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Anal. Chem. 1988, 58, 1340-1344
(7) Miiier, D. D.;Van Campen, D. M . J. Clin. Nutr. 1979, 32, 2354. (8) Carni, J. J.; James, W. D.;Koirtyohann, S. R.; Morrix, E. R. Anal. Chem. 1960, 52, 216. (9) Janghorbanl, M.; Ting, B. T. G.; Fomon, S. J. Am. J . Hematol. 1986, 21, 277. (IO) Gorten, M. K.; Hepner. R.; Workman, J. 8 . J . Pediatr. (St. Louls) 1963, 83, 1063. (11) Dubach, R.; Moore, C. V.; Minnich, V. J . Lab. Clin. Med. 1946, 31, 1201. (12) Finch, C. A.; Gibson, J. G.; Peacock, W. C.; Fiuharty, R. G. Blood 1949, 4, 905. (13) Gorky, L.; Sjoiin, S. Acta Paediatr. 48 Scand. Suppl. 1959, 177, 24.
(14) Chart of the Nuclides; General Electric, Nuclear Energy Division: San Jose, CA, 1972. (15) Janghorbani, M.; Ting, B. T. G.; Young, V. R. Clin. Chim. Acta 1980, 108, 9. (16) National Bureau of Standards, Certificate of Analysis, Standard Reference Material 1577a,Washington, DC, June 15, 1982. (17) Meites, L., Ed. Handbook of Analytical Chemistry, 1st ed.; McGrawHili: New York, 1963;Table 1-17, pp 1-39.
RECEIVED for review October 10, 1985. Accepted January 9, 1986.
High-Performance Liquid Chromatographic Separation of Biologically Important Arsenic Species Utilizing On-Line Inductively Coupled Argon Plasma Atomic Emission Spectrometric Detection W. D. Spall,* J. G. Lynn,J. L. Andersen, J. G. Valdez, and L. R. Gurley Toxicology Group, Life Science Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545
An anlon exchange, high-performance liquid chromatography technlque uslng a l 5 m I n linear gradient from water to 0.5 M ammonium carbonate to separate arsenlte, arsenate, methylarsonlc acid, and dimethylarsinic acld from neutral arsenlc containlng compounds was developed for application to a study of arsenlc metabolism In cultured cell suspensions. Arsenlc detection was accomplished by the direct coupilng of the column effluent to an inductively coupled argon plasma atomic emisslon spectrometer (ICAP-AES) set to monltor the arsenic emisslon line at 197.19 nm. The analysts requlres 20 min and is sensltlve to as low as 60 ng of arsenlc Injected to the column.
Arsenic, an important environmental toxicant, enters the environment from a variety of sources, including nonferrous smelting operations, coal-fired power plants, and agricultural organomenical herbicide applications ( I , 2). Arsenic residues are generally reported as total arsenic with no designation of the molecular form of the arsenic. From a biological and toxicological perspective, it is necessary to know the chemical species distribution of the arsenic, since the toxicological properties of these compounds vary widely (3-5). It is well-establishedthat many organisms, including man, chemically transform arsenic ( I ) . Inorganic arsenite and arsenate ions, methylarsonic acid (MAA),dimethylarsinic acid (DMA, cacodylic acid), phenylarsonic acid, several organic esters of arsenic oxyacids, and volatile alkyl arsines are among the known products of these biological transformations (6, 7). It is obvious that an accurate assessment of the toxicological behavior of arsenic is not possible without knowledge of the concentrations, chemical forms, and interactions of the different molecular species comprising the total arsenic load in the environment. Although the toxicology of arsenic is reasonably well-understood, the biochemical aspects of arsenic transformation by mammalian cells has not been determined. We are undertaking an investigation of the fate of arsenic when mammalian cell cultures are exposed to arsenic in different chemical forms. This is a logical extension of our ongoing study of the effects of heavy metals on the cell cycle of mammalian cells in culture (8-11).
