Development of a Validated HPLC Method for the Simultaneous Determination of Anabolic Steroids in Biological Fluids

Abstract

A reversed phase high performance liquid chromatographic (HPLC) method is developed and validated for the simultaneous determination of anabolic steroids: testosterone (TES), epitestosterone (EPI), and nandrolone (NAN). The analytical column, Inertsil C₈, 5 µm, 250 × 4 mm, was operated at ambient temperature. Isocratic elution was performed using a mixture of 50% buffer solution CH₃COOH 0.11% –CH₃COONa 7.5 mmol/L, pH = 4, 45% CH₃CN and 5% CH₃OH, at a flow rate of 1.1 mL/min. UV detection was performed at 238 nm.

The detection limits of the method were 2.4 ng for NAN, 3.6 ng for TES, and 2.6 ng for EPI in blood plasma, and 2.7 ng for NAN, 1.1 ng for TES, and 3.8 ng for EPI in urine, per 20 µL injection volume. Alprazolam 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 × 6) and was found to be satisfactory, with high accuracy and precision results.

Sample preparation involved solid phase extraction on Nexus cartridges with high recoveries. The developed method was successfully applied to the analysis of urine samples of one female and 9 male volunteers.

Keywords:

• Anabolic steroids
• Blood plasma
• Epitestosterone
• Nandrolone
• Testosterone
• Urine

ACKNOWLEDGMENT

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.