Doping control analysis of 46 polar drugs in horse plasma and urine using a ‘dilute-and-shoot’ ultra high performance liquid chromatography-high resolution mass spectrometry approach

• Two ‘dilute-and-shoot’ UHPLC-HRMS methods for equine urine and plasma are developed.
• These methods can detect 46 polar drugs in equine urine and plasma.
• The LoDs could be achieved at sub to low parts per billion levels.

The high sensitivity of ultra high performance liquid chromatography coupled with high resolution mass spectrometry (UHPLC-HRMS) allows the identification of many prohibited substances without pre-concentration, leading to the development of simple and fast ‘dilute-and-shoot’ methods for doping control for human and equine sports. While the detection of polar drugs in plasma and urine is difficult using liquid-liquid or solid-phase extraction as these substances are poorly extracted, the ‘dilute-and-shoot’ approach is plausible. This paper describes a ‘dilute-and-shoot’ UHPLC-HRMS screening method to detect 46 polar drugs in equine urine and plasma, including some angiotensin-converting enzyme (ACE) inhibitors, sympathomimetics, anti-epileptics, hemostatics, the new doping agent 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), as well as two threshold substances, namely dimethyl sulfoxide and theobromine.For plasma, the sample (200 μL) was protein precipitated using trichloroacetic acid, and the resulting supernatant was diluted using Buffer A with an overall dilution factor of 3. For urine, the sample (20 μL) was simply diluted 50-fold with Buffer A. The diluted plasma or urine sample was then analysed using a UHPLC-HRMS system in full-scan ESI mode.The assay was validated for qualitative identification purpose. This straightforward and reliable approach carried out in combination with other screening procedures has increased the efficiency of doping control analysis in the laboratory. Moreover, since the UHPLC-HRMS data were acquired in full-scan mode, the method could theoretically accommodate an unlimited number of existing and new doping agents, and would allow a retrospectively search for drugs that have not been targeted at the time of analysis.