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Anal. Chem. 1980, 52, 2328-2332
Identification of Chlorinated Methoxybiphenyls as Contaminants in Fish and as Potential Interferences in the Determination of Chlorinated Dibenzo-p-dioxins Douglas W. Philllpson' and Bartholomew J. Puma* Division of Chemical Technology, Food and Drug Admlnistration, Washington, DC 20204
Analyses of Sheboygan Rlver (Wlsconsln) carp samples by a procedure used for the determination of polychlorlnated dlbenro-p-dloxlns (PCDDs) revealed the presence of a group of xenoblotlcs ldentlfled by gas-llquld chromatography/mas? spectrometry (GLC/MS) as CIS,C14, and CI, rlng-subslltuted methoxyblphenyls. These compounds gas chromatographed In the reglon of CIS-,C14-, and C15-PCDDsand produced Intense molecular Ions having the same nomlnal masses and chlorine lsotoplc abundances as those observed In the molecular Ion clusters from trlchloro-, tetrachloro-, and pentachlorodlbenzo-p-dloxlns. Synthesls of 3,3',4',5-tetrachloro-4-methoxyblphenyl and 2,3',4,4'-tetrachloro-3-methoxyblphenyl provlded model compounds whlch came through the PCDD cleanup procedure and had GLC and MS properties condstent wlth those of the residues recovered from the flsh. The flndlng of chlorlnated methoxyblphenyls as contamlnants In fish, comblned wlth the potential for their molecular Ions lo be rnlstaken for those from PCDDs, lndlcates a need for reappralsal of reported ldentlflcatlons of PCDD resldues In envlronmental samples by selected-Ion monltorlng GLC/MS methods based on monitoring excluslvely for the lsotoplc molecular Ions from PCDDs.
Shadoff and co-workers (1) have pointed out the possible interference from chlorinated benzyl phenyl ethers in the determination by gas-liquid chromatography/mass spectrometry (GLC/MS) of polychlorinated dibenzo-p-dioxins (PCDDs) in 2,4,5-trichlorophenol and its derivatives. Our analyses of fish have revealed the presence of a group of environmentally incurred contaminants which also have the potential to be mistaken for PCDDs in GLC/MS analyses that are based on selected-ion monitoring (SIM) for the isotopic molecular ions of PCDDs. Methods for determining low levels of 2,3,7,84etrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) and other PCDDs in industrial chemicals and environmental samples generally involve an extensive sample cleanup procedure followed by GLC/MS analysis. In order to attain method sensitivity levels commensurate with the extreme toxicity of these compounds, Crummett and Stehl (2),Camoni et al. (3), and diDomenico et al. ( 4 ) relied on monitoring for up to three ions from the molecular ion cluster of TCDD or other PCDDs by GLC/ low-resolution MS. O'Keefe et al. (5)bypassed the use of gas chromatography and simultaneously monitored one ion from native TCDD and one ion from 37C1-enrichedTCDD internal standard after direct probe introduction of the cleaned-up sample extract into a high-resolution mass spectrometer operated a t 10000 resolution. Shadoff and Hummel (6) and Harless and Oswald (7) employed the m / z 320 and 322 ions Present address: Department of Chemistry, Roger Adams Laboratory, University of Illinois at Urbana-Champaign, Urbana, IL 61801.
