b2
Anal. Chem. 1983, 55, 2155-2159
4
1
I II 15 min. 0 15
0
Figure 5. Determinatlon of N-nitrosocimetidine (NC) in gastric juice: (Trace A) normal human gastric juice; (Trace El) the same gastric Juice spiked with authentic N-nitrosocimetidine. Conditions: mablie phase, NH,H,PO, (30 mM, pH 6):CH,CN, 2O:O.g (vlv) at 0.5 mL/min, colarimetric reagent at 0.5 mL/imin, detector at 541 nm.
fluids, particularly gastric juice, using the procedure described in this paper.
ACKNOWLEDGMENT We gratefully acknowledge the advice and material help provided by P a d Ulcickas of GTE Sylvania,Manchester, NH, in connection with the high intensity discharge lamp. We concerning the thank a reviewer for helpful suggesti,ons evaluation of chromatographic resolution. This investiglition was supported by the National Institute of Environmental Health Science Grant No. 2-Pol-ES00597-13 and by PHS Grant No. 1-Pol-CA26733.-04, awarded by the National Cancer Institute, DHHS. A preliminary account of
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some of this work was given at the 7th International Meeting on the Analysis and Formation of N-Nitroso Compounds, Tokyo, Japan, Sept 18-0ct 1, 1981. Registry No. MNNG, 70-25-7; MNU, 684-93-5; MNA, 7417-67-6;NOTC, 82660-96-6;NOGC, 76757-85-2;NC, 73785-40-7; NP, 86941-92-6.
LITERATURE CITED (1) Magee, P. N.; Montesano, R.; Preussmann, R . "Chemical Carcinogens"; Searle, C. W., Ed.; American Chemical Society: Washington, DC, 1976; American Chemical Society Monograph No. 173; Chapter 11. (2) Fine, D. H.; Roundbehler, D. P. J . Chromatogr. 1975, 109, 271. (3) Hansen, T. J.; Archer, M. C.; Tannenbaum, S.R. Anal. Chem. 1979, 51, 1526. (4) White, E. H.; Woodcock, D. J. "Chemistry of the Amino Group"; Patai, S., Ed.; Wiley: New York, 1966; Chapter 6. (5) Daiber, D.; Preussmann, R. 2.Anal. Chem. 1984, 206, 344. (6) Fan, T.-Y.; Tannenbaum, S. R. J . Agrlc. FoodChem. 1971, 19, 1267. (7) Iwaoka, W.; Tannenbaum, S. R. "Environmental N-Nitroso Compounds. Analysis and Formation"; Walker, E. A., Bogovski, P., GriciUte, L., Eds.; International Agency for Research on Cancer; Lyon, 1976 Sci. Publ. No. 14, p 51. (8) Shuker, D. E. G.; Tannenbaum, S.R.; Wishnok, J. S.J . Org. Chem. 1981, 4 6 , 2092. (9) Foster, A. 8.; Jarman, M.; Manson, D. Cancer Lett. 1980, 9 ,47. ( I O ) Hansen, T. J.; Iwaoka, W. T.; Archer, M. C. J . LabelledCompd. 1974, IO. 669. (11) Piacek-Lianes, B. G.; Tannenbaum, S. R. Carcinogenesis 1982, 3 . 1379. (12) Chow, Y. L. "N-Nitrosamines"; Anseime, J. P., Ed.; American Chumical Society: Washington, DC, 1979; ACS Symposium Series No. I O I , p 13. (13) Challis, B. C.; Kyrtopoulos, S. A. J . Chem. Soc., Perkin Trans. 1 1979, 299. (14) Snyder, L. R.; Kirkland, J. J. "Introduction to Modern Liquid Chromatography", 2nd ed.; Wiiey: New York, 1979, pp 31-33. (15) Ichinotsubo, D.; McKlnnon, E. A,; Liu, C.; Rice, S.;Mower, H. F. Carcinogenesis 1981, 2, 261. (,6) DeFiora, s,; picclotto, A, Carcinogenesis Isso, , 925,
,
RECEIVED for review May 25,1983. Accepted August 12,1983.
