Anal. Chem. 1991, 63,453-456
453
(14) Hillenkamp, F.; Karas, M.; Ingendoh, A,; Stahl, B. Proceedings of the 2nd Int. Symp. on M s s Spectrom. in Health and Life Sciences; Burlingame, A,, McCloskey, J. A,, Eds.; Elsevier: Amsterdam, in press. (15) Heinen, H. J.; Meier, S.;Vogt, H.; Wechsung, R. Int. J . Mass Spectrom. Ion Phys. 1983, 4 7 , 19. (16) Kratzin, H. D.; Wiltfang, J.; Karas, M.; Neuhoff, V.; Hilschmann, N. Anal. Biochsm. 1989, 183, 1.
(5) Beavis, R. C.; Chait, B. T. Rapid Commun. Mass Spectrom. 1989, 3 , 233. (6) Spengler, B.; Cotter, R. J. Anal. Chem. 1990, 62,793. (7) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.;Yoshida, Y.; Yoshida, T. Rapid Commun. Mass Spectrom. 1988, 2 , 151. (8) Karas, M.; Bahr, U.; Ingendoh, A.; Hillenkamp, F. Angew. Chem., Int. Ed. Engl. 1989, 28,760. (9) Karas, M.: Ingendoh, A.; Bahr, U.;Hillenkamp, F. Biomed. Environ. Mass Spectrom. 1989, 18,841. (10) Karas, M.; Bachman, D.; Bahr, U.;Hillenkamp, F. Int. J. Mass Spectrom. Ion Proc. 1987, 78,53. (1 1) Beavis, R. C.; Chait, B. T. Proceedings of the 37th ASMS Conference on Mass Spectrometry and Allied Topics, Miami Beach, FL, 1989; p 1186. (12) Beavis, R. C.;Chait, B. T. R8pid Commun. Mass Spectrom. 1989, 3 , 432. (13) Zhao, S.;Somayajula, K. V.; Sharkey, A. G.; Hercules, D. M. Z . Anal. Chem. 1990, 338. 588.
RECEIVED for review July 16, 1990. Revised manuscript received November 16,1990. Accepted December 7,1990. This work was supported by a grant from the Gustavus and Louise Pfeiffer Research Foundation, the National Science Foundation, the Deutsche Forschungsgemeinschaft, and the Bundesministerium fur Forschung and Technologie.
Determination of Benzo[ a Ipyrene Sulfate Conjugates from Benzo[ a ] pyrene-Treated Cells by Continuous-Flow Fast Atom Bombardment Mass Spectrometry Yohannes Teffera, William M. Baird, and David L. Smith*
Department of Medicinal Chemistry and Pharmacognosy, Purdue University, West Lafuyette, Indiana 47907
The level of certain water-soluble hydrocarbon conjugates, such as benzo[a]pyrene sulfates (BP-SO,), Is a direct measure of carclnogenlc polycyclic aromatic hydrocarbon metabolism and an indication of exposure. A new method, based on continuous-flow high-resolution fast atom bombardment mass spectrometry, has been developed for the analysis of BP-SO, in the medium of cell cultures treated with benro[alpyrene. An organic solvent extract of medium from cultures of the human hepatoma cell line (HepG2) was fractionated by reversed-phase SEP-PAK chromatography and microbore high-performance liquid chromatography (HPLC). The HPLC fraction containing BP-SO, was collected, dried, and injected into a stream of acetonitriie/water/giycerol that was continuously flowing to the tip of the sample probe which was being bombarded continuously by a beam of high-energy xenon atoms. Molecular anions of BP-SO, ( m / z 347) desorbed from the liqoid were analyzed by a high-resolution ( m / A m5000) mass spectrometer and recorded as a function of time. As little as 1.5 pg of BP-SO, could be detected with a S I N ratio of 8. The mass spectrometer response was linear with respect to the quantity of BP-SO, injected over the range from 15 to 625 pg. The results obtained with this method show that the HepG2 cultures metabolized 3% of the benzo[a]pyrene into the BP-SO, conjugate in 24 h. This procedure, which was used to detect and quantify directly BP-SO, in culture medium without the use of a radiolabeledprecursor, should be generally applicable for analyses of sulfated conjugates resulting from the metabolism of different hydrocarbons.