from TCDD in SIM methods using packed GLC columns interfaced with high-resolution maas spectrometers operated a t 9OOO resolution. Harless and Oswald (8)also used SIM for the m/z 320 and 322 ions in a method combining high-resolution capillary column gas chromatography with high-resolution (ca. 6500) mass spectrometry for the determination of 2,3,7,8-TCDD at parts-per-trillion levels in environmental samples. In a recent attempt to detect polychlorinated dibenzofurans (PCDFs) in fish samples containing high levels of polychlorinated biphenyls (PCBs), we used a slightly modified version of the PCDD/PCDF cleanup procedure employed by Firestone (9) for the analysis of gelatin and by Firestone et al. (10) for the analysis of dairy cattle tissues. GLC/MS analysis of the PCDD/PCDF fraction obtained when this cleanup procedure was applied to PCB-contaminated fish from the Sheboygan River (Wisconsin) revealed residues having the same nominal mass and number of chlorine atoms per molecule as trichloro-, tetrachloro-, and pentachlorodibenzo-pdioxins. These residues were identified as polychlorinated methoxybiphenyls (PCMBs). EXPERIMENTAL S E C T I O N Instrumentation. Hewlett-Packard 5730A and 5710A gas chromatographs equipped with constant current s3Ni electron capture (EC) and flame ionization (FI) detectors, respectively, were used with 1.8 m X 2 mm i.d. glass columns containing 3% OV-101 methyl silicone fluid on 80/100 mesh Chromosorb W-HP. EC/GLC analyses were performed with 5% methane in argon carrier gas at 30 mL/min and column, injector, and detector temperatures ("C) of 200,250, and 300, respectively. Synthetic organic reactions were monitored by FI/GLC as follows: nitrogen carrier gas at 30 mL/min, injector and detector temperatures at 250 and 300 "C, respectively, and the column operated isothermally at either 130 or 200 "C, depending on the compounds of interest. Low-resolution maw spectrometry was performed with a Finnigan 1015 S/L instrument coupled to a Varian 1700 gas chromatograph. The coupling was achieved through an all-glass jet separator and a glass-lined stainless steel transfer line equipped with a vacuum diverter valve inserted between the chromatograph and the jet separator. A 1.8 m x 2 mm i.d. glass column packed with 1%OV-101 on 80/100 mesh Chromosorb W-HP was used at a temperature of 200 "C with 20 mL/min of helium carrier gas. Data aquisition was controlled by a Finnigan 6000 data system. Spectra were recorded over a mass range of 33-500 daltons at a 3 ms/dalton integration time. Accurate mass measurements were made with a Varian MAT CH-5 DF mass spectrometerinterfaced to a Varian 1700 gas chromatograph through a dual stage Llewellyn separator. A 0.9 m X 2 mm i.d. glass column packed with 3% SP-2100 methyl silicone fluid on 80/100 mesh Supelcoport was operated at 180 "C with helium carrier gas at 60 mL/min for the high-resolution mass spectrometry. The spectrometer w a ~ tuned to provide resolution of 10OOO base line for accurate mass measurements. Proton nuclear magnetic resonance (NMR) spectra were recorded with a Varian EM-390 90 MHz instrument. Synthesis of Model Tetrachloromethoxybiphenyls (TCMBs). The aromatic iodide coupling method of Clark et al. ( 1 1 ) was used with slight modifications to prepare 3,3',4',5tetrachloro-4-methoxybiphenyl(I) by the palladium acetate catalyzed coupling of 3,4-dichloroiodobenzene(3 mmol) and
This article not subject to U.S. Copyright. Published 1980 by the American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 52, NO. 14, DECEMBER 1980
Table 1. Proton NMR Data for Synthesized Tetrachloromethoxybiphenyls: Chemical Shift Values, Ppm (Coupling Constants, Hz)
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100R E
’’;O7I
proton CH, 2 5 6
2’ 5‘ 6’
TCMB I 3.98 7.49 7.49 7.62 dd (2.1, 0.4) 7.54 dd (8.4, 0.4) 7.33 dd (8.4, 2.1)
TCMB I1 3.97 7.35 d (8.3) 7.01 d (8.3) 7.51 dd (2.1, 0.4) 7.51 dd (8.4, 0.4) 7.24 dd (8.4, 2.1)
