LC-MS/MS-Based Assay for Free and Deconjugated Testosterone and Epitestosterone in Rat Urine and SerumCarl Jenkinson1, Nawed IK Deshmukh2, Iltaf Shah1, Gergely Zachár3, Andrea D Székely3, Andrea Petroczi1 and Declan P Naughton1*
- *Corresponding Author:
- Prof. Declan Naughton
School of Life Sciences, Kingston University
Kingston upon Thames, London KT1 2EE, UK
Tel: +44 208 547 7097
Fax: +44 208 547 7562
E-mail: [email protected]
Received date: December 16, 2013; Accepted date: January 15, 2014; Published date: January 17, 2014
Citation: Jenkinson C, Deshmukh NIK, Shah I, Zachár G, Székely AD, et al. (2014) LC-MS/MS-Based Assay for Free and Deconjugated Testosterone and Epitestosterone in Rat Urine and Serum. J Anal Bioanal Tech S5: 006. doi: 10.4172/2155-9872.S5-006
Copyright: © 2014 Jenkinson C, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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.