Screening of androgen metabolites produced in vitro by high resolution mass spectrometry

Cytochrome P450 aromatase (P450AROM) is a key enzyme in the steroidogenic pathway that catalyses in brain and ovaries the conversion of testosterone to estrogens, and therefore is thought to play an important role in sexual differentiation of neural structures in the developing mammalian brain. To study the role played by AROM in bovine brain a new analytical approach for the identification of androgen metabolite produced in vitro by P450AROM was adopted. High resolution mass spectrometry coupled to liquid chromatography (HPLC-HRMS) was used to evaluate the quali-quantitative metabolic profile of androstenedione and testosterone (AED and TST) incubated with bovine brain subcellular fractions. To this purpose AED and TST (100 M) were incubated in phosphate buffer (pH 7.4) with brain subcellular fractions (2 mg/ml) in presence of 30 mM MgCl2 and 1 mM NADPH regenerating system, in a shaking bath (37°C) for 60 min. The reaction was stopped and the sample was extracted with dichloromethane. Than the organic layer was removed and evaporated to dryness under nitrogen. The extract reconstituted in water/methanol was analysed by HPLC-HRMS. The analytical approach adopted allowed to determine the P450AROM activity through the identification of Estrone (1.08 pmol/mg protein, min -1) produced in the samples incubated with AED. So, the validated HPLC-HRMS method could be a valuable alternative to the quantification of tritiated water (3H2O) produced from radio labelled androgens usually adopted to follow androgen aromatisation in neuronal primary cell culture.