2,6-dichloro-4-iodoanisole (3 mmol). The changes made were to use dimethylformamide, instead of triethylamine or tri-n-butylamine, as the solvent and to increase the quantity of palladium acetate to 3.3 mmol from 0.075 mmol. With these changes, the procedure gave a (2 + 1 + 1) mixture of TCMB I, 3,3’,4,4’tetrachlorobiphenyl, and 3,3’,5,5’-tetrachloro-4,4’-dimethoxybiphenyl in an overall yield of ca. 30%. FI/GLC was used to monitor the separation of the mixture on silica gel (J. T. Baker, 60/200 mesh, Catalog No. 3405) packed to a height of 300 mm in a 28 mm i.d. chromatographic column. The column was eluted first with 800 mL of hexane to remove the tetrachlorobiphenyl and then with 500 mL of 5% (v/v) dichloromethane in hexane to recover TCMB I. Evaporation of the second eluate to dryness followed by recrystallization of the residue from methanol gave ca. 150 mg (15%) of purified product with a melting range of 120-121 “C. Carbon-hydrogen analysis showed 48.28% C and 2.54% H for the synthesized material compared to calculated values of 48.49% C and 2.50% H for the molecular formula C13H&140. An earlier attempt to prepare TCMB I by the aromatic arylation method of Cadogan (12) produced only a small amount of the desired compound, together with a larger quantity of an unexpected TCMB isomer which was determined from its proton (11). NMR spectrum to be 2,3’,4,4’-tetrachlore3-methoxybiphenyl The synthesis involved the dropwise addition of 4 mL of pentyl nitrite to a solution of 2 g of 3,4-dichloroaniline in 20 g of 2,6dichloroanisole. The reaction was maintained at 60 “C and monitored by FI/GLC. When no dichloroaniline remained, the excess dichloroanisole was removed by vacuum distillation and the residue was chromatographed in a 20 mm i.d. column packed to a height of 200 mm with silica gel (J.T. Baker, 60/200 mesh, Catalog No. 3405). Dichloroanisole was eluted from the column with 200 mL of hexane before the monomethoxybiphenyls were eluted with 200 mL of 50% (v/v) dichloromethane in hexane. The second eluate was evaporated to dryness and a hexane solution of the residue was extracted with concentrated sulfuric acid to remove colored byproducts. Final recrystallization from methanol yielded 600 mg (16%) of TCMB I1 with a melting range of 113-115 “C. Proton NMR spectra were obtained for TCMB I and I1 in CDC13 solutions. Chemical shifts (parts-per-millionvalues calculated relative to internal tetramethylsilane) and coupling constants are reported in Table I. The mass spectra of TCMB I and I1 are shown in Figures 1 and 2, respectively. Analysis of Fish. The sample extraction/cleanup procedure used for the determination of PCDDs and PCDFs in gelatin and other animal products by Firestone and co-workers (9,IO), a modification of that described in the method of Baughman and Meselson (13),was applied to the analysis of fish with minor changes in the alumina and Florisil column chromatography steps to accommodate a wider range of C13- through ClB-substituted PCDDs and PCDFs. A 20-g sample of ground edible tissue was solubilized in aqueous ethanolic KOH, and the alkaline hydrolysate was extracted with several portions of hexane. Lipids were removed from the combined hexane extracts by extraction with concentrated sulfuric acid. The hexane solution was then concentrated and chromatographed on a column of alumina as described by Firestone et al. (IO), except that the first 12 mL of eluant was changed to 5% (v/v) CC14/hexane from 20% CC14/
Flgure 1. Mass spectrum of 3,3’,4’,5-tetrachloro-4-methoxybiphenyl (synthesized TCMB I), background subtracted. 100-
32i
2.‘
230P
350
Mass spectrum of 2,3f,4,4f-tetrachloro-3-methoxybiphenyl (synthesized TCMB 11), background subtracted. Flgure 2.
hexane. After the alumina column was eluted with 5 mL of dichloromethane and the solvent replaced with 0.5-1 mL of hexane, the residue was chromatographed on a column of Florisil as described by Firestone (9),with the following changes: (1) the height of adsorbent in the Florisil column was increased to 4.5 cm from 4.0 cm; (2) 10 mL of 2% (v/v) dichloromethane/hexane was used as the fmt eluant (discarded) instead of 12 d of hexane; (3) the volume of dichloromethane used as the second eluant (PCDD/PCDF fraction) was increased to 15 mL from 5 mL. Following several evaporations with small portions of hexane to ensure the removal of dichloromethane, the volume of the PCDD/PCDF fraction was adjusted with hexane to 1 mL for EC/GLC or 50 FL for GLC/MS or accurate mass analysis.
