Simultaneous Determination of Testosterone and its Major Metabolite Epitestosterone in Biological Fluids by HPLC

Abstract

A reversed‐phase high performance liquid chromatographic (HPLC) method is developed and validated for the simultaneous determination of anabolic steroids: testosterone and its major metabolite, epitestosterone. The analytical column, Inertsil ODS‐2, 5 µm, 250×4 mm, was operated at ambient temperature. Isocratic elution was performed using 35% of A=buffer solution 0.11% CH₃COOH‐CH₃COONa 7.5 mmol/L, pH=4 and 65% of B=CH₃CN, at a flow rate of 0.8 mL/min. Inlet pressure was 155 bar. UV detection was performed at 236 nm.

The limit of detection was 0.02 ng per 20 µL injection volume, while linearity held up to 2 ng/µL. p‐Cresol was used as internal standard at a concentration of 2 ng/µL. Validation of the method was performed in terms of accuracy and precision: intra‐day assay (n=6) and inter‐day assay (n=3×5) and was found to be satisfactory, with high accuracy and precision results.

The method was successfully applied to biological fluids after solid phase extraction (SPE) on Nexus cartridges. Recovery rates from blood plasma ranged between 92.0% and 107.5% for testosterone and between 82.5% and 98.8% for epitestosterone, while the analysis of urine provided recovery rates from 85.0% to 108.0% for testosterone and from 85.5% to 103.0% for epitestosterone. The developed method was applied to the analysis of urine samples of one female and four male volunteers.

Keywords:

• Testosterone
• Epitestosterone
• Biological fluids
• Plasma
• Urine
• HPLC

Acknowledgments

The authors would like to thank Professor P. Nikitas (Laboratory of Physical Chemistry, Chemistry Department of University of Thessaloniki) for kindly providing the chromatographic software for data acquisition.