Factors affecting untargeted detection of doping agents in biological samples.

Doping control is essential for sports, and untargeted detection of doping agents (UDDA) is the holy grail for anti-doping strategies. The present study examined major factors impacting UDDA with metabolomic data processing, including the use of blank samples, signal-to-noise ratio thresholds, and the minimum chromatographic peak intensity. Contrary to data processing in metabolomics studies, both blank sample use (either blank solvent or plasma) and marking of background compounds were found to be unnecessary for UDDA in biological samples, the first such report to the authors' knowledge. The minimum peak intensity required to detect chromatographic peaks affected the limit of detection (LOD) and data processing time for untargeted detection of 57 drugs spiked into equine plasma. The ratio of the mean (ROM) of the extracted ion chromatographic peak area of a compound in the sample group (SG) to that in the control group (CG) impacted its LOD, and a small ROM value such as 2 is recommended for UDDA. Mathematical modeling of the required signal-to-noise ratio (S/N) for UDDA provided insights into the effect of the number of samples in the SG, the number of positive samples, and the ROM on the required S/N, highlighting the power of mathematics in addressing issues in analytical chemistry. The UDDA method was validated by its successful identification of untargeted doping agents in real-world post-competition equine plasma samples. This advancement in UDDA methodology will be a useful addition to the arsenal of approaches used to combat doping in sports.