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Anal. Chem. 1988, 58, 1344-1348
volume at approximately 1.75 min. The phenylarsonic acid elutes after the arsenate peak, which indicates it may interact with the column matrix. It may be possible to improve the resolution of the arsenate and phenylmonic acid by gradient manipulation. I t is also reasonable to assume that similar modifications in the gradient would allow separation of a wide variety of arsenic-containing compounds. The applicability of the method for determination of arsenic-containing species in the culture cell medium was determined by analysis of a mixture of the four metabolites in the medium. An aliquot of the F-10 culture growth medium containing cadet calf serum was mixed with a concentrated solution of the standard metabolites, and the solution was immediately injected. Under these conditions, the recoveries of the standards were excellent. The average of six determinations for arsenite was 87.4% (u = 1.3), DMA was 95.9% (u = 1.8), MAA was 96.5% (u = 1.9), and arsenate was 97.6% (a = 1.9). Although there was reason to expect lowered recoveries for specific compounds over long exposure to the growth medium due either to association of the arsenic metabolites with complex organic compounds such as proteins in the growth medium or to interconversion of arsenite and arsenate by redox reactions, a 3-month survey of the arsenic metabolites stored at 4 "C in the growth medium did not show any statistically significant concentration variations. Longterm stability of the metabolites in growth medium is not a serious problem as long as normal sterile handling procedures are observed. Registry No. MAA2-, 124-58-3;DMA-, 75-60-5;TPO, 115305-5; PAA2-, 98-05-5; AsO;, 15502-74-6; HAs04'-, 15584-04-0.
LITERATURE CITED (1) Medlcal and Blologlcal Effects of Environmental Pollutants -Arsenic ; National Academy of Sciences: Washington, DC, 1977: ISBN 0-30902604-0.
Fowler, B. A. EHP, Environ. Health Perspect. 1977, 19, 239. Fowler, B. A. Proceedings of the Internatlonal Conference on EHP, Environ . Health Persp 1977, 79. Brlnckman, F. E.; Bellma, J. M., Eds. ACS Symp. Ser. 1978, No. 8 2 . Frost, D. V. Fed. Proc. 1987, 2 6 , 194. Wood, J. M. Science (Washington,D . C . ) 1974, 783, 1049. Knowles, F. C.; Benson, A. A. TIBS 1983, 778. Gurley, L. R.; Walters, R. A.; Jett, J. H.; Tobey, R. A. J . Toxicol. Environ . Health 1980, 6, 87. Gurley, L. R.; Walters, R. A.; Jett, J. H.; Tobey, R. A. LA-8063-MS
.
1979.
Gurley, L. R.; Tobey, R. A.; Valdez, J. G.; Halleck, M. S.;Barham, S . S. Scl. Total Environ. 1983, 2 8 , 415. Gurley, L. R.; Tobey, R. A.; Valdez, J. G.; Halleck, M. S.; Barham, S. S. L A - 8 9 9 5 4 s 1981. Crecelius, E. A. Anal. Chem. 1978, 5 0 , 826. Brinckman, F. E.; Blair, W. R.; Jewett, K. L.; Iverson, W. P. J . Chromatogr. Sci. 1977, 75, 493. Stockton, R. A.: Irgolic, K. J. Int. J . Environ. Anal. Chem. 1979, 6, 313.
Brinckman, F. E.; Jewett, K. L.; Iverson, W. P.; Irgolic, K. J.; Chrhardt, K. C.; Stockton, R. A. J . Chromatogr. 1980, 797, 31. Woolson, E. A.; Aharonson, N. J . Assoc. Off. Anal. Chem. 1980, 63, 523.
Irgolic, K. J.; Stockton, R. A.; Chakraborti, D. Spectrochim. Acta, Part B 1983, 3 8 8 , 437. Gast, C. H.; Kraak, J. C.; Poppe, H.; Maessen, F. J. M. J. J . Chroma t o p . 1979, 785, 549. Morita, M.; Uehiro, T.; Fuwa, K. Anal. Chem. 1981, 5 3 , 1806. Jinno, K.; Tsuchida, H.; Nakanishi, S.; Hirata, Y.; Fujimoto, C. Appl. Spectrosc. 1983, 3 7 , 258. Iadevaia, R.; Ahronson, N.; Woolson, E. A. J . Assoc. Off. Anal. Chem. 1980, 6 3 , 742. Woolson, E. A. Waters Associates Technical Bulletin. Trace Level Speciation of Arsenical Compounds: Jan 1982, private communication, Apr 1984. Fassel, V. F. Anal. Chem. 1979, 5 1 , 1290A.
