Direct measurement of steroid sulfate and glucuronide conjugates with high-performance liquid chromatography mass spectrometry

Direct detection of several steroid glucuronide and sulfate conjugates was achieved with electrospray reversed-phase HPLC-mass spectrometry. Separation of steroid 17-OH or 5-H epimers conjugated with glucuronide or sulfate could be achieved using gradient elution. Testosterone glucuronide, testosterone sulfate, epitestosterone sulfate and epitestosterone glucuronide were chromatographically resolved, although significant variation in solvent strength was observed between methanol and acetonitrile. Positive ionization mode MS and MS-MS spectra were employed to obtain both quantitative and structural information. Some differences were noted with respect to steroid structure and adduct formation, including significant differences in the stability of epimers in the declustering region of the interface. Negative ionization mode, although having lower limits of detection, did not provide useful structural information in either the MS or MS-MS mode. Using a packed capillary column (300 mu m I.D.), a detection limit of 25 pg was achieved for epitestosterone glucuronide.