Optimization of Solid-Phase Extraction for the Liquid Chromatography-Mass Spectrometry Analysis of Harpagoside, 8-para-Coumaroyl Harpagide, and Harpagide in Equine Plasma and Urine

Solid-phase extraction cartridges among those usually used for screening in horse doping analyses are tested to optimize the extraction of harpagoside (HS), harpagide (HG), and 8-paracoumaroyl harpagide (8PCHG) from plasma and urine. Extracts are analyzed by liquid chromatography coupled with multi-step tandem mass spectrometry. The extraction process retained for plasma applies BondElut PPL cartridges and provides extraction recoveries between 91% and 93%, with RSD values between 8 and 13% at 0.5 ng/mL. Two different procedures are needed to extract analytes from urine. HS and 8PCHG are extracted using AbsElut Nexus cartridges, with recoveries of 85% and 77%, respectively (RSD between 7% and 19%). The extraction of HG involves the use of two cartridges: BondElut PPL and BondElut C18 HF, with recovery of 75% and RSD between 14% and 19%. The applicability of the extraction methods is determined on authentic equine plasma and urine samples after harpagophytum or harpagoside administration.