Rapid HPLC-Electrospray Tandem Mass Spectrometric Assay for Urinary Testosterone and Dihydrotestosterone Glucuronides from Patients with Benign Prostate Hyperplasia

Extract

The development of benign prostatic hyperplasia (BPH) is dependent on androgens, primarily dihydrotestosterone (DHT) (1). To estimate the activity of 5α-reductase, which catalyzes the conversion of testosterone to DHT, testosterone and DHT have been quantified in biological samples (2)(3)(4). Because of their lipophilic properties, they are usually found in the conjugated form, i.e., linked to a hydrophilic sulfuric moiety or β-glucuronic acid, which are excreted mainly (>95%) in human urine. Despite reports of a close association between BPH and testosterone and DHT (1)(2)(3)(4), to our knowledge, there is no published simultaneous quantitative data for these metabolites as their glucuronides in the urine of patients with BPH.

The major problem associated with quantification of total steroids is incomplete hydrolysis, attributable mainly to the matrix effects of urine on the efficiency of enzymatic hydrolysis, which reduces the reproducibility of the assay (5)(6). Given that it is often necessary to analyze many samples in a short time with good sensitivity and reproducibility, system throughput is a critical issue for many clinical mass spectrometry (MS) groups. In continuation of studies of steroid profiles in diseases related to steroid enzymes (7)(8), we here demonstrate a sensitive and precise HPLC-tandem MS method for quantification of testosterone and DHT glucuronides by use of a three-column, two-switching valve system and the presence of the indicative biomarker in urine samples from BPH patients.