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.

The study explores the optimization of solid-phase extraction, specifically in the analysis of harpagoside, harpagide, and 8-para-coumaroyl harpagide in horse plasma and urine. These substances are extracted from the samples, purified, and then analyzed using liquid chromatography and mass spectrometry techniques.

Methodology

• The researchers used solid-phase extraction cartridges, common tools in horse doping analysis, designed to optimize the extraction of three specific substances: harpagoside (HS), harpagide (HG), and 8-para-coumaroyl harpagide (8PCHG).
• Following extraction, the resultant extracts were analyzed using a process called liquid chromatography, linked with multi-step tandem mass spectrometry.

Key Findings

• The extraction process for plasma employed BondElut PPL cartridges, which achieved extraction recoveries of between 91% and 93%. RSD (relative standard deviation), a measure of precision in the extraction, was recorded between 8% and 13% at 0.5 ng/mL concentration.
• For analytes extraction from urine, two distinct procedures were required. HS and 8PCHG were extracted using AbsElut Nexus cartridges. The recoveries for HS and 8PCHG were 85% and 77%, respectively, with RSD values lying between 7% and 19%.
• HG extraction required the use of two types of cartridges – BondElut PPL and BondElut C18 HF. The recovery in this case was 75%, with the RSD ranged between 14% and 19%.

Applicability

• The researchers tested the extraction methods on actual equine plasma and urine samples following the administration of harpagophytum or harpagoside, suggesting that this method could have practical applications in the field of equine performance analysis.