RESULTS AND DISCUSSION Commercial PCBs have been reported to contain partsper-million levels of PCDFs (14). As part of our effort to find PCDF residues in PCB-contaminated fish, four Sheboygan River carp samples containing from 50 to 790 ppm of PCB were cleaned up by the modified Firestone procedure. Electron capture gas chromatograms of the PCDD/PCDF fractions of all four samples exhibited very similar chromatographic patterns containing two major peaks. A typical chromatogram, with the major peaks labeled A and B, is shown in Figure 3. GLC/MS analysis showed that peak A was due to a mixture of heptachlor epoxide and octachlor epoxide. The residue eluting at the retention time of peak B produced the mass spectrum shown in Figure 4. Although this mass spectrum, like that for TCDD, has as its most prominent feature an apparent molecular ion cluster containing four chlorine atoms and starting a t m / z 320, the lack of a fragment ion cluster containing three chlorine atoms starting at m l t 257, which would result from the characteristic loss of CO + C1 from a TCDD molecular ion ( I @ , shows that peak B is not due t o a TCDD isomer.
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ANALYTICAL CHEMISTRY, VOL. 52, NO. 14, DECEMBER 1980
I
I
h
E
I
I/
6 u Y
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I-,' 0
2
4
6
8
10
12
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Figure 3. Electron capture gas chromatogram of the PCDD/PCDF fraction of a Sheboygan River carp sample containing 370 ppm of PCB. Residues giving peaks A and B are identified in text. 3% OV-101 GLC column at 200 OC. 100 1
/I
Figure 4. Background-subtracted mass spectrum of the fish sample residue giving peak B in the chromatogram shown in Figure 3. The characteristics of the mass spectra observed for a number of other peaks in the PCDD/PCDF fraction chromatograms suggested the presence of a series of compounds congeneric with the material giving peak B. In this series, each compound had the same nominal mass and number of chlorine atoms per molecule as a trichloro-, tetrachloro-, or pentachlorodizenzo-p-dioxin. Reconstructed mass chromatograms (i.e., specific ion chromatograms) for the apparent molecular ions of these C13, C14,and C15 compounds a t m/z 286,320, and 354, respectively, are shown in Figure 5. The compounds observed as peaks in the specific ion chromatograms were easily distinguished from PCDDs on the basis of their complete mass spectra. In addition to parent ion clusters which included the base peak and showed the typical isotopic ratios for three, four, or five chlorine atoms, these mass spectra exhibited significant fragment ion clusters reflecting losses of 43 and 113 daltons from the molecular ions as well as other fragment ions which conformed to the mass spectral data reported for polychlorinated methoxybiphenyls (PCMBs) by Jansson and Sundstrom (16). Support for the identification of the residues as PCMBs was obtained through an accurate mass measurement for the m / z 320 ion observed from peak B. The experimentally determined mass of 319.9316 was in good agreement with the calculated value of 319.9329 for the molecular formula CI3-