Identification of Moriomethylated Polycyclic Aromatic Hydrocarbons in Crude Oils by Liquid Chromatography and High-Resolution Shpol'skii Effect Fluorescence Spectrometry Philippe Garrigues* and Marc Ewatld Groupe d'oce'anographie Physico-Chimique d u LA 348 (CNRS), Laboratoire de Chimie Physique A, UniversitB de Bordeaux I , 33405 Taleiltce Cedex, France High-resolution spectrometry (HRS) of polycyclic aromatic compounds (PAC) In n-alkanes frozen at 15 K (Shpol'skli effect) is applied to the Identification of monomethyiated Isomers In pyrene, phenanthrene, and chrysene. Best results for identification and estimation of the relatlve dlstrikution of Isomers are obtained after fractlonatlon of the crude oil by a now classlcal way, the high-performance llquid chromatography (HPLC) procedure which includes two steps: first, to Isolate compounds according to the degree of aromatlclty, and second, to separate parent compounds accordlng to the degree of alkylation. The chromatographic fractions suspected of contalnlng the studled PAH are then analyzed by HRS at dHferenX levels of fractlonatlon. The results presented here illustrate the capability of this technique for the complete ldentlflcation off methylated Isomers In natural extracts, ailowlng further quantification.
An increasing number of studies on polycyclic aromatic
compounds (PAC) by high-resolution spectrometry (HRS) in frozen n-alkane matrices (Shpol'skii effect) have been reported during the last years (1-3). The high sensitivity and selectivity of such a technique offers an alternative approach to other analytical methodologies (i.e., gas capillary chromatography coupled or not with mass spectrometry (GC/MS) or highperformance liquid chromatography (HPLC)). However, preliminary chromatographic separations of natural samples are often required for specific molecular identification of PAC by low-temperature spectrometry (2, 4). Recent developments in HPLC (normal and reverse phase) allow rapid and powerful separations of the studied series of PAC (5, 6) even if the raw material is very complex, as for crude oils. Complete identification and relative quantification of each isomer are important, because the toxic activity of PAC is often related, for example, to a specific position of the methyl group on the parent molecule. For instance, 5-methylchrysene is one of the stxongest carcinogenic products while the other isomers are only moderate carcinogens (7). In the same way,
0003-2700/63/0355-2155$01.50/00 1963 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 13, NOVEMBER 1983
PYRENE
Aromatic series studied in this work. The black arrows indicate the "Bay Region".
Figure 1.
1-and 9-methylphenanthrene are mutagenic with regard to the other isomers which are inactive (8). As a consequence, specific molecular identification and relative quantification of biologically active molecules are required to characterize the true potential toxicity of natural samples. These aims have been shown to be partially accessible by GC/MS analysis. In fact isomeric compounds usually give a similar fragmentation pattern and proper identification will be difficult unless retention times of each isomer on GC are very different. In these cases, HRS has been shown to be a very powerful alternative technique ( 4 , 9). Therefore, we have developed in our laboratory a combined HPLC/HRS procedure to attain the molecular level of identification in complex natural samples containing PAC. The utility of each HPLC step is demonstrated in this paper by viewing HRS results a t different levels of fractionation. Thus, the low-temperature technique appears to be a general methodology for identification of fluorescent PAC. The neutral PAC presented here have been studied with regard to their toxic power in relation with ecological problems. The importance of methylphenanthrenes and methylchrysenes for environmental considerations (see above) has been extensively mentioned; however, methylpyrenes have not been fully investigated. It seems interesting to study these aromatics simultaneously with the two series mentioned above, for the following reasons. Indeed chrysene and phenanthrene contain an area which is called a "bay region", located between positions 4 and 5 on the aromatic ring (Figure 1). On the contrary, the pyrenic ring does not possess such a region. Recent studies have shown that a "bay region" methyl group is one of the structural requirements favoring the tumorigenic activity of methylated phenanthrenes (8). So, the accurate characterization of the methylated polyarenes is of importance to carcinogenesis research. EXPERIMENTAL SECTION Chromatographic Procedure. Sample aliquots of crude oil (v = 1 mL) were submitted initially to adsorption liquid chromatography on a Florisil (SiOz and MgO mixture) column (5 cm length, 1 cm i.d.) to remove most of the resins and asphaltenes (trapped on the column). Elution was made with n-pentane (u = 300 mL); saturates were eluted first and then the aromatics. The pentane solution was collected and concentrated to approximately 2 mL. Further analyses were performed with luminescence techniques, so that the presence of residual saturated molecules is never a problem. The normal-phase HPLC analyses were performed on a Spherisorb 5-pm aminosilane (NH,) column (20 cm length, 4.7 mm id.) eluted with heptane. The collected fractions were gently evaporated to dryness, dissolved in methanol, and then submitted to reverse-phase HPLC on a Spherisorb 5-pm odadecylsilane (CIS column (20 cm length, 4.7 mm id.) with a mixture of methanol/water (80/20) as eluent. The collected fractions eluted in
Flgure 2. Chromatogrpahic separation of the petroleum aromatic extract on p-silica-",: striped areas indicate the collected fractions which contain respectively phenanthrenes (fraction III,), pyrenes (fraction 1112), and chrysenes (fraction IV).
