Synthesis of pyridine-carboxylate derivatives of hydroxysteroids for liquid chromatography–electrospray ionization-mass spectrometry

Abstract

Synthesis and liquid chromatography-electrospray ionization-mass spectrometric (LC–ESI-MS) behaviors of the picolinoyl, 6-methylpicolinoyl, nicotinoyl, 2-methoxynicotinoyl and isonicotinoyl derivatives of the hydroxysteroids estrone, estradiol, 3β-hydroxyandrost-5-en-17-one (dehydroepiandrosterone) and testosterone in positive mode were investigated. Each steroid was converted to the corresponding pyridine-carboxylate derivative by the acyl chloride method or the mixed anhydride method using the corresponding free acids and 2-methyl-6-nitrobenzoic anhydride; in each case, the latter method principally gave a better yield. The pyridine-carboxylate derivative of each steroid exhibited a clear single peak in liquid chromatography with a reversed phase column and CH 3CN–0.1% CH 3COOH as a mobile phase. The positive-ESI-mass spectra of the picolinoyl, 6-methylpicolinoyl and 2-methoxynicotinoyl derivatives showed a predominance of [M+H] +, whereas [M+H+CH 3CN] + was observed with high intensity in the nicotinoyl and isonicotinoyl derivatives. Even in the case of estradiol, with its two hydroxyl groups, a single charged ion of [M+H] + or [M+H+CH 3CN] + was observed in the positive-ESI-mass spectrum of each derivative. The results revealed that picolinoyl derivatization is a simple and versatile method suitable for the sensitive and specific determination of hydroxysteroids by LC–ESI-MS (selected reaction monitoring).