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Development and validation of UPLC-MS/MS method for detecting nootropic misuse in oral fluids: A step towards combating doping in Esports.

Kassim Ali Salum,Meng-Tsang Hsieh,Shao-Hsin Hung,Hung-Chih Chen,William Chih-Wei Chang

Published 2025 in Analytica Chimica Acta

ABSTRACT

BACKGROUND Esports, now officially recognised by the Olympic movement, requires effective anti-doping measures tailored to its unique performance demands. Traditional tests target substances enhancing strength or endurance, yet Esports performance is often influenced by nootropics or "smart drugs"-compounds improving focus, reaction time, and cognition. This study presents a validated sample preparation protocol and UPLC-MS/MS method for detecting nootropics of concern, including substances currently prohibited, under review, or potentially capable of enhancing Esports performance. Oral fluid is employed as a rapid, non-invasive specimen, offering a practical approach for doping control in Esports. RESULTS Extraction and quantification of 13 nootropics and their metabolites in oral fluid, collected via self-administered oral swabs, were successfully optimised. Solid-phase extraction yielded the highest recoveries for analytes across a wide range of physicochemical properties, outperforming both dilute-and-shoot and supported liquid extraction approaches. The analytes were separated within 4 min using a UPLC BEH C18 column. The analytical method was fully validated, demonstrating good precision, accuracy, and lower quantification limits ranging from 0.01 to 50 ng/mL. Most analytes remained stable for one day at room temperature, three days under refrigeration, and one month when frozen. Oral fluid sampling proved effective in detecting nootropics in authentic volunteer samples for up to 4 h post-administration. Detected concentrations included pentoxifylline (7.9-97.2 ng/mL), piracetam (2.8-215.2 μg/mL), and nicergoline (as its main metabolite; 0.6-5.3 ng/mL). Variability in concentrations was influenced by swab positioning and food intake prior to sampling. SIGNIFICANCE This study establishes a robust and practical analytical method utilising oral fluid for the detection of nootropics relevant to doping control in Esports. The approach enables rapid, non-invasive sample collection and reliable quantification, offering the potential for routine pre-game and in-competition monitoring. While the method is validated, further investigation is warranted to define drug excretion profiles and detection windows in oral fluid under real-use conditions.

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