Background: 5α-Reductase (5αR) catalyses conversion of testosterone to its more potent metabolite dihydrotestosterone (DHT) and is inhibited when treating benign prostatic hyperplasia. 5αR has two isozymes; dual isozyme inhibition with dutasteride gives greater DHT suppression than finasteride, which inhibits only 5αR type 2.
Aim: To develop a novel assay to simultaneously analyse testosterone, epitestosterone, DHT, androstenedione, dutasteride and finasteride in human serum.
Methods & Results: Liquid chromatography tandem mass spectrometry was performed on an ABSciex QTRAP® 5500, with a Waters Acquity UPLC. Positive atmospheric pressure chemical ionisation (APCI) mode was selected over electrospray ionisation to avoid matrix-induced ion suppression. Protonated molecular ions were detected for all analytes and source conditions optimised for DHT, the least abundant analyte (source temperature 450°C, curtain gas 40 psi, collision gas medium, nebuliser current 3 µA, source gas 50 psi). Transitions monitored were: testosterone (m/z289-97), epitestosterone (m/z289-109), DHT (m/z291-91), androstenedione (m/z287-97), dutasteride (m/z529-76), finasteride (m/z373-317). Deuterated internal standards selected were d3-testosterone, d3-DHT, d7-androstenedione, d9-finasteride (in-house synthesis). d5-Testosterone and d4-DHT were unsuitable due to loss of A-ring deuteriums during sample processing. Chromatographic separation was optimised on a Kinetex C18 column (150×3 mm; 2.6 µm) at 35°C. A gradient was applied with methanol/ water+0.1% FA, at a flow rate of 0.25 mL/min, permitting baseline resolution of analytes in a 19 minute run. Several extraction methods were trialled including protein crash (Biotage, Phenomenex), phospholipid crash (Waters), liquid-liquid, supported liquid (SLE; Biotage), and solid phase (Phenomenex, Waters). While recovery of analytes from spiked aqueous extracts were >60% with most extraction methods, efficient extraction of endogenous DHT from serum was only possible with SLE.
Conclusion: This method overcomes challenges of extracting analytes with different chemical properties in the same assay and enables detection of androgens in only 200 µl of male serum. The analytical method presented will provide a research assessment tool of compliance with, and biochemical effects of, 5αR inhibitors.
Declaration of interest: There is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding: No specific grant from any funding agency in the public, commercial or not-for-profit sector.