha[ 1,2d ]thiophene benzo[b]naphtho[2,3dlthiophene cyclopenta[ cd Ipyrene benz [ a ]anthracene chrysene benzofluoranthenes benzo [ e Ipyrene benzo[a]pyrene perylene indeno [ 1,2,3-cd Ipyrene dibenzanthracenes benzo [ghilperylene
1 1 180 43 7
246.557 252.792 258.546 269.476
270 2700
2
21 36
295.323 300.000 301.162
17 316.350 321.789 333.516 1800
4
78 1500
4
9 16
31
344.372 347.669 348.644 351.263 366.811 369.458 389.063
6
34 35 36
389.768 391.245 392.546
37
395.594
38 39 40 41 42 43 44 45 46 47
22 61
6
4 3
396.341 398.782 400.000
18
14 7
1
8
near-base-line resolution of the isomer pair benz[a]anthracene and chrysene, the splitting of the benzofluoranthenes, and the separation between the benzopyrene isomers. The PAH content of three other bullhead catfish from different locations in the same river was similar. A bullhead catfish from a pristine area (resort lake, no industry, agricultural) was analyzed for a comparison; the gas chromatogram is reproduced in Figure 3. An aliquot of 0.6 pL for GC analysis was taken from a 15-pL sample volume. The lower limit of detection is
