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Determination of Mirex in Fish by Gas Chromatography and Gas Chromatography-Mass Spectrometry-Computer System J. L. Laseter,” I. R. DeLeon, and P. C. Remele Center for Bio-Organic Studies, University of New Orleans, New Orleans, Louisiana 70 122
Four fish species from Lake Ontario were analyzed by various gas chromatographic and gas chromatographic-mass spectrometric-computer methods. Quantltative analyses employing reconstructed mass chromatograms with the mlrex (perchloropentacyclo[5.3.0.02~6.03~s.04~8]decane or dodechlorooctahydro-l.3.4-metheno-2H-cyclobuta[c, dlpentalene) base peak ( m / e 272) and gas chromatography with electron capture and Hall electrolytic detectors provided concentration values ranging from 0.15 to 0.33 pg/g fresh weight of tissue. However, the technique of selective ion monitoring or mass fragmentography using high mass ion fragments exclusively associated with mlrex demonstrates that the actual mirex concentrations are from 3 to 6 times lower. Reasons for the wide range of variability as a function of methodology are dlscussed.
unanswered questions. For example, (a) is the molecule indeed mirex and if so, why is the relative abundance of the high mass fragments depressed, (b) are any impurities co-eluting with the mirex that could interfere withquantitation, and (c) how does the quantification by halogen-specific chromatographic detectors compare with mass spectral findings using SIM? In order to answer these questions, we undertook to investigate the “mirex” component in fish from Lake Ontario in greater detail. T h e techniques of low and high resolution mass spectrometry as well as low resolution and high resolution gas chromatography were employed. The accurate quantification a t trace levels of pesticides in aquatically derived food materials is of great importance. Specific concentration criteria have been established for most potentially toxic materials. Quantities present above these criteria or “action levels” will ban such foods for human consumption.
Gas chromatography is the method commonly employed for the routine analysis of trace quantities of chlorocarbon pesticides isolated from the environment. Detectors such as the electron capture and Hall electrolytic conductivity are frequently used because they show a high degree of specificity and sensitivity for such halogenated, organics. However, confirmatory evidence is required (1,2).Gas chromatography-mass spectrometry-computer instrumentation offers a mechanism which not only partially resolves the chlorocarbon molecules of interest but also provides an opportunity to conduct both qualitative and quantitative analyses. The most powerful of these techniques involves selective ion monitoring (SIM) and offers a far greater sensitivity and accuracy than any other routine analytical method. In recent years it has been applied to drugs, drug metabolites, pesticides, and other compounds present in trace quantities in complex mixtures (3-7). Even with techniques such as SIM or mass fragmentography, great care must be taken because of the possibility that several molecules in the sample may have similar fragmentation patterns and chromatographic retention times. In recent years a number of reports have appeared in the literature regarding the presence of mirex (a perchlorinated, cage-structured hydrocarbon, Cl0Cll2)in low concentrations in fish from Lake Ontario (8, 9). Kaiser presented mass spectral evidence for mirex in two fish samples. However, the parent ion cluster at mle 540 was not detected. Additionally, the abundance of the M - C1+ cluster (mle 505) was less than that observed for the mirex standard. Also of interest was the fact that the ion cluster a t mle 235, resulting from the loss of chlorine from the hexachlorocyclopentadiene fragment (C5Cl6+)was present a t higher relative abundances than the standard. Quantification was accomplished by measuring the height of the base peak for mirex (mle 272) against a calibration curve (10). Preliminary data developed on Lake Ontario fish samples in our own laboratory generally support these findings. However, when the mass spectral data for mirex in a sample are normalized to the base peak, the absence and/or unusually low abundances of key high mass ion clusters leave some
Materials. Authentic mirex was obtained from Hooker Chemicals and Plastics Corporation, Niagara Falls, N.Y. The sample was reported to be 100% pure and, therefore, no attempts were made at further purification. Gas chromatographic and mass spectral analyses indicated that the sample was identical to mirex standards provided by the USDA laboratory in Gulfport, Miss., and the EPA Region I1 laboratory in Edison, N.J. All solvents used were Distilled-in-Glass (Burdick and Jackson, Muskegon, Mich.) or equivalent. All other chemicals and reagents were ACS reagent grade or better. Glassware was cleaned with soap and water followed by multiple washes with chromic acid cleaning solution and distilled water prior to use. Preparation of Fish Samples. A Coho salmon, brown trout, white bass, and rainbow trout were collected from Lake Ontario by Equitable Environmental Health, Inc. (Woodbury,N.Y.) during the fall of 1976. The samples employed for this study were those specimensthat provided high electron capture and Hall electrolytic detector responses for a chromatographic peak with retention characteristics similar to mirex. All analytical work was performed with ground portions of the fish including flesh, scales, and skin but excluding bones and internal organs. All samples were ground by a single passage through a Hobart food grinder followed by 5 min of mixing in a Hobart planetary mixer. The ground fish samples were then kept frozen until extracted. The sample preparation procedure for the analysis of mirex in animal tissue was almost identical to the procedure used by USDA (11, 12) and is described as follows: Extraction Procedure. Twenty grams of ground fish were transferred to a 1-L screw-capped bottle and 400 mL of a 3:l n-hexane-isopropanol solution were added. The mouth of the bottle was covered with aluminum foil, capped, and the bottle rotated for 2 h on a mechanical device. The extract was filtered through previously cleaned glass wool into a 500-mL separatory funnel. Fifty mL of a saturated sodium chloride solution and 50 mL of deionized water were added. The funnel was shaken for 2 min and the layers were allowed to separate. The lower layer containing water and isopropanol was drained and discarded. The saturated sodium chloride-water washing was repeated, and again the lower layer was discarded. The remaining hexane layer was washed with two successsive 50-mL portions of deionized water. The hexane layer was finally filtered through glass wool and anhydrous sodium sulfate. The hexane layer was concentrated to about 10 mL on a Buchi (Flawil,Switzerland) rotary evaporator, and then diluted back to 20 mL with fresh hexane.
