Quantification of testosterone and epitestosterone in biological samples by capillary electrophoresis with immunoaffinity extraction.
Testosterone is one of the most common doping drugs abused by athletes. Therefore, it is necessary to develop a sensitive and simple method to monitor testosterone and its epimer epitestosterone. An off-line immunoaffinity extraction followed by capillary electrophoresis for simultaneous determination of testosterone and epitestosterone has been described in this paper. Anti-epitestosterone monoclonal antibody which is specific to both testosterone and epitestosterone had been prepared and immobilized on a Sepharose 4B stationary phase. The immunoaffinity column was used for sample cleanup, extraction and preconcentration. After elution and reconstitution, testosterone and epitestosterone in the sample were separated and quantified by micellar electrokinetic chromatography(MEKC) using the borate buffer (200 mM borate, pH 8.7) containing 40 mM sodium cholate as a chiral selector. The immunoaffinity column was evaluated in different parameters such as the retention mechanism, selectivity, binding capacity, elution protocol, and reusability. The separation of these two compounds by MEKC was also optimized. Limit of detection for testosterone and epitestosterone were 5 and 23 ng mL(-1), respectively. It was satisfactory to apply this method to analyze testosterone and epitestosterone in spiked urine sample with the recoveries from 78% to 109%.
Chen
Hong-Xu
HX
Beijing National Laboratory for Molecular Sciences, BNLMS, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Institute of Analytical Chemistry, College of Chemistry, Peking University, Beijing 100871, China.
Deng
Qin-Pei
QP
Zhang
Li-Wei
LW
Zhang
Xin-Xiang
XX
D000911:Antibodies, Monoclonal; D002846:Chromatography, Affinity; D020374:Chromatography, Micellar Electrokinetic Capillary / Q000379:methods; D019075:Electrophoresis, Capillary / Q000379:methods*; D004845:Epitestosterone / Q000032:analysis* / Q000652:urine; D006801:Humans; D013739:Testosterone / Q000032:analysis* / Q000652:urine
D000911:Antibodies, Monoclonal; D013739:Testosterone; D004845:Epitestosterone