Determination of 11α-hydroxy-9,15-dioxo-2,3,4,5,20-pentanor-19-carboxyprostanoic acid and 9α,11α-dihydroxy-15-oxo-2,3,4,5,20-pentanor-19-carboxyprostanoic acid by gas chromatography/negative ion chemical ionization triple-stage quadrupole mass spectrometry

Abstract

11α-Hydroxy-9,15-dioxo-2,3,4,5,20-pentanor-19-carboxyprostanoic acid (PGE-M) and 9α,11α-dihydroxy-15-oxo-2,3,4,5,20-pentanor-19-carboxyprostanoic acid (PGF-M) in urine were determined in an isotope dilution assay by gas chromatography/triple-stage quadrupole mass spectrometry. After addition of the 2H 7-labeled internal standard, O-methylhydroxylamine hydrochloride in acetate buffer was added either directly (PGE-M) or after standing overnight at pH 10 (PGF-M) to form the methoxime. The sample was acidified to pH 2.5 and PGE-M and PGF-M were extracted with ethyl acetate/hexane. Then the prostanoids were derivatized to the pentafluorobenzyl ester and purified by thin-layer chromatography and the trimethylsilyl ether was formed. The products were quantified by gas chromatography/triple-stage quadrupole mass spectrometry. For PGE-M, the fragment ions m z 349 and m z 356 ( 2H 7 standard) (daughter ions of m z 637 and m z 644 ( 2H 7 standard)) were used. The results of the PGE-M assay were compared with those of an assay using the [ 2H 3]methoxime as the internal standard. For determination of PGF-M, the daughter ions m z 484 and m z 491 ( 2H 7 standard) with the parent ions m z 682 and m z 689 ( 2H 7 standard) were chosen.