Comprehensive screening of acidic and neutral drugs in equine plasma by liquid chromatography–tandem mass spectrometry

A multi-target high-throughput liquid chromatography–tandem mass spectrometry (LC–MS–MS) method for the detection of low ppt to low ppb levels of anabolic steroids, corticosteroids, anti-diabetics, and non-steroidal anti-inflammatory drugs (NSAIDs) in equine plasma was developed for the purpose of doping control. Plasma samples were first deproteinated by addition of trichloroacetic acid. Drugs were then extracted by solid-phase extraction (SPE) using Bond Elut Certify® cartridges, and the extracts were analysed by a triple-quadrupole/linear ion trap LC–MS–MS instrument in positive electrospray ionization (+ESI) mode with selected reaction monitoring (SRM) scan function. Chromatographic separation of the targeted drugs was achieved using a reverse phase 3.3 cm L × 2.1 mm ID, 3 μm particle size LC column with gradient elution. Plasma samples fortified with 66 targeted drugs including betamethasone, boldione, capsaicin, flunisolide, gestrinone, gliclazide, 17α-hydroxyprogesterone hexanoate, isoflupredone and triamcinolone acetonide, etc. at low ppt to low ppb levels could be consistently detected. No significant matrix interference was observed at the retention time of the targeted ion transitions when blank plasma samples were analysed. The method has been validated for its extraction recoveries, precision and sensitivity, and is used regularly in the authors’ laboratory to screen for the presence of these drugs in plasma samples from racehorses.