Evaluating the Effects of Solid-Phase Cartridge Chemistry on Extraction of Bisphosphonates and ITPP From Equine Plasma.

Overview

• This study evaluates how different solid-phase extraction cartridge chemistries affect the recovery and detection of bisphosphonates and myo-inositol trispyrophosphate (ITPP) in horse plasma samples used in equine sports drug testing.
• The goal is to improve extraction efficiency and detection by comparing current methods with an alternative cartridge chemistry to optimize screening protocols.

Background

• Bisphosphonates and myo-inositol trispyrophosphate (ITPP) are drugs banned in horse racing due to their performance-enhancing potential.
• Detecting these substances accurately in equine plasma is important for maintaining fairness in the sport.
• The Australian Racing Forensic Laboratory currently uses a two-step solid-phase extraction (SPE) process:
  • First, passing plasma samples through a mixed-mode SPE cartridge.
  • Second, samples go through a weak anion exchange cartridge.
• After extraction, the substances are methylated and analyzed using liquid chromatography-mass spectrometry (LC-MS).

Problem Statement

• Some bisphosphonate drugs show poor recovery using the current two-step extraction approach.
• Poor recovery means some drugs may not be detected or quantified accurately, threatening the reliability of drug screening in equine sports.

Research Objective

• To assess whether changing the cartridge chemistry in the second extraction step can improve recovery and detection of bisphosphonates and ITPP.
• Specifically, the study compares:
  • The currently used weak anion exchange cartridges.
  • An alternative cartridge: the Affinisep AttractSPE polymeric phase cartridge.

Methodology

• Samples of equine plasma containing bisphosphonates and ITPP were subjected to extraction using both cartridge types.
• The extraction results were evaluated by:
  • Visual comparison of signal-to-noise ratios in chromatograms obtained via LC-MS analysis.
  • Measurement of the recovery rate of the targeted substances from plasma samples.

Key Findings

• The study found differences in extraction efficiency linked to the cartridge chemistry used.
• Some bisphosphonates showed improved recovery with the AttractSPE polymeric phase cartridge versus the weak anion exchange cartridge.
• Improved signal-to-noise ratios suggest better detection sensitivity using the alternative cartridge.

Implications

• Changing the cartridge chemistry in the SPE protocol can enhance the reliability of detecting banned bisphosphonate drugs and ITPP in equine sports.
• Optimizing this step could lead to more accurate routine screening, ensuring drug violations are identified.
• The findings support considering alternative SPE cartridges for equine forensic toxicology labs to improve method performance.

Conclusion

• The study highlights the importance of selecting appropriate SPE cartridge chemistries for drug extraction from plasma.
• Using the Affinisep AttractSPE polymeric phase cartridge may offer a superior approach compared to traditional weak anion exchange cartridges for bisphosphonate recovery.
• Adopting improved extraction methods can strengthen regulatory enforcement in equine racing by enhancing detection capabilities.