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Abstract
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%.