Sensitive techniques for the determination of some arsenic species exist and are generally based on the differential generation of arsine (12) or on the column chromatographic separation of arsenic species. The most sensitive of these techniques uses graphite furnace atomic absorption (GFAA) spectroscopy for the determination of arsenic in the column effluents (13-16). The disadvantage of GFAA is that the arsenic measurement is discontinuous. Because GFAA is not a flow method, its use requires that small portions of the column effluent be collected periodically and individually analyzed. Therefore, the arsenic concentration determined for each portion of the effluent is essentially an instantpeous arsenic concentration in the column effluent at the time of sampling. The problems of discontinuous analysis are not readily appreciated until one considers the details of the analysis procedure. The volume collected for the analysis is usually small, on the order of 50-100 pL. The time required for the actual analysis is limited by the recycle rate of the graphite furnace and varies from 30 to 60 s/analysis, depending on the furnace programming sequence selected by the analyst. The column effluent that elutes between samples (e.g., during the time required for the analysis) is generally not analyzed, but instead is sent to waste. The concentration of the eluting arsenic compound may be determined as either the area under the peak generated from the discontinuous plot of arsenic concentration at each sample point vs. elution time or as the summation of the histogram of the individual arsenic concentration determinations. In either case, the sampling frequency must be high enough to ensure that the estimation technique produces as small an error as possible. The number of samples required is then determined by the degree of accuracy desired and the recycle rate of the graphite furnace. If the recycle rate is assumed to be 30 s, and at least five samples are deemed necessary for accurate concentration determination, then a minimum peak base width of 2.5 min will be required. In practice, it is desirble to use 10 or more samples to determine peak areas, and recycle times are more commonly 45 s, so the eluting peaks should have at least a 5-min base width. It must be emphasized that this technique does not cause chromatographicpeak broadening, but rather, requires wider peaks for accurate analysis, a condition not
0003-2700/86/0358-1340$01.50/00 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 7, JUNE 1986
desirable for optimum resolution and analysis speed. So long as there are only a few, well-resolved, arsenic-containing compounds in the mixture to be andyzed, peaks with base widths of 5 or 6 min do not severely lengthen the analysis time, nor do the wide base widths interfere with chromatographic resolution. However, the metabolic conversion of arsenic compounds by free-growing cells gives rise to the possibility of a large number of arsenic compounds resulting from both the metabolism of inorganic and organic arsenic and the binding of arsenic to biological molecules. This potential for a large number of arsenic compounds in the analyses indicates that high-resolution chromatography may be necessary for their separation. High-resolution chromatography requires small base width peaks to allow for maximum individual component resolution. The wider chromatographic peaks necessitated by discontinuous sampling with the potential for decreased chromatographic resolution led us to consider continuous monitoring of the chromatographic effluent. The direct coupling of high-performance liquid chromatography (HPLC) to inductively coupled plasma (ICP) detection (more specifically, inductively coupled argon plasma atomic emission spectroscopy (ICAP-AES)) has been accomplished in several laboratories (17-20)and offered the possibility for continuoue monitoring of arsenic. The technique has been applied to the separation of selected arsenic compounds, using reversed-phase ion pairing (17) and anion exchange (16, 19) techniques. In the former case, multiple solvent steps were required to elute the species; in the latter case, the manufacturer of the column material would not make the column available to us for the ion exchange technique. Therefore, for the investigation of the fate of arsenic in biological systems, we felt that a more readily available column material and a single-solvent linear gradient would allow optimal use of equipment and operator time for the number of analyses envisioned. Because the aqueous chemistry of arsenic principally involves either neutral or anionic species, we developed an anion exchange technique using a linear gradient of ammonium carbonate to separate arsenite, arsenate, methylarsonic acid, and dimethylarsinic acid from neutral arsenic-containing species. The base-line resolution and the steep gradient reported here allow ample opportunity to effect separation of other arsenic species by modification of the elution parameters. Arsenic detection is accomplished by the direct coupling of the HPLC column to an ICP system monitoring a selected arsenic emission line. The analysis is rapid (requiring only 20-min run time, with 45 min between sample injections), sensitive, and ideally suited to automatic sample injection techniques for the analysis of large numbers of samples.
