Mass spectrometric analysis of androstan-17β-ol-3-one and androstadiene-17β-ol-3-one isomers

Abstract

Mass spectrometric identification and characterization of steroids using electrospray ionization and tandem mass spectrometry has advantages in drug testing and doping control analysis attributable to limitations of gas chromatography followed by electron ionization mass spectrometry. Steroids with an androstadiene-17␤-ol-3-one nucleus and double bonds located either at C-1 and C-4, C-4 and C-9, or C-4 and C-6 were used to determine characteristic fragmentation pathways. Diagnostic dissociation routes are proposed using deuterium labeling, MS 3 experiments, and analyses of structurally closely related compounds. Steroids such as boldenone (androst-1,4-diene-17␤-ol-3-one) produced characteristic product ions at m/z 121, 135, and 147. Compounds with double bonds at C-4 and C-9 generated abundant product ions at m/z 145 and 147. Conjugated double bonds at C-4 and C-6 gave rise to an intense and characteristic signal at m/z 133. Stereochemical differentiation between 5␣-and 5␤-isomers of androstan-17␤-ol-3-ones was possible because of significant differences in relative abundance of product ions generated by elimination of acetone from ␣,␤-saturated 3-keto steroids. T he identification of natural and synthetic compounds using mass spectrometry is of paramount importance for documentation of use of illicit compounds by amateur and professional athletes. Elucidation of dissociation pathways of illegal substances using a variety of ionization and/or dissociation conditions is a robust technique for classification and identification of drugs and related compounds [1].Mass spectrometric studies on fragmentation of steroids using electron ionization (EI) were performed by Djerassi, Budzikiewicz, and coworkers [2][3][4][5][6][7][8]. Detailed studies have also been made employing steroid derivatives such as trimethylsilylated or acetylated compounds [9 -18]. Liquid chromatography combined with mass spectrometers with an atmospheric pressure ionization interface enables the detection and characterization of steroids that complement conventional gas chromatographic-mass spectrometric (GC-MS) assays, especially for compounds that are less suitable for GC-MS owing to thermo-labile properties [19]. A very recent example is the so-called "designer steroid" tetrahydrogestrinone 9, THG) that has been identified in several doping control samples by means of methods based on liquid chromatography (LC) and tandem mass spectrometry (MS/MS).To determine steroid structures, ESI and collisioninduced dissociation (CID) were investigated. We describe fragmentation patterns of the isomers androst-1,4-diene-17␤-ol-3-one (1), androst-4,9 (11)-diene-17␤-ol-3-one (6), androst-4,6-diene-17␤-ol-3-one (9), and their analogs as well as 5␣-androstan-17␤-ol-3-one (12), 5␤-androstan-17␤-ol-3-one (13), and related derivatives (Scheme 1). Using stable isotope derivatives and H/Dexchange experiments, fragmentation pathways are proposed that provide classification of steroids by characteristic product ions generated from protonated molecules that include ...

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