Method validation for routine screening of 145 WADA prohibited substances by LC-MS/MS

Abstract

LC-MS/MS has been recently a technique essential for analysis of doping substances and currently implemented in all WADA accredited laboratories. The protocols employed for sample preparation and equipment set up vary among different laboratories. In this study, a method has been developed and implemented for analysis of 145 substances in the WADA prohibited list by using the liquid chromatography/triple quadrupole mass spectrometry and multiple reaction monitoring/ electrospray ionization in positive and negative ion modes. The prohibited substances in urine sample were initially prepared by two consecutive liquid-liquid extractions (LLE) with methyl tert- butyl ether (MTBE)(pH 9 and 3) following enzyme hydrolysis. The extracted samples were separately analyzed on two different reverse-phase columns, C-18 column for diuretics, narcotics, beta-blockers, beta2-agonists, corticosteroids, anabolic steroids, aromatase inhibitors, SERM and C-8 column for stimulants. The method validation demonstrated the limit of detection (LOD 0.1-100 ng/ml), and retention time reproducibility (CV% < 1) were appropriate for routine screening the presence of WADA prohibited substances.