title: Direct measurement of urinary testosterone and epitestosterone conjugates using high-performance liquid chromatography/tandem mass spectrometry.
abstract: Measurement of the ratio of testosterone (T) and epitestosterone (E) in urine has been used as an indication of 'natural' steroid supplementation for a decade. The direct measurement of the glucuronide and sulfate conjugates of testosterone and epitestosterone by high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) should resolve a number of issues regarding unusual metabolism due to either genetic disposition or attempts to avoid detection of abuse. Determination of nanomoles per liter (0.1 ppb) concentrations of analytes in a complex biological matrix by HPLC/MS/MS is complicated by sample matrix-specific ion suppression during ESI. Deuterated internal standards of all compounds were used to overcome the effects of suppression. Comparison of the HPLC/MS/MS method with a two-part gas chromatographic/mass spectrometric method showed statistical equivalence in urine samples. Analysis of urine samples with elevated T-glucuronide to E-glucuronide ratios did not show that a significant number could be explained by an elevated excretion of epitestosterone sulfate. The HPLC/MS/MS method was also used further to characterize genetic and metabolic factors that give rise to unusual T/E ratios.
authors.affiliation: Athletic Drug Testing and Toxicology Laboratory, Department of Pathology and Laboratory Medicine, Indiana University Medical Center, Medical Science Building A-128, 635 Barnhill Drive, Indianapolis, Indiana 46202-5120, USA.
authors.forename: D J
authors.initials: DJ
authors.lastname: Borts
authors.forename: L D
authors.initials: LD
authors.lastname: Bowers
mesh_terms: D000293:Adolescent; D000328:Adult; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004845:Epitestosterone / Q000737:chemistry / Q000652:urine*; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D020719:Glucuronides / Q000737:chemistry; D006801:Humans; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008875:Middle Aged; D013431:Sulfates / Q000737:chemistry; D013739:Testosterone / Q000737:chemistry / Q000652:urine*
chemical_list: D020719:Glucuronides; D013431:Sulfates; D013739:Testosterone; D004845:Epitestosterone