Qualitative detection of diuretics and acidic metabolites of other doping agents in human urine by high-performance liquid chromatography–tandem mass spectrometry: Comparison between liquid–liquid extraction and direct injection

Abstract

Direct injection of urine has gained interest in the field of analytical toxicology, including doping control analysis. However, implementation of a direct urinalysis method for the LC–MS/MS detection of 34 diuretics and 9 other doping agents yielded several analytical problems, which were not observed using a traditional liquid–liquid extraction. Therefore a comparative study was made between liquid–liquid extraction and direct injection. Comparison of validation results showed that the liquid–liquid extraction at pH 7 allows to analyze samples without major drawbacks regarding matrix effects. Hence, good sensitivity was observed and detection limits ranged between 1 and 250 ng/mL for all compounds. In the direct injection approach shifted retention times were observed for several acidic and basic compounds due to unwanted matrix effects. This shift was reduced by a 25-fold dilution of the urine samples. Besides the improved retention time stability the diluted samples also exhibited lower ion suppression than the undiluted ones. After 25-fold dilution, detection limits ranged between 10 and 250 ng/mL for all compounds. Since these detection limits are at or below the minimum required performance level, imposed by the World Anti-Doping Agency, the method could be applied to routine anti-doping analysis. Samples, previously declared positive, were reanalysed using both the liquid–liquid extraction and direct injection. With both techniques all 26 samples were found to be positive, showing the applicability of direct injection for the analysis of diuretics.