INTRODUCTION The carcinogen benzo[a]pyrene (BP) is metabolized to reactive metabolites that bind to macromolecules. This interaction of reactive metabolites with cellular DNA is believed
to be the initial step in tumor induction ( I , 2). However, a large proportion of B P is metabolized to water-soluble products. These water-soluble conjugates are formed by enzymatic conjugation of phenols with glucuronic acid and sulfate or conjugation of B P epoxides with glutathione (3-8). The predominance of either of the conjugates depends on the type of the cells used. It is reported that the major conjugates found in rodent cells are glucuronides, while in cultured human tissues the predominant products are sulfate esters and glutathione conjugates ($16). In the human hepatoma cell line (HepGB), sulfate conjugates are one of the major water-soluble metabolites (17). Conjugation can be very important in removal of proximate and ultimate carcinogenic BP metabolites. It has been shown that inhibition of conjugative enzymes results in an increase in the formation of mutagenic B P derivatives (18)and an increase in the extent of covalent binding of BP to DNA (11). Determination of conjugates can be used to study the efficiency of the conjugative enzymes in an organism. Furthermore, the detection of conjugates could be used as an indication of exposure of an organism to BP. The metabolism of B P to form water-soluble benzo[a]pyrene sulfates (BP-S04)in cell culture was first reported by Cohen et al., who used a combination of analytical methods, including radiolabeled precursors (3H BP, and 35SNa2S04), synthetic model compounds, and high-performance liquid chromatography (HPLC) to identify the metabolite as BP3-S04 (4). In subsequent investigations of the conversion of BP into water-soluble conjugates in cells, the BP-S04 metabolites have usually been detected only as radiolabeled substances with chromatographic and chromophoric properties similar to those of synthetic standards. Analytical methods that can be used to detect BP-S04with high specificity and sensitivity are needed to advance our understanding of the mechanisms of carcinogenesis of hydrocarbons. Although radioisotopic data demonstrate that some portion of a metabolite is derived from BP, they convey no direct structural information. Alternatives to radiotracer
0003-2700/91/0363-0453$02.50/0 0 1991 American Chemical Society
454
ANALYTICAL CHEMISTRY, VOL. 63, NO. 5, MARCH 1, 1991
347 267
* i 00,
347
183 267 Y
I
205
mlz Figure 1. Negative-ion CF FAB mass spectrum obtained for direct injection of 6.5 ng of the potassium salt of BP-340, standard. Asterisks denote
glycerol matrix peaks. methods are also desirable because they make possible investigations of metabolism of hydrocarbons for which isotopically labeled forms are not available. In addition, detection methods that rely on molecular structure could, in principle, be used to detect hydrocarbon metabolites in human subjects exposed t o hydrocarbons. T o increase the specificity with which BP-S04 can be detected and quantified, several mass spectrometric methods have been investigated. For example, it has been demonstrated that field desorption (19) and direct liquid injection HPLC (20) mass spectrometry give useful mass spectra of BP-S04. However, there are no reports of investigations in which these methods were used to identify or quantify BP-SO, produced by metabolism of BP in cells. In this paper, we describe a new method, based on direct-injection continuous-flow fast atom bombardment mass spectrometry (CF FABMS), for detecting and quantifying BP-S04present in culture media from cells treated with BP. In a preliminary investigation in which conventional FABMS and direct-injection C F FABMS were compared @ I ) , it was demonstrated that the signal-to-noise ( S I N )ratio (molecular anion of BP-3-S04 to glycerol peaks) in C F FABMS is increased 25-fold and that the absolute intensity of the molecular anion (Le., sensitivity) is 20 times larger than in conventional FABMS. These preliminary results suggested that C F FABMS may be useful for investigations of the metabolism of hydrocarbons to their sulfated conjugates by cells.
