Preventive doping control screening analysis of prohibited substances in human urine using rapid‐resolution liquid chromatography/high‐resolution time‐of‐flight mass spectrometry

Abstract

Unification of the screening protocols for a wide range of doping agents has become an important issue for doping control laboratories. This study presents the development and validation of a generic liquid chromatography/time-of-flight mass spectrometry (LC/TOFMS) screening method of 241 small molecule analytes from various categories of prohibited substances (stimulants, narcotics, diuretics, beta(2)-agonists, beta-blockers, hormone antagonists and modulators, glucocorticosteroids and anabolic agents). It is based on a single-step liquid-liquid extraction of hydrolyzed urine and the use of a rapid-resolution liquid chromatography/high-resolution time-of-flight mass spectrometric system acquiring continuous full scan data. Electrospray ionization in the positive mode was used. Validation parameters consisted of identification capability, limit of detection, specificity, ion suppression, extraction recovery, repeatability and mass accuracy. Detection criteria were established on the basis of retention time reproducibility and mass accuracy. The suitability of the methodology for doping control was demonstrated with positive urine samples. The preventive role of the method was proved by the case where full scan acquisition with accurate mass measurement allowed the retrospective reprocessing of acquired data from past doping control samples for the detection of a designer drug, the stimulant 4-methyl-2-hexanamine, which resulted in re-reporting a number of stored samples as positives for this particular substance, when, initially, they had been reported as negatives.

Comparison between triple quadrupole, time of flight and hybrid quadrupole time of flight analysers coupled to liquid chromatography for the detection of anabolic steroids in doping control analysis

“…The main reason for this absence can be the high sensitivity required in the steroid analysis. Around 30% of the steroids included in some multi-class method could not be detected by TOF at the required levels [29,30]. These sensitivity limitations were not present in a method developed by Georgakopoulos et al for the detection of 64 doping agents including 21 anabolic steroids [14].…”

“…Hence, some approaches have been recently developed for the urinary detection of multi-class doping agents by TOF related instruments [14,[28][29][30]. However, only a few steroids are normally included in these methods.…”

Colorimetric detection and chromatographic analyses of designer drugs in biological materials: a comprehensive review

“…The majority of the drugs have hallucinogenic effects. Recently, steroid designer drugs have been detected in urine samples obtained from athletes [8][9][10][11][12][13].…”

“…In a typical screening method [13], urine hydrolyzed with b-glucuronidase from Escherichia coli was adjusted to pH 9-10 with a mixture of sodium hydrogen carbonate and sodium carbonate (10:1, w/w). LLE with diethyl ether was performed using sodium sulfate as a salting-out agent.…”

The Chromatographic Role in High Resolution Mass Spectrometry for Non-Targeted Analysis

“…To this point, most studies have focused on the targeted screening for compounds based on the accuracy of the mass measurement and resultant chemical formulae. This type of approach has been applied in screening of food [1][2][3][4], environmental applications [5][6][7], and forensic applications [8][9][10], where large numbers of compounds are assessed. An extension of this work is the non-targeted workflow where compounds outside of a target list are searched (i.e., data mining) and chemical formulae are generated from the accurate mass information [11].…”