I
50
'
I ' 100 SCRN II
I
150
I 200
Figure 5. Reconstructed total ion gas chromatogram (RGC) and m / r 286,320,and 354 mass chromatograms from the PCW/PCDF fraction of a Sheboygan River fish sample. HeC140. As a further check on the identity of peak B and related peaks obtained from the samples, 3,3',4',5-tetrachloro-4-methoxybiphenyl (I) and 2,3',4,4'-tetrachloro-3methoxybiphenyl (11) were synthesized for use as model tetrachloromethoxybiphenyls (TCMBs). In tests of their behavior in the modified Firestone cleanup procedure, approximately 80% of each model compound was recovered in the PCDD/PCDF fraction. The mass spectra of TCMB I and I1 (Figures 1 and 2, respectively) were consistent with published data for other PCMBs (16). Both compounds gave large peaks for the molecular ion, M+, and significant fragment ion peaks at [M - 15]+, [M - 43]+, and [M - 113]+,corresponding to losses of CH3, CH3C0, and CH3C0 + 2C1, respectively, from M+ a t 320 daltons. In contrast to TCMB I1 and the PCMB residues in the fish, TCMB I produced its base peak in the [M - 151' cluster, rather than in the molecular ion cluster. Despite this and other differences in the relative abundance of ions from the model compounds and the fish residues, the similarity of the fragmentation patterns for TCMB I and I1 to those observed in the mass spectra of the fish samples provided additional support for the identification of the residues as unspecified isomers of trichloro-, tetrachloro-, and pentachloro-PCMBs. At the EC/GLC conditions used in this work, TCMB I and I1 both eluted later than peak B. As shown by comparison of the chromatograms in Figure 6, however, the retention time for TCMB I was very close to that for 2,3,7,8-TCDD. GLC retention times calculated relative to 1.0 for 2,3,7,8-TCDD on 3% OV-101 column a t 200 "C were 0.98, 0.75, and 0.61 for TCMB I, TCMB 11, and peak B, respectively. If the electron capture response for the TCMB residue giving peak B is assumed to be equivalent to that for an equal amount of TCMB 11, the response observed for peak B would represent about 7, 20,20, and 36 ppb of TCMB in the individual Sheboygan River fish samples. While these levels are several orders of magnitude lower than the PCB residue levels found in the same samples (viz., 50, 286, 370, and 790 ppm, respectively), they are also several orders of magnitude greater than the low concentrations of current interest for PCDDs in environmental samples. In GLC/MS molecular ion monitoring methods for PCDD residues, PCMBs containing the same number of chlorine atoms per molecule as the PCDDs of interest will give false
ANALYTICAL CHEMISTRY, VOL. 52, NO. 14, DECEMBER 1980
8
i
i
1
8
10
YmLIIp
Figure 6. Electron capture gas chromatograms of: (A) mixture of synthesized reference compounds (I) 0.3 ng of 3,3’,4‘,5-tetrachloro4-methoxybiphenyl (TCMB I), (11) 0.2 ng of 2,3’,4,4’-tetrachloro-3methoxybiphenyl (TCMB 11); (B) 0.4 ng of 2,3,7,&tetrachIorodibenzo-p-dioxin. 3 % OV-101 GLC column at 200 OC.