EXPERIMENTAL SECTION Isolation of BP-SO, from Media. Cultures of HepG2 cells grown to confluence in two 175-cm2flasks were used to develop the method for investigating cellular metabolism of BP. One flask was treated with BP (1 pg/mL of media) and incubated for 24 h. The medium in a second flask was analyzed either without treatment (blank) or after quantitative addition (10 ng/mL or 50 ng/mL) of authentic BP-3-S04 (Chemcal Carcinogen Repository, NCI through Midwestern Research Institute, Kansas City, MO). Three 400-pL aliquots of each of the four samples of media were analyzed for BP-SO,. Nonpolar substances were removed by extracting with chloroform/methanol/water (6). The aqueous layer from each extract was dried, dissolved in 2 mL of water, and loaded on a C-18 SEP-PAK cartridge (Waters Associates, Milford, MA). The cartridge was washed with 2 mL of water, followed by 2 mL of methanol. The methanol fraction was filtered (4 mm, 0.45 pm of Nylon 66, Alltech & Associates, Deerfield, IL),
dried, dissolved in 20 pL of 30% acetonitrile (ACN) in water with 0.1% trifluoroacetic acid (TFA), and injected onto a C-18 HPLC column (25 cm X 1 mm, Alltech) by using a Rheodyne 7125 injector with a 20-pL external loop. The solvents used for HPLC fractionation were 5% ACN in water with 0.1% TFA (solvent A) and 10% water in ACN with 0.1% TFA (solvent B). Gradients of 30-60% B in 30 min and 60-100% B in 10 min were used for elution. A dual-syringe-drive HPLC pump system (Brownlee Labs, Santa Clara, CA) was used to maintain a flow rate a t 50 pL/min through the column. The eluent was monitored at 254 nm with an Applied Biosystems 757 UV detector equipped with a 1-pL cell. Chromatographic fractions corresponding to a retention time of 18-20 min were collected and dried for medium extracts from each flask of cells. Direct-Injection Continuous-Flow FABMS. A solvent system containing 5050 ACN:water with 3% glycerol was filtered (25 mm, 0.45 pm of Nylon 66, Alltech) and sonicated for 30 min. A Brownlee Labs microgradient pump was used to drive the freshly prepared solvent at a flow rate of 3 pL/min through a 1.5-m length of 50-gm-i.d. fused silica capillary tubing (Polymicro Technologies, Phoenix, AZ) into the ion source of a Kratos MS50RF double-focusing mass spectrometer operated in the negative ion mode with an 8-kV accelerating voltage. Modifications to the ion source for CF FAB operation and the construction of the continuous-flow probe have been described elsewhere (21). The ion source temperature was 48-50 "C. An Ion Tech FAB gun, operated with xenon, was used to desorb ions from the probe tip. Samples dissolved in the mobile phase were injected into the flowing solvent with a Rheodyne 7013 injector equipped with a 0.5-pL internal loop. The signal from a mass window 200 ppm wide (resolution 5000) and centered at m / z 347.0378 was recorded with an analog strip chart recorder and by an HPLC data acquisition system (Dynamax, Rainin Instruments, Woburn, MA). The areas of peaks were determined with the data acquisition system and used to indicate the mass spectromet,ric response.
RESULTS AND DISCUSSION The molecular structure of BP-3-S04 (see Figure 1)includes a nonpolar hydrocarbon fused ring system and a highly polar sulfate anion. With the hydrocarbon portion virtually insoluble in the matrix, glycerol, BP-340, is probably concentrated along the matrix/vacuum interface. This feature, plus the fact that BP-3-SO4 exists as an anion in solution, suggests that negative-ion FABMS may be the basis of a highly sensitive method for detecting BP-S04. This prediction is supported by the excellent SIN ratio obtained for a full-scan
ANALYTICAL CHEMISTRY, VOL. 63, NO. 5, MARCH 1, 1991
a
0
200
400
d
I
600
Quantity (pg) Figure 2. Standard curve illustrating the CF FABMS response for injection of different quantities (15, 62, 312, and 625 pg) of BP-340,.
2
4
6
8
1
455
'
I/ iz
is is
i4 i 7
Time (minutes)
Time (minutes)
0
Time (minutes) Figure 3. CF FABMS single-ion recording of m l z 347.0378 for direct injection of (a) 1.5 pg of BP-340, and (b) blank injection (resolution
5000).