RECEIVED for review May 14, 1985. Resubmitted February 10,1986. Accepted February 24, 1986. This work was performed under the auspices of the U.S. Department of Energy, Contract W-7405-ENG-36.
High-Performance Liquid Chromatography/Thermospray Mass Spectrometry of Eicosanoids and Novel Oxygenated Metabolites of Docosahexaenoic Acid James A. Yergey,* Hee-Yong Kim, and Norman Salem, Jr. Section of Analytical Chemistry, Laboratory of Clinical Studies, DICBR, National Institute of Alcohol Abuse and Alcoholism, ADAMHA, Bethesda, Maryland 20892
High-performance liquid chromatography coupled to mass spectrometry using a thermospray interface and ionlzation source has proved an effective means of analyzing underlvatlred oxygenated fatty acld metabolltes. The method was particularly useful for the qualitative anaiysls of several novel metabolites of docosahexaenoic acid. Mono-, dl-, and trihydroxylated products were Isolated and analyzed from rat braln homogenate following lncubatlon wlth docosahexaenolc acid. Simple derivatlzatlons of the metabolites followed by analysls wlth thermospray mass spectrometry provlded a unique means of obtalnlng molecular weight confirmatlon and further structural Information.
Docosahexaenoic acid (C22:6w3) is a particularly interesting fatty acid because it is the major polyunsaturate in the brain
and is present in high levels in the highly metabolically active cells in the retina, heart, and testes (1). Only recently has the metabolism of C22:6 been intensively studied (1-12), which is surprising in view of the proliferation of eicosanoid research. It is apparent from these reports that the primary metabolites of C22:6 in trout gill (2,3), rat liver microsomes (4), dog retina (5), mouse peritoneal macrophages (6),human platelets (7, 8), and rat brain (9-12) are hydroxylated compounds. Characterizing the metabolism of C22:6 in mammalian brain with respect to the structure and function of enzymatically oxygenated products is the subject of our investigations. Analysis of oxygenated metabolites of fatty acids in biological systems has typically involved class separation by extraction or column chromatography, followed by one or more high-performance liquid chromatographic separations, several derivatization steps, and final analysis by gas chromatography/mass spectrometry (13-15). Although this approach is
This article not subject to U.S. Copyright. Published 1986 by the American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 7,JUNE 1986
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extremely powerful for analyzing known compounds, it can be very cumbersome for the analysis of unknowns. Studying the metabolism of docosahexaenoic acid provided our laboratory with the opportunity to develop a new approach for analyzing this class of compounds. Reducing the analysis to a single class separation followed by high-performance liquid chromatography/thermospray mass spectrometry appeared to be an ideal means of acquiring molecular weight and simple structural information for the unknowns that we encountered. Thermospray interfacing to the mass spectrometer provides a soft ionization method that is particularly suited to the analysis of polar and thermally labile compounds (16). This methodology was particularly attractive for analysis of unknown compounds, since it eliminated many of the sample cleanup steps and does not require derivatization. Prostaglandin and leukotriene metabolites of 5,8,11,14-eicosatetraenoicacid (C20:4w6) served as reference compounds for these analyses.
