Abstract
An ultra performance liquid chromatography–atmospheric pressure chemical ionization–mass spectrometry (UPLC–APCI–MS) method was developed for the separation and quantification of adrenal steroid metabolites from heterologous expression media. Steroids were extracted by liquid–liquid extraction, separated on a Waters UPLC BEH C18 column, ionized by APCI, and detected using a triple quadrupole mass spectrometer in APCI positive mode with single ion monitoring. The incorporation of UPLC enabled the detection of seven structurally closely related steroids at between 5 and 40 ng/ml using run times of 11 min. The adrenal steroidogenic enzyme cytochrome P450 17-hydroxylase/17,20-lyase (CYP17) was expressed in the yeast Pichia pastoris and in nonsteroidogenic COS-1 cells, and used as a model system to evaluate the detection and quantification of adrenal steroid metabolites by UPLC–APCI–MS.
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