Ultra-Fast Retroactive Processing by MetAlign of Liquid-Chromatography High-Resolution Full-Scan Orbitrap Mass Spectrometry Data in WADA Human Urine Sample Monitoring Program

RATIONALE: The World Antidoping Agency (WADA) Monitoring program concentrates analytical data from the WADA Accredited Laboratories for substances, which are not prohibited, but their potential misuse has to become known. The WADA List of Monitoring substances is updated annually, where substances may be removed, introduced or transferred to the Prohibited List, depending on prevalence of their use. Retroactive processing of old samples datafiles has the potential to create information for the prevalence of use of candidate substances for the Monitoring List in years before. MetAlign is a freeware software with functionality to reduce the size of Liquid Chromatography (LC) high resolution (HR) full scan (FS) mass spectrometry (MS) datafiles and to perform fast search for presence of substances in thousands of reduced datafiles.

METHODS: Validation was performed to the search procedure of MetAlign applied to ADLQ screening LC-HR-FS-MS reduced datafiles originated from antidoping samples for tramadol (TRA), ecdysterone (ECDY) and ECDY metabolite 14-desoxyecdysterone (DESECDY) of the WADA Monitoring List. Searching parameters were related to combinations of accurate masses and retention times (RT).

RESULTS: MetAlign search validation criteria were based on the creation of correct identifications, false positives (FP) and false negatives (FN). The search for TRA in 7,410 ADLQ routine LC-HR-FS-MS datafiles of the years 2017 to 2020 revealed no false identification (FP and FN) compared to the ADLQ WADA reports. ECDY and DESECDY detected by MetAlign search in approximately 5% of the same cohort of antidoping samples.

CONCLUSIONS: MetAlign is a powerful tool for the fast-retroactive processing of old reduced datafiles collected in screening LC-HR-FS-MS to reveal prevalence of use of antidoping substances. The current study proposed the validation scheme of MetAlign search procedure, to be implemented per individual substance of the WADA Monitoring program, for the elimination of FNs and FPs.