EXPERIMENTAL SECTION Equipment. A Waters Associates Model 721 system controller equipped with dual Waters Model 6000A pumps and a Waters Model U6K injector was used for the HPLC separation. The column was a Waters Z-module containing an 8 mm x 100 mm Radial-PAK cartridge flied with Bio-Rad h i n e x A-27 resin. This spherical anion exchange resin is an 8% cross-linked styrenedivinylbenzene copolymer with attached quaternary amine functional groups and has an average particle size of 15 f 2 pm and an exchange capacity of approximately 1.4 mequiv/mL of resin. The column was prepared by Waters Associates and delivered in the carbonate form. Slurry-packed, 30-cm metal columns prepared in this laboratory have produced resulta identical with the radial-packed column. The column effluent line was connected directly into the nebulizer of a Perkin-Elmer Model 5500 ICP instrument, which served as the detector for the arsenic. The standard cross-flow nebulizer supplied with the ICP was used without modification. The rf power was set at 1250 W, with reflected line power never exceeding 5 W.
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The ICP signal taken at the recorder output of the Model 5500 was fed to a Hewlett-Packard Model 3390A recording integrator for determination of peak areas and retention times. Attentuation, chart speed, integration, and digital filtering parameters of the 3390A were adjusted as required. Chemicals. Ammonium carbonate ((NH,),CO,) (Baker,HPLC grade), sodium arsenite (NaAs02),and sodium arsenate (Na2HAsOJ (Mallinckrodt, reagent grade) were used as received; methylarsonic acid and dimethylarsinic acid were 99% purity (Ansul Co., Weslaco, TX); phenylarsonic acid and triphenylarsine oxide (Alfa Products, Danvers, MA) were used as received; water was deionized and glass-distilled. All solutions were degassed under mild vacuum before use. Caution: All arsenic compounds are toxic to some degree and should be handled with appropriate care. Culture Medium, F-10 culture medium supplemented with 15% cadet calf serum, streptomycin, and penicillin (8)was used to determine the applicability of the method to the types of samples anticipated from the biological study. Procedure. The chromatograph was operated with a flow rate of 1mL/min. The most efficient linear gradient found was 100% water to 0.5 M ammonium carbonate in 15 min, followed by 5-min isocratic elution with 0.5 M ammonium carbonate. After recycle to original conditions, system equilibration generally required 15-20 min. The column and injector were held at 45 O C by immersion in a water bath (HaakeModel W-19). These conditions eluted the arsenate in less than 20 rnin and gave a sample turnaround time of approximately 45 min. Injected arsenic concentrations from 100 ng to 10 pg in sample volumes of 10-500 mL gave satisfactory separations. The column was stored in 0.05 M ammonium carbonate when not in use. The arsenic in the column effluent was quantified by monitoring the arsenic emission line at 197.19 nm. This wavelength was selected for ita favorable signal-to-noiseratio (SIN) and ita relative freedom from spectral interference. The signal-to-noiseratio of the system may be operationally defined as the millivolt output of the photomultiplier resulting from aspiration of a 1ppm arsenic solution divided by the photomultiplier output when a blank is aspirated and should not be confused with the background equivalent concentration, which is a measure of the background signal offset. The arsenic emission line at 193.69 nm, with a signal-to-noise ratio of 14.2, has greater sensitivity than the 197.19-nm line (SIN = 9.3) or the 228.8-nm line (SIN = 5.1), but is strongly affected by the carbon content of the solution due to the carbon emission line at 193.09 nm., The 197.19-nm line is slightly affected by the carbon content of the solution, but the interference was not considered to be sufficient to offset the increase in sensitivity of the 197.19-nm line over the 228.8-nm line. The parameters affecting the ICP sensitivity were optimized for this particular instrument. Gas flows and plasma viewing parameters were varied to give the greatest signal-to-noiseratio possible from a 1 ppm arsenic solution aspirated at 1 mL/min. Plasma gas flow varied from 12 to 18L/min argon; nebulizer flow rate varied from 0.4 to 0.9 L/min argon; auxiliary gas flow varied from 0.6 to 1.6 L/min argon; and viewing height above the load coil varied from 13 to 20 mm. At 197.19 nm, optimal values were found to be 14.5 L/min argon plasma gas flow, 0.5 L/min argon for the nebulizer gas flow, 0.4 L/min argon auxiliary gas flow, and a viewing height 15 mm above the load coil.
