An evaluation study of stability and robustness for implementing supercritical fluid chromatography - mass spectrometry in the anti-doping field

Code: T17R02TK

Ultra-high performance supercritical fluid chromatography-mass spectrometry (UHPSFC-MS) could represent in the near future an orthogonal technique to LC-MS and GC-MS for routine doping analysis. This technique now benefits from a broader recognition and interest, thanks to new technological improvements and the recent commercialization of new platforms.

The aim of this project is to evaluate the potential of UHPSFC-MS/MS for screening and confirmation purposes in routine anti-doping analysis. This aim will be achieved through a comprehensive robustness study (different columns chemistries, column batches, instruments and laboratories) by selecting representative compounds of major classes of prohibited substances (approx. 50 compounds) fortified in urine samples. Various aspects will be evaluated such as the stability of retention times and the inter-batch variability of SFC columns. Then, an inter-laboratory study as well as an inter-instrument study will be performed with other academic and/or industrial laboratories equipped with the same brand of SFC instrument (Waters Acquity UPC² system) or equipped with other brands of UHPSFC instruments, including Agilent and Shimadzu, to evaluate the ruggedness of the SFC-MS/MS method.

Main Findings

The aim of this study was to assess the interlaboratory reproducibility of ultra-high performance supercritical fluid chromatography coupled with tandem mass spectrometry method for routine antidoping analyses.To do so, a set of 21 doping agents, spiked in urine and analyzed after dilute and shoot treatment, was used to assess the variability of their retention times between four different laboratories, all equipped with the same chromatographic system and with the same ultra-high performance supercritical fluid chromatography stationary phase chemistry. The average relative standard deviations (RSD%) demonstrated a good reproducibility of the retention times for 19 out of 21 analytes, with RSD% values below 3.0%. Only for two substances, namely fenbutrazate and niketamide, the retention was not repeatable between laboratories, with RSD% of approximately 15% in both cases. This behaviour was associated with (a) the low organic modifier percentage (around 2-4%) in the mobile phase at the corresponding retention times, and (b) the influence of the system volume on poorly retained analytes. An analysis on seven “blind” urines was subsequently carried out in the same four laboratories. In these blind samples, either one, two, or none of the 21 doping agents previously analyzed were present at an unknown concentration. Each laboratory had to perform the identification of the compounds in the samples and estimate their concentrations. All laboratories assigned all target analytes correctly in all blind urine samples and provide a comparable estimation of their concentrations.