Analysis of doping control samples using SFC-MS
Athletes are required to provide urine samples, as part of an anti-doping programme, in a doping control station and the sample collection is witnessed by a Doping Control Officer. As part of the process, a Doping Control Form (example attached) is completed and the samples are transported to the Drug Control Centre (DCC) at King's College London. The part of the Doping Control Form received by the DCC contains no information about the athlete (e.g. name, address). It contains information about sample volume, specific gravity, event code, and any medication declared to have been used by the athlete. A box on the form indicates whether the athlete has consented for his/her samples to be used for the research purposes. Once the athlete’s sample arrives in the DCC, it is split and undergoes the sample preparation steps (e.g. solid
phase extraction) prior to analysis. The usual method for sample analysis is liquid chromatography-high resolution mass spectrometry (LC-HRMS) (please see Musenga and Cowan, Use of ultra-high pressure liquid chromatography coupled to high resolution mass spectrometry for fast screening in high throughput doping control. J. Chromatography A 2013;1288:82-95). In our research project, funded by the World Anti-Doping Agency (WADA), we would like to investigate the potential application of supercritical fluid chromatography-mass spectrometry (SFCMS) in the analysis of doping control samples. We aim to compare the performance of SFC-MS with LC-HRMS. In order to do our research, once the samples have been analysed by LC-HRMS, we will re-analyse them by SFCMS and compare two methods by results obtained. We plan to do our research with approximately 1000 samples and our research refers to secondary data analysis. The DCC is required by WADA to analyse all samples received by LC- HRMS no matter if the athlete had given research consent or not. However, we will only analyse samples by SFC-MS with the research consent provided by the athlete on the Doping Control Form. As a laboratory accredited by the WADA, we have to comply with strict criteria in the use of athlete samples for research. These include: waiting at least 3 months after the anti-doping analytical report has been issued; transference of the sample from the coded bottle to a separate container so that it is impossible for the identity of the athlete who provided the sample to be discoverable by anyone.
This study clearly demonstrated the applicability of SFC-MS for routine antidoping analysis. We analysed approximately 3,000 samples (3 x 1,000) in total using SFC-MS equipment from three different vendors to investigate whether this technology is applicable for routine anti-doping analysis. Our work demonstrated SFC-MS to be a robust analytical technique. It was demonstrated that it may be considered as a complementary technique to LC-MS, which is readily available in anti-doping laboratories. Three SFC-MS instrument manufacturers (Agilent Technologies, Shimadzu UK Ltd and Waters Corporation) generously contributed to our study to investigate the routine applicability of SFC-MS in doping control. All three vendors dedicated instrument time and resource for the project. A large number of anti-doping samples and QCs were delivered to each manufacturer. We worked closely with each manufacturer in agreeing the method of operation and helped with the analysis of the samples, data review and undertook the independent statistical analysis of the data.
All three instruments appeared to meet the requirement for robustness needed for routine use. Most of our compounds showed excellent chromatography. There were only a few examples of co-elution due to interference with endogenous compounds that could benefit from modification of the chromatographic conditions. Retention times were generally remarkably stable over the analysis time. We demonstrated some software features that would facilitate data review especially with the software from one manufacturer. Despite using a “dilute and inject” approach, the SFC columns were found to be stable over more than 1,200 injections of samples, standards and quality controls on three different systems. In general, SFC-MS was found to be similar to LC-MS for routine use but with the advantage of complementarity providing additional information of the chromatographic properties of the analyte thereby further confirming the identity of a doping agent.