Potential of supercritical fluid chromatography for endogenous anabolic agents detection in doping control

Code: 21A06AM

The detection of endogenous anabolic agents still represents one of the major challenges in doping control. Gas chromatographic separation in combination with combustion and isotopic ratio mass spectrometry (GC/C/IRMS) is needed to discriminate between intake and natural production of the endogenous steroids. However, the technique requires a very tedious and lengthy sample preparation that makes it inapplicable to screening analysis and limits its use to those samples that have been flagged suspicious in steroidal module of the Athlete Biological Passport (ABP) after GC-MSn analysis. Historically based on gas-chromatography, the analytical approach for anabolic agents has later expanded to include liquid chromatography, and more recently investigations have considered supercritical fluid chromatography (SFC) as well. The introduction in 2012 of the ACQUITY Ultra Performance Convergence Chromatography (UPC²) system, with improved robustness and performance, suggested that a widespread implementation in routine analysis would follow. However, laboratories have been slow in introducing the change, assumedly at least partly because of the limited research being performed and published so far. We intend to perform an in-depth investigation of the potential of UHPSFC combined with mass spectrometry for the analysis of endogenous anabolic agents in urine samples. In particular, this project will investigate the separation and quantification of endogenous steroids both in the free and conjugated form by UHPSFC-MS and evaluate the various factors influencing sensitivity. The optimal conditions for chromatography and MS will be established, such as the use of co-solvents, the effect of the MS parameters, in combination with an appropriate sample preparation procedure to maximise selectivity and sensitivity as well as achieving the required chromatographic separation. This project will provide an alternative or complementary analytical method to current GC/MSn methods traditionally used in anti-doping laboratories. Additionally, we aim to explore the potential use of UHPSFC combined to fraction collection to simplify and automate sample preparation for GC/C/IRMS analysis.