Anal. Chem. 1902, 5 4 , 1079-1082
1079
Liquid Chromatographic Determination of Intact Leukotriene A, M. A. Wynaida, D. R. Morton, Experimental Sciences
R. C.
Kelly, and F. A. Fitzpatrick'
I Unit 7234, Pharmaceutical Research and Development, The Upjohn Company, Kalamazoo, Michigan
4900 1
trans,ll,l4-cis-eicosatetraenoicacid], leukotriene D4[(5S,6R)5-hydroxy-6-(S-cysteinylglycine)-7,9-trans,l1,14-cis-eicosatetraenoic acid], and 5-hydroxy-6,8,11,14-eicosatetraenoicacid (5HETE) were synthesized as described (6-8). Leukotriene B4 [ (5S,12R)-5,12-dihydroxy-6,14-cis,8,10-tralzs-eicosatetraenoic acid], prepared biosynthetically, was kindly supplied by Pierre Borgeat, Lava1 University, Quebec. Arachidonic acid (NuChek Prep, Elysian, MN), acetonitrile, ethanol, and methanol, distilled-in-glass (Burdick and Jackson, Muskegon, MI), AR grade boric acid and sodium borate (J.T. Baker, Philipsburg, NJ), and mone or dibasic potassium phosphate (Mallinckrodt, St. Louis, MO) were used as received. Other chemicals cited were the highest purity commercially available. Apparatus. Samples were chromatographed with an Altex/Beckman Model llOA single piston pump, a Rheodyne 7120 injection valve fitted with 10,20,50,100, or 200 pL loops, a Tracor 970A variable-wavelength spectrophotometric detector, and a Linear 1 mV potentiometric recorder. Microparticulate reversed-phase chromatographic columns, pBondapak c18 (Waters Associates, Milford, MA), Nucleosil C18 (Rainin Instruments, Wobum, MA), Lichrosorb RP-8 (E. Merck Laboratories, Elmsford, NY), and Ultrasphere ODS (Altex-Beckman, Berkeley, CA) were T h e leukotrienes are a new class of naturally occurring, used as received. biologically active lipids that originate from the oxidative High-Performance Liquid Chromatography. Compounds metabolism of polyunsaturated fatty acids, especially aracited in the text were dissolved in 50/50 (v/v) acetonitrile/O.Ol chidonic acid ( I ) . T h e unstable lipid epoxide (5S)-5,6M, pH 10, borate buffer to yield concentrations of 5-100 pg/mI.. oxido-7,9-trans,ll,l4-cis-eicosatetraenoic acid, designated These solutions were chromatographed isocratically on different microparticulate reversed-phase columns. Mobile phases comleukotriene A,, is the pivotal biosynthetic precursor for other posed of 0.01 M, pH 10, borate buffer containing acetonitrile leukotrienes, designated leukotriene B, [(58,12R)-5,12-di(30%-70% v/v) were tested to determine the composition conhydroxy-6,14-cis,8,1O-trans-eicosatetraenoic acid], leukotriene C4 [(5S,6R)-5-hydroxy-6-(S-glutat~onyl)-7,9-trans,ll,l4-c~~- sistent with maximal stability of leukotriene A4 and optimum separation of leukotrienes, arachidonic acid, and its &lipoxygenme eicosatetraenoic acid], leukotriene D4 [ (5S,GR)-5-hydroxymetabolites. Different reversed-phase columns were evaluated 6-(S-cysteinylglycine)-7,9t ram,11,14-cis-eicosatetraenoicacid], under normalized conditions of mobile phase composition and and so forth (2-4). Quantitation of these latter, stable comflow rate (1.0 mL/min) with UV detection a t 215 nm. The depounds has relied mainly on bioassay for the, so-called, slow tector wavelength was adjusted to 235 nm for 5-hydroxyreacting substance of anaphylaxis (SRS-A), now proven t o 6,8,11,14-eicosatetraenoicacid, 270 nm for 5,12-disubstituted consist of a mixture of leukotrienes that contract specific eicosatetraenoic acid, and 280 nm for leukotrienes in experiments to verify compound identity by chromatographic retention and smooth muscles such as the guinea pig ileum. This bioassay selective UV detection. is sensitive; however, its selectivity, versatility, accuracy, and Quantitative Analysis. Leukotriene A, was quantitated by precision are inadequate for many applications. Continued high-performance liquid chromatography with 40/60 (v/v) acedependence on the bioassay is inconsistent with our present, tonitrile/O.Ol M borate buffer, 1.0 mL/min and UV spectrodetailed knowledge of leukotriene structures (Figure 