Direct measurement of urinary testosterone and epitestosterone conjugates using high-performance liquid chromatography/tandem mass spectrometry

J Mass Spectrom. 2000 Jan;35(1):50-61. doi: 10.1002/(SICI)1096-9888(200001)35:1<50::AID-JMS912>3.0.CO;2-J.


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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adolescent
  • Adult
  • Calibration
  • Chromatography, High Pressure Liquid / methods*
  • Epitestosterone / chemistry
  • Epitestosterone / urine*
  • Female
  • Gas Chromatography-Mass Spectrometry / methods
  • Glucuronides / chemistry
  • Humans
  • Male
  • Mass Spectrometry / methods*
  • Middle Aged
  • Sulfates / chemistry
  • Testosterone / chemistry
  • Testosterone / urine*


  • Glucuronides
  • Sulfates
  • Testosterone
  • Epitestosterone