mass spectrum obtained for 6.5 ng of BP-3-S04 (Figure 1). This mass spectrum has two major peaks ( m / z 347 and 267) corresponding to the molecular anion and a fragment ion formed by the loss of SO3. The intensities of these peaks are similar to those of the matrix peaks a t m / z 183 and 205. Although the same peaks were observed in conventional FABMS, the S I N ratio is substantially improved in CF FABMS. While full-scan spectra may be used to answer some types of questions, high-resolution single ion monitoring is better suited for high-sensitivity measurements. Preliminary studies indicated that a resolution of 3000 was adequate to completely separate the molecular anion of BP-3-S04,m / z 347, from the background signal (21). High resolution is advantageous for these studies because it improves the signal-to-noise ratio and lowers the detection limit, despite a 5-fold loss of sensitivity. In addition, high resolution opens the possibility of determining the elemental composition of the analyte. Unlike conventional FABMS, CF FABMS responds lineraly over a wide range to the quantity of sample injected (22). As a result, CF FABMS can be used to quantify substances without the need for isotopically labeled internal standards. The linearity of the CF FABMS response is indicated by results presented in Figure 2, which show the average response (peak area of m / z 347.0378) for injections of 15-625 pg of BP-3-S04. The response at each concentration is the average of three injections. The relative standard deviation (RSD) a t each concentration was less than 10%. Linear regression analysis shows that the slope and y intercept of the response curve are 0.010 451 and 0.240 48, respectively. The correlation coefficient for the curve was 0.999. Although the slope of the curve varied with several instrumental parameters (Le.,probe temperature, flow rate, solvent composition), the linearity changed little. Injection of quantities greater than 10 ng gave badly tailing peaks and a nonlinear response. The ability to detect subpicogram quantities of BP-3-S04is demonstrated in Figure 3, which shows the FABMS response for an injection of 1.5 pg ( S I N 8:l). Injection of solvent immediately afterwards gave no response, indicating that BP-3-S04was com-
2
4
6
8
10
12
14
16
Time (minutes) Figure 4. UV and CF FABMS detection of BP-SO, in media of HepG2 cells treated with BP for 24 h. Upper: UV recording of HPLC fractionation of medium from cells that were treated wth BP (a) and untreated (d). Arrows indicate the time at which fractions were collected and analyzed by CF FABMS. Lower: Single-ion recordings for (a) medium from cells treated with BP, (b) 15 pg of BP-3-S04 standard, (c) solvent blank, and (d) medium from untreated cells.
pletely washed from the injector. The primary goal of this investigation was to develop a high-specificity method for quantifying BP-SO4 produced by metabolism of B P in cells in culture. Isotope dilution mass spectrometry using internal standards labeled with stable isotopes has proved to be a highly accurate method for quantifying a wide variety of substances. However, an isotopically labeled internal standard was not used in this investigation because it was necessary to develop a method that could be used to analyze sulfate conjugates of different hydrocarbons for which labeled internal standards may not be available. In the present method, the media extract was fractionated through two steps of reversed-phase chromatography followed by direct-injection CF FABMS. Microbore HPLC was used for the final purification step because C X its characteristic high sensitivity and rate of sample recovery. All media samples were filtered prior to fractionation by microbore HPLC to reduce the likelihood of damaging the column. Portions of the UV absorbance traces of the microbore HPLC chromatograms recorded for HepG2 cell cultures that were treated with BP and untreated control cultures are given in the upper portion of Figure 4 (a and d upper, respectively). The arrows indicate the time at which authentic BP-3-S04 elutes under the same chromatographic conditions. It is important to note that there is high UV absorbance at the elution time of BP-S04 in the media blank sample, indicating that another substance with absorbance a t 254 nm elutes at approximately the same time. Thus, analysis for BP-SO4 in media by its UV absorbance may give a false positive response, indicating that a more specific detector is required. Chromatographic fractions collected at the time corresponding to the retention time of BP-S04 were analyzed by high-resolution direct injection CF FABMS. The single ion
456
ANALYTICAL CHEMISTRY, VOL. 63, NO. 5, MARCH 1, 1991
recording of the molecular ion of BP-SO4 ( m / z 347) for the media from cells treated with B P gave a strong signal (Figure 4a lower) indicating that B P was metabolized to BP-SO, in HepG2 cell cultures. Similar analysis by CF FABMS of media from untreated cells (blank) gave no response (Figure 4d lower), substantiating the high specificity of the method. Injection of 15 pg of authentic B P - 3 3 0 , gave the response in Figure 4b, while injection of solvent gave little response (Figure 4c). Analysis of triplicate aliquots of media spiked with 10 or 50 ng/mL authentic B P - 3 4 0 , based on the CF FABMS response of the spiked media and authentic standard gave a recovery rate of 67% (RSD 11%) and 80% (RSD 8%), respectively. Similar analysis of treated media showed that the media from HepG2 cells contained an average of 23 ng/mL BP-SO, (RSD 9%). On the basis of the recovery rates obtained by interpolation (75%), these results show that the cells have metabolized 3% of the B P into BP-SO,. Although the chromatographic procedures used in this study probably do not separate the isomeric sulfate conjugates of B P phenols, they do separate the conjugates of B P phenols from those of BP diols. On the basis of previous studies using radioisotopes and enzymatic cleavage, the majority of BP-SO4 formed in HepG2 cells is BP-3-S04 (17). These results demonstrate that the present method can be used to identify and quantify BP-SO, formed in cells in culture with an uncertainty of less than approximately 13%. Higher accuracy could likely be achieved by using an internal standard labeled with a stable isotope. Further studies could be carried out with enzymatic cleavage and HPLC to identify the specific B P phenols in these cells. The accurate quantitation of hydrocarbon metabolites in a cell culture medium is difficult because their concentrations may be low and because the cell culture medium is a complex mixture of many substances. As a result, analytical methods suitable for quantifying hydrocarbon metabolites must have both high sensitivity and specificity. The present results demonstrate that high-resolution CF FABMS can be used to detect and quantify BP-S04 in culture media and will serve as an important tool for investigations of hydrocarbon metabolism in biological samples.