EXPERIMENTAL SECTION Samples analyzed by thermospray liquid chromatography/mass spectrometry were separated with a Beckman HPLC system comprised of two Model 114M pumps, a Model 450 data system used to control the pump gradients, and an Altex Model 210 injector. The Du Pont 25 cm X 4.6 mm, 5-km Zorbax ODS column was connected directly to the mass spectrometer interface. The thermospray interface utilized was a standard Vestec design (Vestec, Inc., Houston, TX) supplied with the mass spectrometer. The interface probe and mass spectrometer source temperatures were optimized by using standards and typically operated at 210 and 275 "C, respectively. Thermospray mass spectrometry was performed on an Extrel 400-2 quadrupole instrument using a thermospray ionization source. The source and analyzer were equipped with 360 L/s turbomolecular pumps that maintained approximately 1.5 X torr pressure in the analyzer region during thermospray operation. Auxiliary pumping was connected to the source opposite the thermospray probe through an isopropyl alcohol/dry ice cold trap. Positive ion mass spectra were obtained by scanning from 110 to 510 daltons without an external ionization source; i.e., the filament remained off. Negative ion spectra of pentafluorobenzyl derivatives were obtained by using the filament to generate a source of low-energy electrons for electron capture. Separations for comparison to thermospray data were carried out with a Hewlett-Packard Model 1090 liquid chromatographic system, equipped with an autoinjector and a diode-array ultraviolet-visible detector. Separations were done on a Du Pont 25 cm X 4.6mm, 5-pm Zorbax ODS column. All chromatographic separations were performed by using a flow rate of 1 mL/min and identical mobile phases and gradient conditions. The initial mobile phase of 75:25 0.1 M ammonium acetate:acetonitrile was ramped to 58:42 in 20 min and then to 3070 in 10 min. Injections of 50 pL were made for both standards and samples dissolved in acetonitrile. Representative standards that are reported in this article included 15-ketoprostaglandin E2 (Sigma Chemical Co., St. Louis, MO), 5-, 12-, and 15-hydroxyeicosatetraenoic acid (Seragen, Boston, MA), and leukotriene B4 (Calbiochem,La Jolla, CA). Rat brains are rapidly excised and homogenized in 50 mM Tris (pH 7.4). Aliquots of 226 fatty acid solution in ethanol are evaporated to dryness in a homogenizing tube with a stream of nitrogen. Rat brain homogenate is added and the mixture homogenized to disperse the substrate (final substrate concentration is 0.1-1 mM). This mixture is incubated at 37 O C for 30-60 min, as appropriate. Ethanol is added to make a 15% final concentration; the mixture is centrifuged at 3000 rpm to remove excess tissue, and the supernatant is applied to a C-18 Sep-PAK cartridge. The cartridge is then sequentially eluted with 20 mL of 15% ethanol, benzene, and ethyl acetate. This separation method is similar to the one used by Powell (17) except that the mixture is not acidified. Hydroxylated derivatives are found in the ethyl acetate fraction, which is concentrated for HPLC analysis. Dried samples were redissolved in acetonitrile and stored at -70 "C. Ethyl acetate fractions were analyzed by HPLC and thermospray HPLC/MS.
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Samples of brain homogenate were boiled and analyzed in a similar fashion to control for nonenzymatic oxidation of C22:6. All solvents were "distilled-in-glass" HPLC grade (Burdick and Jackson, Muskegon, MI). Ammonium acetate (J. T. Baker, Phillipsburg, NJ) solutions were filtered with 0.45-km Millipore filters. Derivatization of fatty acids to pentafluorobenzyl esters and methoximation of ketones have been previously described (18). Pentafluorobenzyl bromide, diisopropylethylamine, and methoxylamine hydrochloride in pyridine were purchased from Pierce Chemical Co. (Rockford, IL). Hydrogenation (99.9998% reagent grade hydrogen, Matheson Products, Dorsey, MD) was performed at room temperature over PtO, (ICN Pharmaceuticals, Plainview, NY) for 60 min.
RESULTS AND DISCUSSION Data are presented for representative eicosanoid standards from which some generalizations can be deduced for application to the analysis of C22:6 metabolites. A more complete compilation of thermospray spectra of eicosanoids can be found elsewhere (19). Details of the biochemistry of docosahexaenoate metabolite formation including work on structure elucidation are beyond the scope of this report; such studies are in progress and will be communicated elsewhere. The purpose of this article is to provide an example of the power of the thermospray technique for this type of qualitative analysis. An important requirement for evaluating an HPLC/MS system is its ability to maintain the integrity of the chromatographic resolution. Figure 1illustrates the chromatographic data obtained by using ultraviolet detection in comparison to the total ion trace obtained for the same sample by positive ion thermospray mass spectrometry. It is clear that although the relative intensities for .the two detection systems are different, the chromatographic quality is comparable. Mass spectra of oxygenated fatty acid standards were dominated by protonated and ammoniated molecules and
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 7, JUNE 1986
