Screening method of mildronate and over 300 doping agents by reversed-phase liquid chromatography-high resolution mass spectrometry

Considering the huge amount of substances associated with athletic performance improvement, current doping control analysis requires a comprehensive screening method, which leads to the detection of prohibited substances of different physico-chemical properties. This comprehensiveness associated with instrumental approaches based on high resolution mass spectrometry has allowed the development of extremely sensitive and selective detection methods. Furthermore, it is desirable the method to be simple, fast and straightforward. Mildronate is a highly polar quaternary amine, classified as metabolic modulator by the World Anti-Doping Agency (WADA). The inclusion of mildronate in the screening strategy is a challenge considering its singular physicochemical properties, compared to numerous doping agents of low and medium polarity. For this purpose, a method combining solid-phase extraction (SPE) and dilute-and-shoot approach has been developed and validated, allowing the detection of mildronate and Other 332 prohibited substances. In the sample preparation protocol, the enzymatic deconjugation step and SPE conditions were stressed to enable the recovery of mildronate without jeopardizing the detection of Other doping agents. The C₁₈/18% SPE cartridge without any type of ionic interaction, associated with the dilute-and-shoot approach proved to be effective for all monitored substances. The instrumental method employed was based on liquid chromatography using a reversed-phase column in a 12-minute gradient coupled to a high-resolution mass spectrometry in full scan with positive and negative switching and fragmentation in the positive mode, for the most critical detection compounds. The performance of the method was evaluated regarding selectivity, precision, recovery, carry-over, limit of detection and stability, following the recommendations of WADA.