Profiling of bisenoic prostaglandins and thromboxane B 2 in bronchoalveolar fluid from the lower respiratory tract of human subjects by combined capillary gas chromatography-mass spectrometry

Abstract

Methods for the profiling of prostaglandin D 2 (PGD 2), prostaglandin E 2 (PGE 2), prostaglandin F 2α (PGF 2α), 9α, 11β-trihydroxyprosta-5z,13E-dien-1-oic acid (9α,11β-PGF 2), 6-keto-prostaglandin F 1α (6kPGF 1α), and thromboxane B 2 (TxB 2) in bronchoalveolar lavage (BAL) fluids from human subjects by combined capillary gas chromatography-mass spectrometry are described. Alquots (5 ml) of BAL fluid obtained using a standardized lavage protocol were extracted on octadecylsilyl silica cartidges after addition of 0.8 to 2.0 nanograms of tetradeuterated analogs of PGE 2, PGF 2α, and 6kPGF 1α as internal standards. Eluted analytes and internal standards were prepared for vapor phase analysis by sequential reactions resulting in the formation of methyloxime-pentafluorobenzyl ester-trimethylsilylether derivatives. The derivitized analytes were detected by simultaneous monitoring of ions at six different masses characteristic for each of the derivatized prostanoids. The samples were of adequate purity for identification and quantitation of each of the prostanoids with detection limits of 0.1 to 0.2 picograms of each analyte per milliliter of BAL fluid. The time required for analysis of each sample was approximately 30 minutes. Standard curves of unlabeled species of the six prostanoids extracted after addition to BAL fluid were linear over a range from subpicogram to nanogram quantities. The differences between the amounts of prostanoid added and the amount of prostanoid measured were typically less than 19%, and the intra-assay coefficients of variation for repeated measurement of a single sample were less than 20%. PGE 2, PGD 2, PGF 2α, and TxB 2 were detectable in BAL fluids from normal subjects with levels of each of these compounds being less than 2.6 picograms/ml. BAL fluids from patients with lung disease presented qualitative and quantitative profiles of prostanoids markedly different than those from normal subjects. These analytical methods provide a basis for in vivo comparisons of prostanoid profiles in the lower respiratory tract of man and should be readily adaptable for use in a variety of clinical studies.