Analysis of oxosteroids by nano-electrospray mass spectrometry of their oximes

A method for the analysis of neutral oxosteroids by electrospray mass spectrometry is described. The oxosteroids are converted into their oximes by treatment with hydroxyammonium chloride in aqueous methanol, Intense peaks corresponding to protonated oxime molecules are observed in nano-electrospray mass spectra, The detection limits for the oximes of progesterone, pregnenolone and dehydroepiandrosterone were 2.5, 5 and 25 pg/mu L, respectively, approximately 20 times lower than for the underivatised steroids. The signal intensities were proportional to the concentration of the steroids in the range of 500 to 2.5 pg/mu L. Fragmentation by collision-induced dissociation (CID) was studied using oximes of 28 model steroids carrying an oxo group at C-3, C-17 or C-20, Some of the steroid oximes were labelled with deuterium or N-15. Fragment ions were observed which yielded useful structural information. Upon CID, protonated oximes of 3-oxo-Delta(4)-steroids produced abundant ions by cleavage through the B-ring and by loss of the side chain, while protonated oximes of saturated 3-oxosteroids did not give abundant ions by cleavage through the B-ring, Protonated oximes of 20-oxosteroids unsubstituted at C-21, C-17 or C-16 produced a characteristic ion at m/z 86 containing the side chain, C-16 and C-17, Protonated oximes of steroids containing only a 17-oxo group gave fewer ions of diagnostic value. Coupled with the selective isolation of steroid oximes from a biological matrix this method of derivatisation and CID may be used for the analysis of neutral oxosteroids in biological samples. Copyright (C) 2000 John Wiley & Sons, Ltd.