High resolution accurate mass screening of prohibited substances in equine plasma using liquid chromatography – Orbitrap mass spectrometry

Abstract

A recent trend in the use of high resolution accurate mass screening (HRAMS) for doping control testing in both human and animal sports has emerged due to significant improvement in high resolution mass spectrometry in terms of sensitivity, mass accuracy, mass resolution, and mass stability. A number of HRAMS methods have been reported for the detection of multi-drug residues in human or equine urine. As blood has become a common matrix for doping control analysis, especially in equine sports, a sensitive, fast and wide coverage screening method for detecting a large number of drugs in equine blood samples would be desirable. This paper presents the development of a liquid chromatography-high resolution mass spectrometry (LC-HRMS) screening method for equine plasma samples to cover over 320 prohibited substances in a single analytical run. Plasma samples were diluted and processed by solid-phase extraction. The extracts were then analyzed with LC-HRMS in full-scan positive electrospray ionization mode. A mass resolution of 60 000 was employed. Benzyldimethylphenylammonium was used as an internal lock mass. Drug targets were identified by retention time and accurate mass, with a mass tolerance window of ±3 ppm. Over 320 drug targets could be detected in a 13-min run. Validation data including sensitivity, specificity, extraction recovery and precision are presented. As the method employs full-scan mass spectrometry, an unlimited number of drug targets can theoretically be incorporated. Moreover, the HRAMS data acquired can be re-processed retrospectively to search for drugs which have not been targeted at the time of analysis.