Validation of a Profiling Method for Urinary Neurochemicals as Potential Diagnostic Markers for Brain Doping

Abstract

Brain doping, involving the electrical stimulation of targeted brain regions, has the potential to enhance athletic performance; however, it remains unregulated by the World Anti-Doping Agency (WADA). We argue that brain doping violates the spirit of sport by enabling temporary performance enhancement without effort, and that uncontrolled stimulation intensity may pose risks to athlete health. Accordingly, this study aims to establish a diagnostic strategy for brain doping by focusing on neurochemicals as mediators between brain stimulation and behavioral outcomes. To simulate a brain doping scenario, transcranial direct current stimulation was applied to healthy male volunteers, and urine samples were collected before and after stimulation. To effectively profile 36 neurochemicals present in urine, we optimized sample preparation procedures and liquid chromatography-tandem mass spectrometry conditions. In particular, to enable simultaneous quantification of neurochemicals present at varying concentrations, a rigorous matrix-matched validation was conducted that included the actual concentration ranges of each compound. The limits of quantification for each analyte ranged from 0.002 to 2.61 ng/mL. Linearity (R² > 0.99) was achieved within the actual detectable range in urine, and within this range, intra-day accuracy (%) and precision (%RSD) were confirmed to be 83.2-117.9 and ≤16.5, respectively, while inter-day accuracy and precision were 73.9-112.1 and ≤25.5, respectively. These results demonstrate the effectiveness of the method for profiling neurochemicals in human urine. Based on the profiling results induced by stimulation, both parametric and non-parametric statistical analyses were performed to identify potential markers capable of distinguishing brain doping. We present a strategy for diagnosing brain doping through the identified markers based on neurochemical changes. The results will serve as foundational data for WADA in establishing regulations related to brain doping, promote awareness of brain stimulation in sports, ensure fair competition, and contribute to minimizing potential side effects.