methanol/water were then extracted with a suitable Shpol'skii n-alkane solvent for the analyzed PAH. Elution of the aromatic compounds was monitored at 254 nm with UV-Vis monochromatic absorption detector (LDC Spectromonitor 111). Low-Temperature Spectrofluorimetry. Low-temperature luminescence experiments were performed with a homemade spectrofluorimeter previously described (IO). Fused silica tubes containing the solutions were attached to the cold head of a closed cycle cryogenerator (CTI, Cryodyne, 21 S) operating at 15 K. Preliminary fast freezing of the solutions at 77 K in liquid nitrogen gave satisfactory conditions for avoiding aggregate formation and for observing sharp emission spectra ( 1 , I O ) . The total cohcentration of solutions of PAH mixtures were adjusted at about lo4 M. In this concentration range, the formation of aggregates or microcrystallites is minimized and the reproducibility of fluorescence intensity is not altered (1, 11). Excitation was provided by the light of a Xenon-lamp (XBO Osram 450 W) dispersed by a monochromator (Model H20, Jobin Yvon) with a bandwidth of about 2.5 nm and focused on the front surface of the analyzed frozen solution. Luminescence emission was observed at 90Dthrough a 1-m scanning spectrometer (Model HR 1000, Jobin Yvon) operating at a band-pass of 0.1 nm. Detection was provided by a photomultiplier (EM1 9789 QB) and spectra were recorded on a chart recorder (Model Servotrace, Sefram). Chemicals and Reagents. The studied PAH were from different sources: four methylphenanthrenes (lMP, 3MP, 4MP, 9MP) and 1-and 4-methylpyrenes were synthesized in the laboratory by R. Lapouyade (12) and 2-methylpyrene (2MPy) and the six methylchrysenes were kindly donated by A. Colmsjo, Arrhenius laboratory, Stockholm, Sweden. 2-Methylphenanthrene (2MP) was provided by "K and K" pharmaceuticals. Purity of all these PAHs was checked by capillary gas chromatography (GC) or GC/MS. AU the used solvents (spectroscopicgrade from Fluka or Merck) were purified by distillationand then dried and kept on molecular sieves; residual emission of solvents was verified by room-temperature spectrofluorimetry (MPF-44 spectrofluorimeter; Perkin-Elmer, Norwalk CT). The crude oil sample studied came from a North Sea petroleum well. RESULTS AND DISCUSSION Normal Phase HPLC. One representative petroleum chromatogram is shown in Figure 2. In this first step, three fractions (1111,111,, and IV) were collected, containing, respectively, phenanthrenic, pyrenic, and chrysenic derivatives, according to the retention times determined on a synthetic mixture containing the three aromatic parent compounds. However, some high-resolution experiments performed on
ANALYTICAL CHEMISTRY, VOL.
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-EKE
FLW-EWE
I M C M TOTAL A T I C I ,
EXTRACT
Flgure 3. Chromintographic separation of the fraction I1 I (phenanthrene and pyrene compounds) on p-silica-C,,: striped areas indicate the collected fralctions which contain respectively phenanthrene (fraction III,), methylphenanthrenes (fraction IIIJ,and methylpyrenes (fraction I I[ IC).
I-
I
I I
I I
I
I
I
I
Flgure 4. Chromatographic separation of the fraction I V (tetracyclic compounds) on p-silica-C striped area indicates the collected fraction IV, which contains methylchrysenes. Fraction IV, contains triphenylene and lraction IV, benzanthracene and chrysene.