RESULTS AND DISCUSSION The most frequently described compounds arising from biological transformation of arsenic are arsenite, arsenate, methylarsonic acid (MAA), dimethylarsinic acid (DMA), phenylarsonic acid, trimethylarsine oxide, and phenylarsine oxide, with the first four compounds being by far the most important metabolites determined to date (7). It was decided to develop the separation method for the main metabolites. The retention times of other compounds would then be determined by using that system. The common metabolites all exist in a charged form in aqueous solutions, so anion exchange chromatography is a logical choice for their separation. Furthermore, neutral arsenic-containing species would elute from the column in the column void volume, and higher
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 7, JUNE 1986
Table I. Effects of Temperature on Capacity Factor and Plate Numbers' temp,OC 25 35 45
arsenite k' N
DMAb
N
k'
MAAC
k'
N
arsenate k' N
3.03 150 4.23 370 7.21 825 10.32 1300 2.55 215 4.09 880 6.79 2460 8.39 2200 2.51 500 4.05 3720 6.24 7400 8.05 10800
k' = Capacity factor = (retention time of compound dead volume elution time)/(dead volume elution time); N = number of theoretical plates = 16(retention time/base width)2. Values are average of five determinations. DMA, dimethylarsinicacid anion. MAA, methylarsonic acid anion. T I M E (MIN)
Flgure 1. Separation of major arsenic metabolites: peak identification, As02- = arsenite, DMA- = dimethylarsinic acid, MAA2- = methylarsonic acM, HAs0,'- = arsenate. Conditions for the separation are as follows: flow rate, 1 mL/min: column, 8 mm X 100 mm Aminex A-27 RadiaCPAK; linear elution gradient from water to 0.5 M (NH4),C03 in 15 min, followed by isocratic flow of 0.5 M (NH,),CO, for 5 min, and recycle to initial conditions: ICP detection at 197.19 nm. charged compounds or compounds that would interact with the column matrix should elute after the arsenate, thus providing a reasonable separation of other potential arseniccontaining compounds, The Aminex resin was selected on the basis of the work of Woolsen et al. (16,21,22). Several carbonate solvent systems were examined, and ammonium carbonate was chosen for several reasons. First, solutions of ammonium carbonate are quite pH stable once their concentration is greater than 0.001 M, which ensures a constant degree of ionization of the various arsenic species. Second, the 8.92 pH of the ammonium carbonate solution is such that the four main arsenic species will exist to a degree as anions in solution. Finally, the predominant ionic species in ammonium carbonate solution are singly charged, i.e., the ammonium and bicarbonate ions, which should lead to a better selectivity for the elution of multiply charged species when using a gradient. A linear gradient from water to 0.5 M ammonium carbonate was selected to elute the four compounds. The pH of the aqueous sample was adjusted to pH 9-10 with one drop of 5 M ammonium hydroxide/mL of sample, thus ensuring that the metabolites were ionized upon injection into the chromatograph. In order to improve the exchange kinetics of the column and give sharper peaks, the temperature of the column and injector was elevated to 45 OC by water bath immersion. Under these conditions, a steep gradient of 15 min could be used to produce the chromatogram shown in Figure 1. The retention order for the compounds is as anticipated, with the singly charged arsenite and DMA eluting before the doubly charged MAA and arsenate. Because retention times of some compounds in ion exchange are quite temperature sensitive, we investigated the temperture dependence of each compound's retention time over a range of temperatures. The results of that study are given in Table I. None of the compounds displayed extreme sensitivity to temperature variations, but the increase in theoretical plates for each compound a t 45 "C indicated a significant advantage in using this temperature. Steep gradients often cause column shock in resin-based packing materials-swelling and shrinking of the resin and pressure fluctuations in the column that may lead to shortened column life and variable column performance. In an attempt to reduce the column shock, we investigated the effects of starting the gradient at nonzero ammonium carbonate levels. The fiial solvent composition and the total gradient time were held constant, while the starting solvent composition was
'
A.0;
HhO;
0.06M
Z
0
0
5
10
