Received on January 22, 2008
Accepted on August 27, 2008

Endocrinology and Metabolism

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

¹ General Clinical Research Center; Division of Endocrinology and Metabolism, Harbor–UCLA Medical Center and Los Angeles Biomedical Research Institute, Torrance, CA
² General Clinical Research Center; Division of Endocrinology and Metabolism, Harbor–UCLA Medical Center and Los Angeles Biomedical Research Institute, Torrance, CA, and Department of Pediatrics, Harbor–UCLA Medical Center and Los Angeles Biomedical Research Institute, Torrance, CA
³ Department of Medicine, Harbor–UCLA Medical Center and Los Angeles Biomedical Research Institute, Torrance, CA
⁴ General Clinical Research Center; Division of Endocrinology and Metabolism, Harbor–UCLA Medical Center and Los Angeles Biomedical Research Institute, Torrance, CA, and Department of Medicine, Harbor–UCLA Medical Center and Los Angeles Biomedical Research Institute, Torrance, CA

^* To whom correspondence should be addressed. E-mail: wang{at}labiomed.org.

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 5-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.