EXPERIMENTAL
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 8, JULY 1978
Sulfuric Acid Wash. A 10-mL aliquot of the hexane extract (representing 10 g of fish) was placed in a 250-mL separatory funnel and 40 mL of fresh hexane were added. To this mixture were carefully added 10 mL of concentrated sulfuric acid with gentle swirling. The mixture was shaken and the layers were allowed to separate. The acid layer was drained and discarded. This sulfuric acid wash was repeated two additional times. The remaining hexane layer was then washed with three successive 25-mL portions of deionized water and the washings were discarded. The hexane layer was filtered through glass wool and anhydrous sodium sulfate and then concentrated to about 2 mL. Column Clean-up. A chromatographic column was prepared by placing in an 11 X 300 mm tube with a fritted disk and a 50-mL flask reservoir, 2.5 cm of anhydrous sodium sulfate followed by 25 cm of 60-120 mesh nonactivated Florisil, and topped with another 2.5 cm of sodium sulfate. The column was prewashed with 100 mL of hexane, allowing it to drain to the top of the upper sodium sulfate layer. The sample was transferred onto the top of the column and chromatographed with 100 mL of hexane. The hexane fraction was concentrated t o about 2 mL and stored in a freezer until analyzed. Immediately prior to analysis the sample extract volumes were carefully reduced to dryness under a stream of N2 and the residue redissolved in 20 to 40 FL of hexane. Samples that were used for electron capture gas chromatographic analyses were further cleaned up by use of the silicic acid column chromatography procedure developed by Armour and Burke (1) as modified by Gaul and Cruz-LaGrange (13). Combined Gas ChromatographylMass Spectrometry. Three instruments were employed in this study. Most analyses were performed on a Hewlett-Packard (HP) 5982A mass spectrometer equipped with an H P Model 5700A gas chromatograph and an HP 5933A dual-disk data system. The gas chromatography was equipped with a 70 cm X 20 mm (id.) glass column packed with 2 % OV-101/0.2% Carbowax 20M on 100-120 mesh Chromsorb W-HP. Analyses were performed under the following conditions: the helium flow rate was 15 mL/min; the injection port was maintained at 250 "C; the oven temperature was at 200 "C; and the effluent was passed through a silicone membrane separator maintained at 200 "C and was directed through a glass-lined transfer line into the ion source. The transfer line was maintained at 250 "C. Mass spectra of the chromatographic effluents were measured in both the computer and manual modes of operation. In the computer mode of operation, the data system was used for mass spectrometer control. Under a pre-selected data command, 70-eV spectra were scanned from mle 100 to mle 600. The mass range was scanned at a rate of 162.5 amu/s every 3.1 s. Scanning was initiated about 2 min after injection to allow for passage of the solvent front. During each analysis, the total ion current was monitored as well as the mass chromatograms of two selected ions, mle 272 and mle 274. In conducting the SIM quantitative analyses, ion fragments at mle 272, 439, 509, and 546 were measured and quantified at the expected retention time for mirex by use of the H P selective ion monitoring computer routine. The dwell-time for a given ion was 200 ms. In the manual mode of operation, 70-eV spectra were scanned repetitively from mle 200 to mle 600 at 100 amu/s, with a band width of 112 Hz. Scanning was usually initiated about 1 min before the expected time of elution for mirex and was continuously monitored on the oscilloscope. When the mle 235 and mle 270 ion clusters were observed on the oscilloscope, the scans were recorded on a Datagraph 154-5light beam oscillograph with a chart speed of 11 cm/s. See Figure 1 for typical scans. Some samples were qualitatively analyzed at a resolution of -1200 on a DuPont 21-491 mass spectrometer interfaced with an H P Model 5750 gas chromatograph and equipped with a 76 cm X 2.3 mm (id.) stainless steel column packed with 3% OV-101 on 100-120 mesh Chromsorb WAWIDMCS. The helium flow rate was 10 mL/min. The injection port was maintained at 200 "C, and the oven temperature was programmed from 175 to 250 "C a t 4 "Clmin. The GC effluents were directed through a stainless steel jet separator via stainless steel transfer lines. The jet separator was maintained at 220 "C; the transfer lines, at 190 "C. The ion source was kept at 210 "C. During an analysis, the total ion current was monitored and at a time just before mirex
