Enzymic conversion of 3β-hydroxy-5-ene-steroids and their sulfates to 3-oxo-4-ene-steroids for increasing sensitivity in LC-APCI-MS

A method of increasing the sensitivity of 3β-hydroxy-5-ene (Δ5)-steroids in liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS) based on the structural conversion by cholesterol oxidase (ChO) was demonstrated. The Δ5-steroids were rapidly converted to their 3-oxo-4-ene (Δ4)-forms by the treatment with ChO and the obtained Δ4-forms provided 3-14-fold higher sensitivity compared to intact steroids in the positive-APCI-MS. This enzymic conversion method was also applied to the sulfated conjugates of Δ5-steroids after solvolysis. The method enabled the detection of trace levels of dehydroepiandrosterone and androstenediol 3-sulfate in human serum, which could not be detected by the usual LC-MS.