positive responses unless (A) the operating resolution of the mass spectrometer is sufficient to separate the molecular ions of PCDDs from the molecular ions of PCMBs or (B) the GLC column resolves the PCMBs from the PCDDs of interest or (C) the PCMBs are removed by the sample cleanup procedure. The following considerations relate to these possibilities: (A) Since the difference in mass between the molecular ions for PCDDs and PCMBs containing the same number of chlorine atoms per molecule is about 0.036 daltons, the resolution ( R )required to separate peaks of approximately equal intensity for these ions may be calculated from the equation R = m10.036, where m is the nominal mass of the molecular ion in daltons. Thus, the resolution required to separate these ions is >6000 for the monochloro compounds, ca. 8800 for the tetrachloro compounds, and >12 000 for the octachloro compounds, or clearly more than that provided by low-resolution mass spectrometry. All but one (8) of the previously cited high-resolution MS methods (5-8) for determining TCDD provide sufficient resolution to separate the M+ of TCDD from the M+ of TCMB when the peaks from these ions are approximately equal in intensity. However, the work of Baughman and Meselson (I 7) in an analogous situation involving the separation of the DDE ion a t m / z 321.930 from the TCDD ion a t m / z 321.894 (where the difference in mass between the two ions was also equal to 0.036 daltons) indicates that high concentrations of TCMB would obscure or otherwise interfere with the determination of low concentrations of TCDD, even with the mass spectrometer operating at 12 000 resolution. Thus, even if high-resolution MS is used, PCMBs, if present in the cleaned up sample, may interfere in molecular ion SIM analyses for PCDDs and may indeed lead to false positive results if the ion current is recorded as a chromatogram. (B) With respect to the GLC separation of PCDDs from PCMBs, it is noteworthy that of the 198 theoretically possible TCMB isomers, one of the two isomers synthesized for this work had a retention time within 2% of that for 2,3,7,8TCDD on a widely used GLC column. In view of the large number of potentially interfering TCMBs, reliance on the uniqueness of the GLC retention time for 2,3,7,8-TCDD does not appear to be warranted. Furthermore, in a study ( 4 ) which specified
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the GLC retention time criterion used for identification of 2,3,7,8-TCDD from the SIM peaks of the m / z 320, 322, and 324 ions, a difference of kO.1 min from the retention time of less than 4 min for the 2,3,7,8-TCDD standard was tolerated. On a percentage basis, this tolerance exceeds the retention time difference between 2,3,7,8-TCDD and TCMB I. (C) At least one of the published PCDD cleanup procedures is known to recover PCMBs, and it is probable that PCMBs would come through other procedures which depend on alumina and/or Florisil column chromatography for isolating PCDDs from PCBs and chlorinated hydrocarbon pesticides. Further work in our laboratory has shown that hexane elutes PCDDs and PCDFs from silica gel while TCMB I, TCMB 11, and the PCMB, heptachlor epoxide, and octachlor epoxide residues found in Sheboygan River fish are retained. Thus, methods which employ silica gel chromatography may reduce or eliminate the potential interference from PCMBs in the determination of PCDDs. However, existing silica gel based cleanup procedures for PCDDs employ various volumes of a variety of solvents to elute PCDDs from diverse amounts of silica gel and the effectiveness of these procedures has not been determined for isolating PCDDs from PCMBs. A search of the literature failed to reveal other findings of PCMBs as environmental contaminants in fish. However, Herbst et al. (18) found mono-, di-, and trihydroxylated methoxytrichlorobiphenyls in the water and plants of an aquarium used in a study of the uptake of 2,4‘,5-trichlorobiphenyl-14C by goldfish. Chlorinated methoxybiphenylols were also found as metabolites of chlorobiphenyls in rat, rabbit, and frog studies cited by Sundstrom et al. (19) in a review of the metabolism of chlorobiphenyls. In addition, Maass et al. (20) identified 4-chloro-4’-methoxybiphenylas a metabolite of 4-chlorobiphenyl in a study on lichens, and Lay et al. (21) found that the metabolism of 2,2‘,4,4’,5pentachlorobiphenyl in the rat produced a methoxypentachlorobiphenyl. These findings of methoxylated metabolites of chlorobiphenyls in a variety of biological systems suggest that the PCMBs and PCBs found in the Sheboygan River fish might be related. Another indication of a possible relationship between these residues is that the concentration of the TCMB residue found a t the retention time of peak B in the four fish samples is approximately proportional to the PCB concentration in the fish, varying from 0.005% to 0.014% of the PCB residue level. Two contravening indications should be pointed out, however. The first is that the magnitude of the residue giving peak A (heptachlor epoxide f octachlor epoxide) also appeared to be proportional to the concentration of PCB in the fish. Thus, the seeming relationship of the PCMB and PCB residue levels may reflect nothing more than the age or fat content of the fish. Second, we have used the modified Firestone procedure for the analysis of only one other fish sample bearing environmentally incurred PCBs at a level comparable to those found in the Sheboygan River fish, and this sample, a Waukegan Harbor (Lake Michigan) carp containing 190 ppm of PCB, did not show PCMB residues when examined a t GLC/MS conditions sensitive to these compounds a t the 1-ppb level. Recognition of the presence of PCMBs in the environment and of their potential to be mistaken for PCDDs makes evident the need for using more definitive means to confirm the presence of PCDDs in environmental samples than can be provided by SIM methods in which only the isotopic molecular ions are monitored. The degree of certainty for the identification of PCDD residues by SIM methods would be greatly enhanced if supported by data for peaks from the fragment ion cluster corresponding to [M - COCl]’ in addition to the peaks from the M+ cluster. For reliable identification and quantitation of PCDDs by GLC/low-resolution MS, it might
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Anal. Chem. 1980, 52, 2332-2336
be necessary to examine a sufficient segment of the mass spectrum of the suspect residue to rule out the presence of PCMBs (by the absence of fragment ion clusters resulting from losses of CH3, CHSCO, and CH3C0 + 2C1 from the M+ cluster) and other potential interferences not yet observed.
