Screening method for stimulants in urine by UHPLC-MS/MS: identification of isomeric compounds

A fast screening method for the detection of more than 60 stimulants in urine was developed. The method consisted of a dilution of the urine (1:5 v/v) and analysis by ultra high performance liquid chromatography coupled to tandem mass spectrometry, using a C18 column (1.7 µm particle size), a mobile phase containing deionized water and acetonitrile with formic acid, and gradient elution. The chromatographic run time was 5 min. The detection was performed in positive mode electrospray ionization, monitoring one or two specific ion transitions for each analyte. Appropriate repeatability was obtained, with relative standard deviation (RSD) values below 25% for most of the analytes. Regarding intermediate precision, estimated during routine work, higher RSDs were obtained, probably due to between-day differences in the status of the mass spectrometer and in the chromatographic system. Matrix effect ranged from 60 to 255% with RSD lower than 35% for the majority of compounds. Despite the matrix effect observed, the signal/noise ratio of the analytes spiked at 50 ng/mL was greater than three in all tested samples, allowing a correct detection of all substances at the minimum required performance levels required by the World Anti-Doping Agency and demonstrating the suitability of the method. The method was tested in administration study samples and satisfactorily in operation for more than one year with routine doping samples. The presence of isomeric stimulants with closely similar chromatographic and/or mass spectrometric properties did not allow the unequivocal identification of these compounds after the first analysis. Different possibilities for separation and identification of isomeric compounds are presented.