A rapid non‐target screening method for determining prohibited substances in human urine using liquid chromatography/high‐resolution tandem mass spectrometry

Within the complex construct of today's antidoping work, continuously updated routine doping controls, as well as advancements in sampling and analysis have been of particular relevance and importance. New analytes of existing classes of prohibited substances are frequently included into sports drug testing assays, analytical approaches are optimized to allow for better sensitivities, selectivity, and/or faster turnaround times, and research dedicated to addressing analytical issues concerning scenarios of both (potentially) inadvertent doping and new emerging doping agents is constantly conducted. By way of reviewing and summarizing, this annual banned‐substance review evaluates the literature published between October 2018 and September 2019 offering an in‐depth evaluation of developments in these arenas and their potential application to substances reported in WADA's 2019 Prohibited List.

Section: Diuretics and Other Masking Agents Stimulants Narcotics Amentioning

confidence: 99%

Annual banned‐substance review – Analytical approaches in human sports drug testing

Thevis

Kuuranne

Geyer

2020

“…These fall into several categories, each with distinct pharmacological profiles, chemical properties, and year‐by‐year increasingly specific requirements for their detection 2 . With recent technological advances in both liquid chromatography and mass spectrometry (LC‐MS), a great deal of effort has been directed toward development of multiclass initial testing procedures (ITP) to streamline analytical workflows in antidoping laboratories 3–32 …”

Section: Introductionmentioning

confidence: 99%

“…On the other hand, the second group does require preconcentration via liquid–liquid extraction (LLE) or solid phase extraction (SPE), which may or may not be preceded by enzymatic deconjugation. For this reason, multiclass ITPs had initially been developed utilizing triple quadrupole mass spectrometry (LC‐MS/MS) and two complimentary preparations such as dilute‐and‐shoot 3–12 and extraction‐based total fraction assays 13–32 . A comprehensive review has been provided by Thevis et al 33 wherein the former methods would typically cover diuretics, stimulants, narcotics, β‐blockers, and some of the β 2 ‐agonists, whereas the latter would focus on anabolic agents, hormone modulators, fentanyl analogs, synthetic cannabinoids, and glucocorticosteroids.…”

Section: Introductionmentioning

confidence: 99%

High‐throughput liquid chromatography tandem mass spectrometry assay as initial testing procedure for analysis of total urinary fraction

Sobolevsky¹,

Ahrens²

2020

In the recent years, a lot of effort was put into the development of multiclass initial testing procedures (ITP) to streamline analytical workflow in antidoping laboratories. Here, a high‐throughput assay based on liquid chromatography–triple quadrupole mass spectrometry suitable for use as initial testing procedure covering multiple classes of compounds prohibited in sports is described. Employing a 96‐well plate packed with 10 mg of weak cation exchange polymeric sorbent, up to 94 urine samples and their associated positive and negative controls can be processed in less than 3 h with minimal labor. The assay requires a 0.5‐ml urine aliquot, which is subjected to enzymatic hydrolysis followed by solid phase extraction, evaporation, and reconstitution in a 96‐well collection plate. With a 10‐min run time, more than 100 analytes can be detected using electrospray ionization with polarity switching. The assay can be run nearly 24/7 with minimal downtime for instrument maintenance while detecting picogram amounts for the majority of analytes. Having analyzed approximately 28,000 samples, nearly 400 adverse analytical findings were found of which only one tenth were at or above 50% of the minimum required performance level established by the World Anti‐Doping Agency. Compounds most often identified were stanozolol, GW1516, ostarine, LGD4033, and clomiphene, with median estimated concentrations in the range of 0.02–0.09 ng/ml (either as parent drug or a metabolite). Our data demonstrate the importance of using a highly sensitive ITP to ensure efficient antidoping testing.

“…In the past decade, there had been a move towards using high‐resolution mass spectrometry (HRMS) for the detection of target analytes, leveraging on mass accuracy to filter out unwanted background peaks or noise and at the same time having the option to retrospectively analyse the stored data for untargeted drugs 11,13,16,18,19,22–27 . This paper describes a sensitive ultra‐high‐performance liquid chromatography–high‐resolution mass spectrometry (UPLC–HRMS) method performed on equine urine after SLE for the detection of 124 drugs and/or their metabolites.…”

Section: Introductionmentioning

confidence: 99%

A high‐throughput and broad‐spectrum screening method for analysing over 120 drugs in horse urine using liquid chromatography–high‐resolution mass spectrometry

Wong

Chan

Choi

et al. 2020

A high‐throughput method has been developed for the doping control analysis of 124 drug targets, processing up to 154 horse urine samples in as short as 4.5 h, from the time the samples arrive at the laboratory to the reporting deadline of 30 min before the first race, including sample receipt and registration, preparation and instrument analysis and data vetting time. Sample preparation involves a brief enzyme hydrolysis step (30 min) to detect both free and glucuronide‐conjugated drug targets. This is followed by extraction using solid‐supported liquid extraction (SLE) and analysis using liquid chromatography–high‐resolution mass spectrometry (LC–HRMS). The entire set‐up comprised of four sets of Biotage Extrahera automation systems for conducting SLE and five to six sets of Orbitrap for instrumental screening using LC–HRMS. Suspicious samples flagged were subject to confirmatory analyses using liquid chromatography–triple quadrupole mass spectrometry. The method comprises 124 drug targets from a spectrum of 41 drug classes covering acidic, basic and neutral drugs. More than 85% of the targets had limits of detection at or below 5 ng/mL in horse urine, with the lowest at 0.02 ng/mL. The method was validated for qualitative identification, including specificity, sensitivity, extraction recovery and precision. Method applicability was demonstrated by the successful detection of different drugs, namely (a) butorphanol, (b) dexamethasone, (c) diclofenac, (d) flunixin and (e) phenylbutazone, in post‐race or out‐of‐competition urine samples collected from racehorses. This method was developed for pre‐race urine testing in Hong Kong; however, it is also suitable for testing post‐race or out‐of‐competition urine samples, especially when a quick total analysis time is desired.