DETERMINATION OF STEROIDS BY LIQUID-CHROMATOGRAPHY MASS-SPECTROMETRY

On-line atmospheric pressure chemical ionization (APCI) and electrospr ay ionization (ESI) liquid chromatography/mass spectrometry (LC/MS) we re evaluated for the analysis of a variety of steroids. Steroids were classified into three major groups based on the spectra and the sensit ivities observed: (I) those containing a 3-one, 4-ene functional group , (LI) those containing at least one ketone group without conjugation, and (III) those containing hydroxy group(s) only. In the APCI mode, t he best sensitivity and the lowest detection limit for all three group s were obtained by using a mobile phase consisting of methanal and 1%- 2% acetic acid in water. The APCI spectra were characterized by MH+, M H+-H2O, MH+-2H(2)O, etc., with the degree of H2O loss being compound d ependent: group I steroids produced stable MH+ and group III steroids showed extensive water loss. In the electrospray mode the best sensiti vity and the lowest detection limit for the first two groups were obta ined when pure methanal and water were used as the mobile phase. This condition produced abundant stable MNa+ due to ubiquitous sodium. Dete ction limits in the 5-15 pg range can be easily achieved using ESI LC/ MS. Addition of ammonium acetate or use of acetonitrile in the mobile phase, common in the LC/MS analysis of steroids, decreased the sensiti vity for the group I and II steroids and thus should be avoided. For g roup III steroids, the detection limit can be improved by the addition of acetic acid to the mobile phase. (C) 1997 American Society for Mas s Spectrometry.