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

A method for the analysis of neutral oxosteroids by electrospray mass spect rometry is described. The oxosteroids are converted into their oximes by tr eatment with hydroxyammonium chloride in aqueous methanol, Intense peaks co rresponding to protonated oxime molecules are observed in nano-electrospray mass spectra, The detection limits for the oximes of progesterone, pregnen olone and dehydroepiandrosterone were 2.5, 5 and 25 pg/mu L, respectively, approximately 20 times lower than for the underivatised steroids. The signa l intensities were proportional to the concentration of the steroids in the range of 500 to 2.5 pg/mu L. Fragmentation by collision-induced dissociati on (CID) was studied using oximes of 28 model steroids carrying an oxo grou p at C-3, C-17 or C-20, Some of the steroid oximes were labelled with deute rium or N-15. Fragment ions were observed which yielded useful structural i nformation. 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 un substituted at C-21, C-17 or C-16 produced a characteristic ion at m/z 86 c ontaining the side chain, C-16 and C-17, Protonated oximes of steroids cont aining only a 17-oxo group gave fewer ions of diagnostic value. Coupled wit h the selective isolation of steroid oximes from a biological matrix this m ethod of derivatisation and CID may be used for the analysis of neutral oxo steroids in biological samples. Copyright (C) 2000 John Wiley & Sons, Ltd.