Screening analysis of doping agents in horse urine and plasma with dilute and shoot using liquid chromatography high resolution mass spectrometry

Various technical methodologies are required to accurately detect substances of different chemical and pharmacological properties in biological samples, which are increasing in number and variety daily. Therefore, laboratories where many samples and different factors are analyzed simultaneously need methods with easy sample preparation, short analysis times and low analysis costs. In this study, the objective was to scan substances susceptible to chemical degradation, amenable to analysis without hydrolysis, and exhibiting short-term stability by employing a straightforward, expeditious, and cost-efficient method. For this purpose, a high-throughput dilute and shoot screening protocol was developed and validated utilizing high-performance liquid chromatography coupled with high-resolution mass spectrometry to analyze various pharmacological compounds in horse urine and plasma. Over 200 prohibited substances across multiple categories were scanned within a 13 minute run. Chromatographic separation was performed on a C18 column using an elution gradient of mobile phase A, 5 mM ammonium bicarbonate at pH 9, and mobile phase B, methanol, at a flow rate of 0.3 mL min⁻¹. The method was validated according to the specifications of 2002/657/EC multi-screening requirements. The detection capability ranged from ≤1 to 200 ng mL⁻¹ for prohibited substances. The implementation of the screening method in doping analysis, and the analysis of real positive case samples served to underscore the practical applicability of the developed method. To the best of our knowledge, this is a rare method that can be applied to both urine and plasma samples and provides a rapid, practical, broad-spectrum, and high-throughput analysis of prohibited substances in horse plasma and urine cost-effectively.