15
20
25
TIMECMIN) Flgure 2. Effectsof initial ammonium carbonate concentrations on retention times. The initial (NH4)2C03concentrations for each chromatogram are shown on the rlght. Peak Identification is the same as in Figure 1. varied from pure water to 0.08 M ammonium carbonate. The results show the effects of initial ammonium carbonate concentration on the retention times of the individual species. The retention time of DMA is quite sensitive to the initial concentration of ammonium carbonate and, in fact, moves toward the void position for the system as starting ammonium carbonate concentration is increased (Figure 2). At the same time the other retentiom times decreased slightly, as would be expected from the increased ammonium carbonate concentration. This results in a change in the order of elution of arsenite and DMA as the initial concentration of ammonium carbonate increasgs. The cause for the behavior of DMA is not known, but may involve ion pairing interactions with the solvent. In order to determine the accuracy of precision of the analysis, the ICP response was optimized for viewing height, analytical wavelength, gas flows in the torch, and photomultiplier gain. Photomultipliergain was manually set as high as possible without overloading the amplifier with a 1 ppm arsenic solution in 0.5 M ammonium carbonate aspirated at 1mL/min into the nebulizer. All parameters were checked and reoptimized each day before and after samples were run. The emission value of the 1 ppm standard using a fixed photomultiplier gain was recorded for each day's determinations and did not vary by more than &7% over a period of 2 months. With all ICP parameters optimized, the output signal showed a 1.4-mV peak-to-peak noise level with 0.5 M ammonium carbonate running through the chromatograph and into the ICP. The signal was fed to the Hewlett-Packard 3390A recording integrator, where it was digitally filtered before plotting and integration of peaks. Detection limits were taken to be that concentration of compound such that the maximum signal resulting from the compound elution without
ANALYTICAL CHEMISTRY, VOL. 58,NO. 7, JUNE 1986
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Table 11. Detection Limits and Response Data €or the Major Arsenic Metabolites parameter
arsenite
DMA
MMA
arsenate
detection limit (pg of As injected)O
0.390
0.060
0.057
0.126
linear range (pg of As)* slope of linear response curvec (integrator counts/kg of As)
0.4-10
0.3-6
0.3-6
0.3-5
219.1
232.1
384.1
254.9
6.2
7.7
6.1
8.2
251.9
145.5
confidence interval of sloped intercept of response
83.87
curve corr coeff no. of determinations
0.841 21
0.818 21
0.950 19
-14.60
0.831 25
Detection limit defined as amount injected producing a peak signal-to-noiseratio of 2. bLinearrange may exceed this value, but was not determined at other concentrations. Linear least-squares fit of integrated peak area vs. pg of As injected. dConfidence interval = t u / n , where t = value of the t-distribution for 95% of sample with n - 1 degrees of freedom, u2 = variance of the slope of the linear least-squares fit of the data and, n = number of determinations. (I
signal filtering was twice the background noise. For concentrations above the detection limit, and for peak area measurement, digital filtering was always used; the amount of filtering was determined by the automatic threshold selection feature of the 3390A. Response curves were determined for each compound by injection of serial dilutions of solutions prepared by direct weighing of the compound. Peak areas were plotted against the micrograms of arsenic injected for each of the metabolites. The results of linear least-squares fitting of the data for all compounds are given in Table 11. The data were collected over a period of time, which is reflected in the scatter of the data. The calibration data for any single day were linear and showed correlation coefficients greater than 0.92. Deviation in optical alignment and small variations in gas flow rates for the ICP easily account for the day-to-day variations. One might expect that for any given compound the response of the ICP would be a constant function of the amount of arsenic injected. In our study, peak area response, measured by the slope of the peak area vs. amount of injected plots, is a function of the compound. This response is due in part to different chromatographic peak widths and shapes as well as possible molecular effects. The peaks of