A simple and rapid ESI-LC-MS/MS method for simultaneous screening of doping agents in urine samples

Objective: The use of performance enhancing substances is banned in sports by the World Anti-Doping Agency (WADA). Though most prohibited substances can be detected by GC/MS, inclusion of corticosteroids and designer drugs has made it essential to detect these critical doping agents on LC/MS/MS due to their better separation and detection. Materials and Methods: A common extraction procedure for the isolation of acidic, basic and neutral drugs from urine samples was developed. A total of 28 doping drugs were analyzed on API 3200 Triple quadrupole mass spectrometer using C18 column in atmospheric pressure electrospray ionization. The mobile phase composition was a mixture of 1 formic acid and acetonitrile with gradient time period. Results: The method developed was very sensitive for detection of 28 doping agents. The linearity was performed for each drug and the total recovery percentage ranged from 57 to 114. Limit of detection is found to be 0.5 ng/ml for carboxy finasteride and 1-5 ng/ml for other drugs. The method was successfully used to detect positive urine samples of 3-OH-stanozolol, methyl phenidate, mesocarb, clomiphene metabolite and carboxy finasteride. Conclusion: The method developed based on controlled pH extraction method and HPLC-mass spectrometry analysis allowed better identification and confirmation of glucocorticosteroids and a few other drugs in different categories. The validated method has been used successfully for testing of 1000 In-competition samples. The method helped in detection of chemically and pharmacologically different banned drugs in urine in a single short run at a minimum required performance limit set by WADA.