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

Abstract

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.

LC‐MS/MS method for the quantification of new psychoactive substances and evaluation of their urinary detection in humans for doping control analysis

“…The sensitivity of the strategy (LLOQ of 1 ng/mL for most of the analytes), the specificity (LOI of 1 ng/mL for most of the analytes), and the fast sample preparation makes this method ideal for its implementation in different application fields such as forensic or doping control. In this last field, the proposed strategy is compatible with the methods commonly used for the detection of other stimulants . The suitability of the methodology has been tested by evaluating the current strategy in the doping control field for four NPS (MDMC, 6‐APB, MEPH, and 2C‐E).…”

Photocatalysis combined with chromatographic methods as a new promising tool in drug metabolism studies – a review

“…For this reason, high pressure pumps and columns packed with <2 μm particle size are being increasingly used. The high separation power is an important property of ultrahigh-performance liquid chromatography (UHPLC), because of the ability to resolve isomeric compounds [66,67]. Additionally, the use of LC coupled with MS system allows the structural identification of the analyzed HPLC-ESI/IT MS HLM (1 h incubation) [64] metabolites.…”

Annual banned‐substance review: analytical approaches in human sports drug testing

“…While several compounds are also considered relevant for doping controls, the enormous comprehensiveness of forensic toxicology methods is not (yet) reflected in sports drug testing assays, arguably due to the different clientele. An LC-MS/MS-based initial testing procedure covering a total of 63 analytes was presented by Monfort et al [200] The method allowed for meeting the MRPL criteria for all compounds (50 ng/mL), and urine samples producing suspicious test results regarding potential isomers (e.g. ephedrine/pseudoephedrine) are subjected to dedicated confirmatory analyses enabling the chromatographic separation of the compounds.…”

“…--2 Diuretics - [182] --S6 Stimulants [63,191,203,205] [189,[192][193][194][196][197][198][199][200]202,204] [24, [186][187][188][189][190][191]195,198,201] S7 Narcotics ----S8 Cannabinoids [208][209][210] [ 95,[208][209][210][211][212][214][215][216][217] - [206][207][208][209][210]213,216] S9 Glucocorticosteroids - [221][222][223][224][225] - [24,...…”