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present is necessary to obtain mass spectra, in contrast to the lengthy derivatization scheme needed for analysis of these compounds by gas chromatography/mass spectrometry. The observation that the number of fragments due to losses of water is directly related to the number of oxygenated sites on the fatty acid chain should prove useful in analyzing oxygenated fatty acid metabolites of unknown structure. The dominance of molecular species and the overall simplicity of the spectra, including the virtual absence of background ions beyond the solvent ion region, should make interpretation of spectra of compounds of this type straightforward. The information gained in the chromatography and thermospray mass spectrometry of cyclized and hydroxylated fatty acids was extended in our investigations to the novel metabolites of docosahexaenoic acid. The chromatograms produced by either ultraviolet detection or thermospray mass spectrometry showed numerous peaks in the rat brain ethyl acetate fraction that were not present in boiled controls. This implied that the peaks represented enzymatically produced metabolites of docosahexaenoic acid. An example of a total ion chromatogram of docosahexaenoic acid metabolites formed from rat brain in vitro is shown in Figure 3. The peaks are labeled according to assignments made based on thermospray mass spectra. Boiled control chromatograms showed varying amounts of the monohydroxylated metabolite, indicating that some of this metabolite may be formed as a result of autooxidation, whereas all other labeled peaks were not present in detectable amounts in the boiled control samples. Initial experiments indicated that approximately 1-2% of incubated C22:6 is recovered in the ethyl acetate fraction. This corresponds to approximately 20-200 ng for each peak presented in Figure 3. The mass spectra of each of several peaks labeled with the same heading (e.g., trihydroxy) were virtually identical in terms of the masses observed; however, reproducible differences were observed in the relative intensities of the various ions. It is reasonable to assume, therefore, that these represent several isomers of the same basic structure. The positive ion mass spectra observed for the docosahexaenoic acid metabolites contained fragmentation patterns similar to those observed for eicosanoid standards. Figure 4 illustrates an example of a spectrum for trihydroxylated species showing peaks corresponding to the expected ammoniated species and losses of water from the ammoniated and protonated molecule. The spectrum is virtually free of background or other fragmentation. Note that two series of peaks are observed separated by two mass units. These are due to the superposition of the spectra of trihydroxylated
ANALYTICAL CHEMISTRY, VOL. 58, NO. 7, JUNE 1986 REL.IN1
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approach, we attempted to significantly increase the negative ion abundance by making the pentafluorobenzyl (PFB) ester derivative of the samples. When analyzed by GC/MS in the negative ion mode, these esters yield an abundant (M - PFB)ion due to electron capture followed by elimination of the PFB group. We found that eicosanoid standards that were derivatized in this fashion indeed showed the expected (M PFB)- ion when analyzed by thermospray mass spectrometry, but this ion was observed only when operating with the auxiliary filament on. Therefore, the various peaks observed in the rat brain incubate were purified by HPLC, derivatized to the PFB ester, and analyzed in the negative ion mode with the auxiliary filament on. An example of this type of analysis is shown in Figure 5. The positive ion spectrum for a monohydroxylated C22:6 shows a typical (M H - H20)+ion as base peak, whereas the negative ion spectrum of the PFB derivative is dominated by the (M - PFB)- ion. Negative ion spectra were obtained for each of the C22:6 metabolites, which confirmed the molecular weight assingments made previously by analysis of the positive ion data. Further structural information was also obtained by thermospray mass spectrometric analysis of the hydrogenated derivatives of the docosahexaenoic acid metabolites. Following collection of HPLC fractions, samples were hydrogenated and reanalyzed. An example for the dihydroxylated C22:5 species is presented in Figure 6. The underivatized sample exhibits the usual ammoniated molecule and apparent losses of water from the ammoniated and protonated molecule. The spectrum for the hydrogenated sample exhibits exactly the same peaks but shifted by 10 daltons, implying that five double
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C22:6 and C22:5. On the basis of the observations made for the eicosanoid reference compounds, the number of oxygenated functionalities is readily deduced from the spectrum, as are the molecular weights of 376 and 378 daltons. Negative ion spectra were investigated in order to further substantiate the molecular weight of the metabolites. Spectra obtained with the filament off were somewhat less intense than in the positive ion mode, but contained analagous ions with approximately the same relative abundance. As an alternative
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 7, JUNE 1986
bonds had been reduced. Similar results were obtained for the monohydroxylated species; however, we were unsuccessful in hydrogenating and rerunning the trihydroxylated species as these fractions appeared to be too labile for this type of analysis. The positive ion spectra for the dihydroxylated C22:5 species were quite different from that of the mono- and trihydroxylated metabolites in that they contained an intense MH+ ion. The fact that we observed a similar phenomenon for reference compounds containing ketone groups (Figure 2a, 15-keto-PGE2) suggested the possibility that the dihydroxylated C22:5 might contain a similar group. We therefore attempted to analyze the methoximated derivative of the dihydroxylated C22:5 sample along with appropriate standards. In the case of standards, we observed the expected 29-dalton shift, but for the dihydroxylated C22:5 sample, no shift was observed. Based on these data we concluded that some functional group other than a ketone accounts for the MH+ ion observed in these particular metabolites.