these fractions by gas capillary chromatography or low-temperature spectrofluorimetry (13) show that some phenanthrenic compounds can also be eluted in fraction IIIzwith the pyrenic series. So fractions 1111and IIIz were collected together to avoid the loss of triaromatic hydrocarbons. This whole fraction will be called fraction I11 in the following text. We can note that with the UV detector set at 254 nm, the observation of tetracyclic aromatic compounds (other than pyrenes) (fraction IV) is not favored (maximum absorption at about 270 nm). Therefore the weak chromatographic peak of fraction IV does not reflect the real abundance of tetracyclics vs. triaromatics. Reverse-Phase Chromatography. This step has been performed on firaction 111 (tricyclic and pyrenic rings) and fraction IV containing tetracyclics without pyrenic compounds (see above and Figure 2). Each chromatographic peak noted in Figure 3 (related to fraction 111) has been collected. According to the retention time determined for synthetic mixtures containing pyrene, phenanthrene, 9-methylphenanthrene, 11-methylpyrene,and 3,6-dimethylphenanthreneit appears that phenanthrene is eluted in fraction 111,, pyrene and monomethylphenanthrenes are eluted in fraction IIIb, and dimethylphenanthrenes and monomethylpyrenes are eluted in fraction 111,. Chromatographic separation of the fraction IV is presented in Figure 4. Each noted peak has been collected. According to retention times determined on the synthetic mixture of all the six monomiethylchrysenes, it appears that these cornpounds are contained in the chromatographic fractions noted IVd. This collected firaction presents a poorly resolved
CMP'J MP
2Mp
9MP
1MF
I '
34s
3% (nm)
C& 46b(n WLENCTH Flgure 5. Emission spectra of monomethylphenanthrenes(MP) in an equimolar synthetic mixture (each MP at C = 2 X M) and in the crude oil extracts, at different levels of fractionation, frozen in n-hexane at 15 K. Excitation at 298 nm for fluorescence spectra. Excitation at 297 nm for phosphorescence spectra. Peaks noted "Ph" indicate the emission of phenanthrene. The attributed peaks are only due to pure bands related respectively to identified compounds by reference with emission bands from synthesized molecules.
structure of four peaks. We also note, as mentioned by previous works, that more resolved chromatograms can be obtained by using an elution gradient (6). Nevertheless, two methylchrysenes (5MC and 6MC) will present the same retention time and cannot be differentiated by using such a LC procedure (6). In addition, methylated derivatives of other tetracyclic series (Le., benzanthracene, triphenylene, benzo[clphenanthrene) perhaps present in the fraction are eluted with the same retention times as some methylchrysenes (14). For all these considerations, rough reverse-phase chromatography was performed without gradient since the ultimate molecular and individual identification is made by HRS. Low-Temperature Spectrometry. (a) Phenanthrenic Compouds. The total aromatic extract and the two HPLC fractions (I11and I&) have been dissolved in normal hexane (n-C,) which is a suitable Shpol'skii solvent for tricyclic compounds. The utility of each HPLC separation is shown by the comparison of the emission spectra of each fraction with those of a n equimolar synthetic mixture of the five MP isomers (Figure 5). By use of an appropriate excitation wavelength, each MP can be identified in the synthetic mixture, either from the fluorescence spectrum (3MP, 4MP, and 9MP) or from the phosphorescence spectrum (1MP and
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 13, NOVEMBER 1983 FLUORESCENCE
PHOSPHORESCENCE
3MC bMC FRACTIONATION ON P-SILICA-NH2
i Fmction IV )
I M C 5MC 1 MC
2 MC
-
ii
a'
d
FRACTIONATION ON
1
>-
t
Y IW -
z
w
Y W
ci 60
365
495
370 WAVELENGTH ( nm
500
I
Emission spectra of monomethylchrysenes (MC) in an equimolar synthetic mixture (each MC at c = M) and in the crude 011 extracts frozen in n-octane at 15 K. Excitation at 327 nm for fluorescence and phosphorescence spectra. Flgure 8.
