Sport Doping Screening in Biological Matrices by Multi-Dimensional LC-QToF

As seen in the past few decades, the reputations of many professional sports leagues and players have been tarnished due to cheating in the form of sports doping. As such, it is commonplace to have organizations, protocols and methods dedicated to detecting performance enhancing agents in biological samples such as urine. While doping commonly refers to steroids, the World Anti-Doping Agency (WADA)’s list of prohibited substances contains a wide variety of compounds with anabolic agents being only one of many classes. The wide variety of compound, low levels and the short time frame in which results are expected all contribute to the challenges of developing a comprehensive screening method for sports doping agents.

All target drugs in this screening study were classified in 9 classes based on their chemical structure (79 total). Once in solution, target analytes were individually infused to acquire their respective precursor and product ions. Once the quantitative and qualitative transitions for all drugs were identified, the chromatography conditions were optimized for each class using a 6x6 automated methods development protocol. Each target analyte was subjected to a total of 108 LC/MS/MS methods which were carried out within 72 hours. The multi-dimensional LC was configured for "Trap & Elute" with At-Column Dilution. The total run time was 10 minutes. The mass spectrometer was a XEVO G2 QToF operated in positive ionization mode.

Current analytical techniques use a combination of extraction procedures, often requiring an enrichment process and accurate detection for any given target analyte. As such, new analytical strategies are needed to reach those goals. This work evaluated the performance of 2D LC variant using a QToF setup instead of a triple quadrupole mass spectrometer for the analysis of drug of abuse in urine targeting low and sub ppb level. From a previous study focusing on the analysis illicits drugs in urine by 2D LC/MS/MS, target LOD at 100 ppt was reported from a 1 mL urine sample. Considering the level of complexity of urine samples and also ensuring the retention of all target analyte in a clean extract, several strategic extraction protocols were evaluated to determine which one can reach a target detection level between 0.1 ppb and 1 pbb.