ACKNOWLEDGMENT The authors thank the following members of the Food and Drug Administration for their contributions to this report: Peter A. Dreifuss, Samuel W. Page, Albert M. Gardner, John W. Butler, James D. Link, William J. Trotter, and Lee J. Miller.
LITERATURE CITED (1) Shadoff, L. A.; Blaser, W. W.; Kocher, C. W.; Fravel, H. G. Anal. Cbem. 1978, 50, 1588-1588. (2) Crummett, W. B.; Stehl, R. H. EHP, Environ. Health Perspect. 1973, 5, 15-25. (3) Camoni, I.; DiMuccio, A.; Pontecorvo, D.; Vergori, L. J. Cbromatogr. 1978, 153,233-238. (4) diDomenico, A.; Merll. F.; Boniforti, L.; Camoni, I.; Di Muccio, A,; Taggi. F.; Vergori, L.; Colli, G.; Elli, G.; et al. Anal. Chem. 1979, 51,735-740. (5) O'Keefe, P. W.: Meselson, M. S.; Baughman, R. W. J. Assoc. Off. Anal. Cbem. 1978, 67, 621-626. (6) Shadoff, L. A.; Hummel, R. A. Bbmed. Mass Spectrom. 1978, 5, 7-13. (7) Harless, R. L.; Oswald, E. 0. 24th Annual Conference on Mass Spectrometry and Allied Toplcs, San Dlego, CA, May 9-13, 1976; pp 26-28.
(8) Harless, R. L.; Oswald, E. 0. 25th Annual Conference on Mass Spectromeby and Allied Topics, Washington, DC, May 29-June 3, 1977; pp 592-594. (9) Firestone, D. J. Agric. Food Cbem. 1977, 25, 1274-1280. (10) Firestone, D.; Clower, M.. Jr.; Borsetti, A. P.; Teske, R. H.; Long, P. E. J. Agric. Food Cbern. 1979, 27, 1171-1177. (1 1) Clark, F. R. S.; Norman, R. 0. C.; Thomas, C. B. J. Chem. Soc., Perkin Trans. 11975, 121-125. (12) Cadogan, J. I. G. J. Cbem. SOC. 1982, 4257-4258. (13) Baughman, R.; Meselson, M. EHP, Environ. Health Perspect. 1973, 5 , 27-35. (14) Bowes, G. W.; Mulvihill, M. J.; Simoneit, B. R.; Burlingame, A. L.; Risebrough, R. W. Nature (London) 1975, 256, 305-307. (15) Safe, S.; Hutzinger. 0. "MassSpectrometry of Pesticides and Pollutants"; CRC Press: Cleveland, OH, 1973; pp 72-73. (16) Jansson, B.; Sundstrom, G. Biomed. Mass. Spectrom. 1974, 1 , 389-392. (17) Baughman, R.; Meselson, M. I n "Chlorodioxins-Origin and Fate"; Blair, E. H., Ed.; American Chemical Society: Washlngton, OC, 1973; pp 92-104. (18) Herbst, E.; Scheunert, I.; Klein, W.; Korte, F. Chemosphere 1978, 7, 22 1-230. (19) Sundswom, G.; Hutzinger, 0.; Safe, S. Chemosphere1978, 5,267-298. (20) Maass, W. S. G.; Hutzinger, 0.; Safe, S. Arch. Environ. Contam. Toxicol. 1975, 3 , 470-478. (21) Lay, J. P.; Klein, W.; Korte, F. Chemosphere 1975, 4 , 161-168.