2MP). In the total aromatic extract, only the phenanthrene (Ph) and the 9-methylphenanthrene (9MP) can be detected. After the first HPLC step according to the size of aromatics, most of the methylated derivatives of phenanthrene (1-,3-, 9MP) are detected by reference with the artificial mixture. After the reverse-phase HPLC on p-silica-C18,according to the degree of alkyl substitution, only the emission peaks attributed to methylphenanthrenes are observed in fraction IIIb. Then, the emission spectra of the natural extracts look like those of the synthetic mixture, i.e., convenient signal to noise ratio, no undesirable emission. We observe the lack of the 4MP in the crude oil extract (Figure 5). (b) Chrysenic Compounds. For these tetracyclic compounds, n-octane (n-C,) appears to be the most suitable solvent for observing well resolved emission spectra, as shown in Figure 6 for the synthetic mixture of the methylchrysenes. Each isomer can be unambigously identified either by fluorescence spectrometry (1-,6-, 4-, and 5MC) or by phosphorescence (2-, 3MC). Studies on the total aromatic fraction of the crude oil show that the methylchrysenes cannot be detected, certainly due to the complexity of the natural matrix. After one HPLC step on p-silica-NH2,quasi-linear fluorescence peaks are observed in fraction IV and can be attributed respectively to 1-,3-, and 6-methylchrysene. With the sensitivity of our setup, phosphorescence emission of any methylchrysene cannot be detected. Further, liquid chromatography on p silica-C18leads us to collect the fraction noted 'IVd" suspected to contain alkyl derivatives of chrysene. HRS spectra of this fraction allow then a clear identification of 1-,2-, 3-, and 6-methylchrysene. We can point out the relative absence of 4MC and 5MC in the analyzed crude oil sample. (c) Methylpyrene Series. As compared with the PAH isomers studied above, the monomethylated pyrenes (MPy) constitute one of the simplest series to be identified by the low-temperature spectrometry in n-alkane matrices, since the fluorescence spectra of individual compounds do not overlap
372
375
378 (nml
Emission spectra of monomethylpyrenes (MPy) in an M) and in equimolar synthetic mixture (each MPy at C = 3.3 X the crude oil extracts frozen in n-hexane at 15 K. Excitation at 342 nm. Figure 7.
(Figure 7). In an equimolar synthetic mixture of the three isomers frozen at 15 K in n-hexane, 1- and 2MPy can be identified by one specific pure emission peak and the 4MPy by three peaks. Total aromatic extract and the two fractions (111and 111,) have been dissolved in n-hexane and fluorescence spectra were recorded a t a temperature of 15 K. Without fractionation, a complete identification of each isomer can be done, but further chromatographic separations remove specially undesirable fluorescent compounds in order to obtain a flat base line which will be of interest if quantitative measurements are required. We observe that all three isomers are equally present in the natural extract. Works presented here on three series of monomethylated PAH show that a clear identification of each methyl PAH isomer is possible by HRS a t 15 K after a suitable two-step HPLC procedure. We can note that a such complete resolution obtained here on mixtures of methylchrysenes has never been reached by any analytical methods (6,15),i.e., liquid chromatography (6), matrix isolation spectroscopy (19),or capillary gas chromatography even by the recent use of high-performance stationary phases (16). Indeed numerous tetracyclic alkyl derivatives are present in natural samples and have quite the same chromatographic retention indexes in liquid (14) or gas phase (17) and also the same mass spectrum. This partial
ANALYTICAL CHEMISTRY, VOL. 55, NO. 13, NOVEMBER 1983
resdt demonstrates that on a critical analytical problem such as the identification of methylchrysenes, the Shpol'skii effect is one of the most sensitive techniques for analysis of natural samples. As a consequence, it is now possible to discuss the relative abundance of isomers in each examined PAH series. In the phenanthrene and chryiaene series, isomers which bear substituents on the 4 or 5 position (4MP, 4MC, and 5MC) are lacking in the examined crude oils. The steric hindrance can be invoked as in previous work (18) to explain the low abundance of such compounds in the crude oil examined here. In the pyrenic series, all the isomers are quite equally present in the crude oil extract. As shown in Figure 1, the pyrenic ring does not possess a "bay region" where some of the substitution sites can be oveircrowded this fact could explain that the abundance of each methylated homologue of pyrene appears to be quile the same. In conclusion we wish to emphasize tlhat the results presented here have been obtained with routine procedures employed in HPLC and HRS. The use of a dye laser for excitation could be ,avoided. Broad band excitation with almost no "site selection" effect, allows us to observe at the same time all the isomers in a series. This way will be of interest for quantifying the relative (distribution of islomers (19). The real evaluationOf the toxicO1Odcal Of can be then directly related to the quantification of the effective biolcrgically active isomers in H methyl-PAH series. Further quantitative developments on neutral and heterocyclic PAH are in progress in our laboratory, using the HPLC/HRS procedwe described here.