ACKNOWLEDGMENT We thank Robert Polzer for treating the HepG2 cell cultures.
LITERATURE CITED (1) Dipple, A. I n Polycyclic Hydrocarbons and Carcinogenesis; Harvey, R. G., Ed.: American Chemical Society: Washington, D.C., 1985; pp 1-18. (2) Geacintov, N. E. I n Polycyclic Aromatic Hydrocarbon Carcinogenesis: Structure-Activity Relafionships; Yang, S. K., Sllverman, B. D., Eds.; CRC Press: Boca Raton, FL 1988; Vol. 11, pp 181-206. (3) Nemoto, N.; Gelboin. H. V. Biochem. Pharmacol. 1976, 2 5 , 1221- 1226. (4) Cohen, G. M.; Haws, S. M.; Moore, B. P.; Bridges, J. W. Biochem. PharmaCOl. 1976, 2 5 , 2561-2570. (5) Nemoto, N.; Gelboin, H. V.; Habig, W. H.; Ketley, J. N.; Jakoby, W. B. Nature (London) 1975, 255, 512. (6) Baird, W. M.; Chern, C. J.; Diamond, L. Cancer Res. 1977, 37, 3 190-3 197. (7) Autrup, H. Biochem. Pharmacol. 1979, 2 8 , 1727-1730. (8) Gmur, D. J.; Varanasi. U. Carcinogenesis 1982, 3 , 1397-1403. (9) Baird, W. M.; Chemerys, R.; Erickson, A. A,; Chern, C. J.; Diamond, L. Polynuclear Aromatic Hydrocarbons ; Science Publishers: Ann Arbor, MI, 1979: pp 507-516. (10) Zaleski, J.; Bansal, S. K.; Gessner, T. Carcinogenesis 1983, 4 , 1359-1366. (11) Burke, M. D.; Vadi, H.; Jernstrom, B.; Orrenius, S. J. Biol. Chem. 1877, 252, 6424-6431. (12) b i r d , W. M.; O'Brien, T. G.; Diamond, L. Carcinogenesis 1981, 2 , 81-88. (13) Merrick, B. A.; Manfield, B. K.; Nikbakht, P. A,; Selkirk, J. K. Cancer Lett. 1985, 2 9 , 139-150. (14) Autrup, H. Drug Metab. Rev. 1982, 13, 603-646. (15) Mass, J. J.; Rodgers, N. T.; Kaufman, D. G. Chem.-Biol. Interact. 1881, 33, 195-205. (16) Mehta, R.; Cohen, G. M. Biochem. Pharmacol. 1978, 2 8 , 2479-2484. (17) Plakunov, I.; Smobrek, T.A.; Fischer, D. L.; Wiley, J. C. Jr.; Baird, W. M. Carcinogenesis 1987, 59-66. (18) Bock, K. W.; Bock-Henning, B. S.; Lilienblum, W.; Volp, R. F. Chem.Biol. Interact. 1981, 36, 167-177. (19) Deutsch, J.; Gelboin, H. V. Biomed. Mass Spectrom. 1882, 9 , 99-101. (20) Bieri, R. H.: Greaves, J. Biomed. Mass Spectrom. 1987, 1 4 , 555-56 1. (21) Smith, D. L. I n Continuous-flow Fast Atom Bombardment Mass Spectrometry; Caprioli, R. M.; Ed.; Wiley & Sons: New York, 1990. (22) Caprioli, R . M.; Fan, T.; Cotrell, J. S. Anal. Chem. 1988, 5 8 , 2949-2954.
RECEIVED for review August 20, 1990. Accepted December 10, 1990. This investigation was supported by the following grants from the National Institutes of Health; CA 28825 (W.M.B.) and GM 40384 (D.L.S.). Y. Teffera was partially supported by a David Ross Fellowship from Purdue University. The B P - 3 4 0 , was purchased through the Chemical Carcinogen Repository, National Cancer Institute at the Midwest Research Institute, Kansas City, MO.
