LC-MS/MS-based assay for free and deconjugated testosterone and epitestosterone in rat urine and serum

Testosterone and epitestosterone are mainly excreted as glucuronides. The aim of this study was to develop and validate a method using liquid chromatography tandem mass spectrometry (LC-MS/MS) to analyse testosterone and epitestosterone in rat serum and urine to assist in vivo studies on steroid metabolism. The method was developed by spiking charcoal stripped rat plasma and urine with the analytes. The developed method was then applied to serum (n=6) and urine samples (n=6) from young male brown Norway rats to determine testosterone and epitestosterone concentrations. The assay showed linearity within quantification range coefficient (r2) values above 0.991. Optimum conditions were determined for the deconjugation of glucuronidated testosterone and epitestosterone along with the internal standard stanozolol D3. Accuracy, precision and extraction recovery for both compounds was satisfactory in both matrices. The method was capable of quantifying 0.250 ng/mL concentrations of testosterone and epitestosterone in 100 μL of serum and urine. The average concentrations of free and deconjugated testosterone and epitestosterone found in the rat samples were: urine–201.68 ± 90.16 ng/mL and 85.37 ± 21.20 ng/mL; serum– 363.40 ± 11.615 ng/mL and 1.75 ± 0.118 ng/mL, respectively. This method is sensitive, specific and reproducible for the determination of free and deconjugated testosterone and epitestosterone in rat serum and urine. The method can be used for in vivo analysis for further investigations of testosterone and epitestosterone concentrations in studies monitoring endocrine dysfunctions and doping.