arsenite and arsenate ions are wider than those of the organic species, which gives rise to higher detection limits for the inorganic species. The MAA peak has the smallest half-width, thus the lowest detection limit and the highest response slope for the metabolites. Although the arsenate response slope is fairly large, the ability to detect the arsenate peak is limited by the sloping base line on which it lies. This sloping base line is apparent in Figure 1and dominatesthe chromatogram at concentrations near the detection limit. The sloping base line arises from the spectral broadening of the carbon emission line at 199.4 nm associated with increasing carbonate concentration in the eluate. In order to determine whether the observed functional molecular response difference was due to chromatographic or ICP analysis conditions, solutions containing known amounts of each metabolite were introduced into the ICP by a peristaltic pump, bypassing the chromatographic column and pumping system. Once differences in response measurement were taken into account, the response curves generated in this fashion showed concentration dependencies identical to those obtained by passage through the chromatograph. The use of different ammonium carbonate concen-
TIME (MINI Figure 3. Separation of major metabolites from neutral and column
interacting arsenic compounds. Chromatographic conditlons are the same as those in Figure 1. Peak Identification is as follows: TPO = triphenylarsine oxide, As0,- = arsenite; DMA- = dlmethylarsinic acid, = arsenate, PAA2- = phenMAA2- = methylarsonic acid, HASO:ylarsonic acid. trations did not change the ICP response slopes. There appears to be a molecular functional dependence of arsenic response for these compounds using this set of ICP analytical conditions, and this dependence arises from the ICP conditions. The sensitivity of the technique is limited to a large degree by the inability of the nebulizer to admit a greater quantity of sample into the plasma. Typically, only 6-10% of the aspirated sample reaches the plasma (23). Sample introduction techniques that would allow a greater quantity of the nebulized sample to reach the plasma would produce a greater apparent sensitivity, but the ease of connection for the HPLC to the ICP using the existing nebulization apparatus was an important consideration to the development of the method. If future applications require greater sensitivity, alternate sample introduction techniques will be investigated. The sensitivity of the method is also affected by the shape of the peaks, which is not uniform for the four metabolites. The arsenite and arsenate peaks have a broader base and wider half-widths than the organic arsenic compounds. The MAA and DMA peaks show a leading shoulder that could not be resolved by altering the run conditions. The contribution of the shoulder to the total areas of the peak was independent of the sample load for either compound. Introduction of the compounds by themselves indicated that the shoulders were associated with the individual compound and not derived from other metabolites. Using samples from other sources or using more highly purified samples did not change either the location or contribution of the shoulders to the main peaks. We have assumed that the shoulders are derived from the exchange kinetics, since decreasing the operation temperature increases the relative size of the shoulders and begins to resolve them from the main peak. At temperatures above 45 "C we found that gas bubbles from the decomposition of the ammonium carbonate began to interfere with the analysis so that the shoulders could not be merged with the peaks any further by temperature increases. Other buffers with better temperature stability should be investigated for use at increased temperatures in an attempt to decrease the peak width of all of the metabolites. Once the method had been developed, other arsenic-containing compounds were run to determine their retention times. The results of a run containing phenylarsonic acid, triphenylarsine oxide, and the four metabolites are shown in Figure 3. Retention times for the additional two compounds were 17.72 and 1.79 min, respectively. Uncharged arsenic species, such as triphenylarsineoxide, elute at the column void
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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.
Docosahexaenoicacid (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