ACKNOWLEDGMENT We Joussot-Dubien for continuous support' interest, and valuable discussions. J. BeXlocq has performed HPLC extractions. R. Lapouyade and A. Colmsjo have kindly given us methylphenanthrenes (R.L.), mlethylpyrenes (R.L,.), and methylchrysenes (A.C.). We thank J. L. Oudin (TOTAL-CFI') and D. Jonathan (SNEA(P)) for providing several crude oils. J*
2159
Registry No. Ph, 85-01-8; 9MP, 883-20-5;lMP, 832-69-9;3 N ! , 832-71-3;1MC, 3351-28-8; 2MC, 3351-32-4;3MC, 3351-31-3;6M[C, 1705-86-7;1MPy, 2381-21-7;~ M P Y3442-78-2; , 4MPy, 3353-12-6.
LITERATURE CITED (1) Colmsjo, A.; Stenberg, U. Anal. Chem. 1979, 5 1 , 145-150. (2) Yang, Y.; D'Silva, A. P.; Fassel, V. A. Anal. Chem. 1981, 5 3 , 894-899. (3) Inman, E. L . ; Jurgensen, A.; Wlnefordner. J. D. Analyst (London) 1982, 107, 538-543. (4) Ewald, M.; Lamotte, M.; Garrigues, P.; Rima, J.: Veyres, A.; Lapouyade, R.; Bourgeois, G. I n "Advances In Organlc Geochemistry 1981"; Bjmoy, M., et al., Eds.; Wlley: New York, 1983; pp 705-709. (5) Wlse, S. A.; Cheder, S. N.; Hertz, H. S.; Hilpert, L. R.; May, W. E. Anal. Chem. 1977, 49, 2306-2310. (6) Wlse, S. A.; Bonnett, &. J.; May W. E. I n "Polynuclear Aromatic Hydrocarbons: Chemistry and Bloiogical Effects"; BJorseth, A,, Deninis, A. J., Ed$.; Rattelle Press: Columbus, OH, 1980; pp 791-806. (7) Hecht, S, S : Bondineli, W. E.; Hoffmann, D. J . Natl. Cancer Irist. ( U . S . ) 1974, 5 2 , 1121-1133.. (8) Lavoie, E. J.; Tuliey-Freiler, L.; Bedenko, V.; Hoffman, D. Cancer Res. 1981, 41, 3441-3447. (9) Garrlgues, P.; Ewald, M.; Lamotte, M.; Rima, J.; Veyres, A,, Lapouyade, R.; Joussot-Dubien, J. Int. J . Environ. Anal. Chem. 1982, 1 1 , 305-312. (10) Garrigues, P.; Lamotte, M.; Ewald, M.; Joussot-Dubien, J. C . R . Seances Acad. Scl., Ser. 2 1981, 293, 567-571. (11) Rima, J.; Lamotte, M.; Joussot-Dublen, J. Anal. Chem. 1982, 5 4 , 1059-1064. (12) Lapouyade, R.; veyles, A.; Hanafi, N.; Couture, A.; Lablache-Combler, A. J . Org. Chem. 1982, 47, 1361-1364. (13) De Vazelhes, R.; Angelin, M. L. Laboratoire de Chimie Physique A, Universltd de Bordeaux I,France, unpublished results. (14) Wise, S, A.; Bennett, W, J,; Guenther, F, R,; May, W, E. J , Chromatogr. Scl. 1982, 19, 457-465. (15) Lee, M. P.; Novotny, M.; Battle, K. D. I n "Analytical Chemistry of Polycyclic Aromatic Compounds"; Academlc Press: London, 1981; pp 353-362. (16) Kong, R. C.; Lee, M. L.; Tominaga; Pratap, R.; Iwao, M.; Castle, R. Anal. Chem. 1982, 5 4 , 1802-1806. (17) Lee, M. L.; Vassilaros, D. L.; Whlte, C. M.; Novotny, M. Anal. Chem. 1979, 5 1 , 1802-1806. (16) Mair, B. J.; Martinez-Pico, J. L. Proc., Annu. Meet., Am. Pet. Inst. 1982, 4 2 , 173-185. (19) Tokousbaldios, P.; Hinton, E. R., Jr.; Dlckinson, R. B., Jr.; Bilotta, P. V.; Wehry, E. L ; Mamantov, G. Anal. Chem. 1978, 5 0 , 1189-1193. (20) Garrigues, P.; Ewald, M. Org. Geochem., in press.
RECEIVED for review May 24, 1983. Accepted August 1, 1983. This work has been supported by grants from CNRS, TOTAL-CFP, and SNEA(P).
