Simultaneous Measurement of Serum Testosterone and Dihydrotestosterone by Liquid Chromatography–Tandem Mass Spectrometry

BACKGROUND: Recent reports have described inherent problems with androgen immunoassays compared with mass spectrometry analyses. METHODS: We developed a method for measuring serum testosterone (T) and 5alpha-dihydrotestosterone (DHT) simultaneously via liquid-liquid extraction followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with positive-mode electrospray ionization. RESULTS: The DHT and T calibrators showed a linear response from 0.069 nmol/L to 34.4 nmol/L and 69.3 nmol/L, respectively. T interference in the DHT assay and vice versa were negligible. Within- and between-run imprecision values were <5% for both analytes. Percent recoveries of T and DHT spiked into samples at concentrations spanning the calibration curve were 100%-113% and 98%-107%, respectively. The lower limit of quantification was 0.069 nmol/L for both steroids. Serum T concentrations measured by LC-MS/MS were different from those obtained by RIA, especially at lower T concentrations. Serum DHT concentrations measured by LC-MS/MS were markedly lower than those generated by RIA because of the nonselectivity of the RIA without chromatography. The reference intervals (mean +/- 2 SDs) determined for T and DHT were 9.2-33.7 nmol/L and 0.47-2.65 nmol/L, respectively, for 113 healthy adult men and 0.33-2.02 nmol/L and 0.09-0.91 nmol/L, respectively, for 133 healthy premenopausal women. CONCLUSIONS: We have developed and validated a selective and precise method for simultaneous measurements of serum T and DHT that can be adopted for routine measurements of these androgens in health and disease in men and women.