400
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Figure 1. Oscillographic recording of the high mass end of a mirex standard (lower trace) and mirex extracted from a rainbow trout from Lake Ontario (upper trace) at an ionization energy of 70 eV and a resolution of 8O% and >90% recoveries, respectively. Prior to any attempt to quantify the mirex in a contaminated fish sample, it was necessary to establish unequivocally its presence by mass spectrometry and determine further the nature and degree of interference from spurious sources. In Figure 1, the analogue output from m l e 320 to m l e 560 for a mirex standard is compared to a spectrum obtained for a chromatographic peak that elutes a t the equivalent retention time in an extract isolated from a rainbow trout. To the author's knowledge, these are the first published data to show the parent ion cluster necessary to confirm the presence of mirex in a fish sample from Lake Ontario. In both traces the base peaks ( m J e272) of the mirex component are approximately the same intensity. As can be seen, the high mass ions originating from mirex in the environmentally derived sample are of somewhat lower abundance than those in the corresponding standard. When such data are normalized by computer to the base peak, the absence of most of the high mass ion fragments, including the parent ion cluster, frequently occurs resulting in the inability t o confirm the presence of mirex in an actual sample. This observation is consistent with the previous extractions and analyses of 23 separate fish specimens from Lake Ontario by our laboratory.
ANALYTICAL CHEMISTRY, VOL. 50, NO. 8, JULY 1978
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265 m/e Flgure 2. Mass spectra at 70 eV from approximately m l e 260 to m / e 280 obtained by successive scans A through D at low resolution (-1200) through a peak that elutes at the retention time of mlrex. Spectra were obtained on a DuPont 21-491 mass spectrometer under conditions described In the text. A scan of a mirex standard (lower trace) is included as a reference. The fragments associated with mirex as identified by high resolution mass spectrometry are marked with the letter “M”
However, it is important to note that when any of the above fish samples were spiked with low concentrations (- 100 to 200 ng/g) of authentic mirex and then analyzed by mass spectrometry, they clearly demonstrated the full complement of fragments, including the parent ion cluster, The ion fragments relating to mirex were enhanced by the expected relative abundance increments over those already present. This latter finding greatly reduces the possibility that the mass spectrometry instrumentation is discriminating against selected mass regions of the mirex spectrum under actual data acquisition conditions. There remain a t least two other possibilities to explain the anomalous ion fragment production observed. First, the m / e 270 ion cluster, as measured at low resolution, is actually a mixture of ion fragments of sufficient similarity in mass as to interfere with computer quantitation. A second possibility is that a molecule of a cyclic chlorocarbon type is co-eluting with mirex and, upon electron bombardment, contributes to the abundance of the mle 270 and mle 236 ion clusters. For purposes of quantification, the use of ion fragments subject to strong interference could result in unreliable data.
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Table I. Reproducibility for Multiple Analyses of 100 ng of Mirex by SIM at m/e 646 % deviation from the Trial Area units mean‘“ 1 402 7.1 2 47 0 8.6 3 400 7.5 4 41 9 3.1 5 467 I .9 6 439 1.4 ‘“ The mean is 432.8; standard deviation is i: 31; and the coefficient of variance is 7.1%. T o test for interference at the m l e 270 ion cluster, a series of mass spectra were obtained at slightly higher resolution ( N 1200). Figure 2 illustrates a scan of a mirex standard, followed by a series of scans prior to, during, and immediately followingthe elution of mirex in an actual fish extract sample. Ion fragments associated with mirex are designated by use of the letter “M”. It can be seen that there are a t least two other sets of ion fragments derived from molecules having similar gas chromatographic retention times as mirex. In this particular extract, they fall within a few tenths of an amu of the base peak at m l e 271.8. These findings suggest that when analyses are carried out by low resolution mass spectrometry (