CONCLUSIONS High-performance liquid chromatography coupled with thermospray mass spectrometry is a useful tool for the analysis of oxygenated fatty acid metabolites. The chromatographic integrity of the system is maintained and thermospray ionization of these molecules is effected without the need for an external ionization source. Mass spectra contain primarily ammoniated molecules and simple but informative losses of water from the ammoniated and protonated molecule, making molecular weight assignments straightforward. The relative simplicity of the spectra and lack of appreciable background is ideally suited for the initial analysis of unknown metabolites. This technique has proved to be extremely valuable in the identification of novel metabolites of C22:6 produced by the rat brain, including mono-, di-, and trihydroxylated species. The relative ease of obtaining the molecular weights and basic functional group information contained in these metabolites using thermospray mass spectrometry when compared to off-line HPLC mass spectrometric or GC/MS methods was evident. It has also been clearly demonstrated that simple derivatizations of samples followed by reanalysis by thermospray mass spectrometry can yield important structural information. Pentafluorobenzyl esterification of free acids was useful in
confirming molecular weights, since the negative ion mass spectra contained intense (M - PFB)- ions. This PFB derivatization was also effective in improving the sensitivity of the technique in the negative ion mode. Methoximation of ketones was used to demonstrate the absence of a ketone functionality in the dihydroxylated C22:6 metabolites. Similarly, hydrogenation of samples and reinjection into the thermospray system confirmed our assignment of the number of double bonds present. Registry No. 15-oxo-PGE,, 26441-05-4; C22:6w3 (monohydroxylated), 100313-25-5;C22:6w3 (trihydroxylated),10033410-9;C225 (trihydroxylated), 100313-28-8;C22:5 (dihydroxylated), 100313-30-2;LTB4,71160-24-2;15-HETE, 73180-00-4; 5-HETE, 100313-26-6; 12-HETE, 100313-27-7.
LITERATURE CITED (1) Salem, N., Jr.; Kim, H.-Y.; Yergey, J. "Health Effects of Polyunsaturated Fatty Acids in Seafoods"; Simopouios, A. P., Kifer, R. R., Eds.; Academlc Press: New York, in press, 1985. (2) Mai, J.; Goswaml, S. K.; Bruckner, G.; Kinsella, J. E. Prostaglandins 1981, 2 1 , 691-698. (3) German, B.; Bruckner, G.; Kinsella, J. Prostaglandins 1983, 2 6 , 207-210. (4) Van Rollins, M.; Baker, R. C.; Sprecher, H. W.; Murphy, R. C. J. Blol. Chem. 1984, 259, 5776-5783. (5) Bazan, N. G.; Birkle, D. L.; Reddy, T. S. Biochem. Blophys. Res. Commun. 1984, 125, 741-747. (6) Rigaud, M. Prostaglandins 1984, 2 7 , 358-361. (7) Aveidano, M. I.; Sprecher, H. J . Blol. Chem. 1983, 258, 9339-9343. (8) Fischer, S.; Schacky, C. V.; Siess, W.; Strasser, T. H.; Weber, P. C. Biochem. Biophys. Res. Commun. 1984, 120, 907-918. (9) Salem, N., Jr. Trans Am. SOC.Neurochem. 1983, 14, 105. (10) Stojanov, M.; Yergey, J.; Salem, N., Jr. Proc. Fed. Am. SOC. Exp. Blol. 1984, 4 3 , 1463. (11) Yergey, J.; Salem, N., Jr. Annu. Conf. Mass Spectrom. Allled Top. 32nd 1984, 104-105. (12) Salem, N., Jr.; Stojanov, M.; Yergey, J.; Kim, H.-Y. Trans. Am. SOC. Neurochem. 1085, 16, 108. (13) Hubbard, W. C.; Watson, J. T. Prostaglandins 1978, 12, 21-35. (14) Froilch, J., Ed. "Methods in Prostaglandin Research"; Raven Press: New York, 1979. (15) Whorton, A. R.; Carr, K.; Smingal, M.: Walker, L.; Ellis, K.; Oates, J. A. J. Chromatogr. 1979, 163, 64-71. (16) Blakely, C. R.; Vestal, M. L. Anal. Chem. 1983, 5 5 , 750-752. (17) Powell, W. S.Prostaglandins 1980, 157, 947-957. (18) Waddell, K. A,; Blair, I . A.; Wellby, J. Biomed. Mass Spectrom. 1983, 10, 83-88. (19) Pace-Asciak, C. K. "Atlas of Prostaglandins and Related Compounds"; Raven Press: New York, in press, 1986. (20) Yergey, J.; Salem, N., Jr. Annu. Conf. Mass Spectrom. Allled Top. 33rd 1985. I
RECEIVED for review September 16,1985. Accepted November 22, 1985.