1). photometric detection at 280 nm to maximize sensitivity and We have evaluated t h e high-performance liquid chromaselectivity. Calibration standards were prepared from an ethanolic tography of leukotrienes and other 5-lipoxygenme metabolites stock solution (5.0 mL) of leukotriene A4 lithium salt (500 wg/mL) of arachidonic acid, with the specific aim to develop a quanstabilized with 30 equiv of lithium hyroxide. The stock solution titative method for intact leukotriene A4. Currently, there was stored at -70 "C and held on dry ice except for brief periods a t room temperature to transfer samples for dilution. The apare no quantitative methods, either bioassay or chemical, for pearance of (12RS)-O-ethyl-(5S)-hydroxy-6,8,11,14-eicosatetraethis compound despite its central importance to leukotriene noic acid in the chromatogram indicated the gradual decompoenzymology; attempts at direct quantitation have been limited sition of the stock solution; this decomposition was less than 10% by its instability. The unique adaptability of reversed-phase for 7-10 days. Whenever appropriate, new stock solutions of high-performance liquid chromatography permitted a solution leukotriene A4lithium salt were prepared by saponifying leukot o this problem. We report a quantitative method for intact triene A4 methyl ester (6 pmol) in 8.2 mL of 41/1 (v/v) THF/water leukotriene A, based on high-performance liquid chromacontaining 0.2 mL 1.0 M LiOH. After 16 h at 23 "C under Ar tography with mobile phases that stabilize the allylic 5,6-epthe solvents were removed and the residue was reconstituted in oxide. This method may complement semiquantitative absolute ethanol. Working standards ranging from 50 to 0.50 bioassays or high-performance liquid chromatography assays pg/mL were prepared by diluting the leukotriene A, stock solution with mobile phase immediately prior to injection. Quantitation for stable leukotrienes (5) and clarify the role of these lipids was based on comparative peak height measurements between in allergic and bronchial disorders. standards and unknowns. For 20-pL injections the detector EXPERIMENTAL SECTION sensitivity was varied between 0.02 and 0.32 absorbance units full Materials. Leukotriene A4 methyl ester, and its lithium salt scale, 280 nm, to maximize the peak height response. [ (5S)-5,6-oxido-7,9-tram,ll,14-cis-eicosatetraenoic acid, lithium To determine the stability of leukotriene A, under different salt], leukotriene C4 [ (5S,6R)-5-hydroxy-6-(S-glutathionyl)-7,9- conditions, buffers (10.0 mL) containing various ingredients were Closely related members of a new class of naturally occurrlng llplds, named leukotrlenes, were separated by reversedphase hlgh-performance llquld chromatography using blends of acetonltrlle/O.OI M pH 10 borate buffer as the moblle phase. An alkaline eluent was essentlal because leukotrlene A,, a compound of central Importance to thls new enzymatlc pathway, contains an allyllc epoxide group which can be stablllzed for chromatography and analysis In Its intact form only under bask condltlons. As llttle as 500 ng/mL of Intact leukotrlene A, was determlned with a preclslon of f8-9% relative standard devlatlon, using UV detection at 280 nm. The alkallne moblle phase requlred to stabllize leukotrlene A, was also appllcable to the chromatography of other leukotrlenes and Hpoxygenase metabolites of arachidonic acid. The stablllty and In vttro cellular blosynthesls of leukotrlene A, were characterized by thls method.
0003-2700/82/0354-1079$01.25/0
0 1982 American Chemical Society
1080
ANALYTICAL CHEMISTRY, VOL. 54,
NO. 7, JUNE 1982
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Table I. Capacity Factors for Leukotrienes and 5-Lipoxygenase Products compound
A
leukotriene A, methyl ester 5-hydroxyeicosatetraenoic acid 6 -lactone 5-hydroxyeicosatetraenoic acid methyl ester arachidonic acid 5-hydroxyeicosatetraenoic acid leukotriene A, leukotriene B, leukotriene C, leukotriene D,
25.0 18.1
14.2