for review October l7, l979. Accepted September 2, 1980.
Continuous-Flow Analysis for Uric Acid in Biological Fluids, with Immobilized Uricase in a Closed-Loop System Asfaha Iob and Horacio A. Mottola" Department of Chemistry, Oklahoma State University, Stillwater, Oklahoma 74078
Relatively Inexpensive and/or stable enzymes can be directly used in solution for repetitive determinations in closed-flow systems. Important ciinlcal determlnatlons (e.g., uric acld In bloiogical fluids), however, require enzymes that are not sufflclently stable and/or inexpensive to be used in solution for relatively long periods of tlme and at the high actlvlty levels needed for success In unsegmented closed-flow systems. Thls paper reports on the chemlcal Immobilization of uricase on controlledpore glass, certain characterlstics of the lmmobllized enzyme preparation, and application of it as packing In mixing-delay coils for the determination of uric acld In blologlcal fluids. Determinations can be peformed at a 100 samples/h rate with satlsfactory precision (24% RSD). The method was compared wlth a regularly used coiormetric procedure in the SMA 18/90 analyzer [correlation coefficient = 0.98 for 22 samples of human blood serum]. The lmmoblllzed enzyme preparatlon retains over 70 % of its initial activity afler repetltive use for more than 10 months.
The use of enzymes as analytical reagents has increased in recent years. As biological catalysts which work in complex living systems, enzymes offer two characteristics of paramount importance in analytical chemistry: (1) generally high selectivity (and occasional specificity); (2) capability of selfregeneration via the catalytic cycle. The first of these properties is widely recognized and used; the second usually restricts the use of enzymes to immobilized ones. Recently (I), however, we have shown that if the enzyme is relatively 0003-2700/80/0352-2332$0 1 .OO/O
inexpensive and relatively stable [so that it retains its activity toward the substrate of interest a t reasonable constant level at room temperature and with time] it can be directly used in solution for repetitive determinations in closed-flow systems; the closed loop affords enzyme recycling and reutilization. For enzymes relatively inexpensive and stable this approach has definite advantages over systems utilizing immobilized enzymes packed in chromatographic-type columns inserted a t a given point (before detection) in the flow train (21, namely, simpler and faster determinations. Unfortunately important clinical determinations employ enzymes that are not sufficiently stable and/or inexpensive to be used in solution for relatively long periods of time and at the high levels required for success in flow systems. For the development of determinations competitive in cost and number of determinations per hour in almost unattended continuous-flow analysis, enzymes such as uricase [used for specific uric acid determination in biological fluids] must be rendered stable and present in high local concentration. Immobilization, in these cases, offers a real advantage. This journal has devoted regular attention to the analytical applications of immobilized enzymes in the form of feature articles (3-5). There are four principal methods for enzyme immobilization: (1) containment by membrane; (2) entrapment; (3) adsorption; (4) covalent bonding. Immobilization by covalent bonding is best for continuous-flow analysis and either packed-bed reactors (6, 7) or open tubes with enzymes immobilized on the wall (7-9) can be used, In the case of packed beds, because of its mechanical stability, controlled-pore glass (and other types of porous glass beads) constitutes a convenient matrix for enzyme immobilization. This paper describes the immobilization of 0 1980 American Chemical Society
