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.
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.
Deventer
K
K
Doping control laboratory (DoCoLab), Ghent University (UGent), Department of Clinical Chemistry, Microbiology and Immunology, Technologiepark 30, B-9052 Zwijnaarde, Belgium. Koen.Deventer@UGent.be
Pozo
O J
OJ
Van Eenoo
P
P
Delbeke
F T
FT
D000097:Acetonitriles / Q000737:chemistry; D005591:Chemical Fractionation / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004232:Diuretics / Q000378:metabolism / Q000652:urine*; D004300:Doping in Sports; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D016482:Urinalysis / Q000379:methods
D000097:Acetonitriles; D004232:Diuretics; C032159:acetonitrile