A fully automated method to quantitate total carbon dioxide in equine plasma by headspace GCMS

Pre-race dosing of horses with alkalinising agents to manipulate performance has been evident in racing worldwide for over 30 years. To regulate the use of alkalinising agents, racing authorities adopted thresholds for total plasma carbon dioxide (TCO₂ ) in racehorses. Traditionally, racing laboratories have measured plasma TCO₂ using ion selective electrode (ISE) technology, with the Association of Official Racing Chemists (AORC) approving the use of only three ISE instruments for measurement. Because of the manufacture and support of these instruments ceasing, racing laboratories have explored alternative techniques to measure plasma TCO₂ . In this study, headspace gas chromatography mass spectrometry (HSGCMS) with fully automated sample preparation was investigated as an alternative technique to ISE. Sample preparation was carried out online on a Gerstel robot, where plasma was aspirated directly from sealed vacutainer tubes before further treatment and headspace injection into a GCMS. The method was successfully cross validated against a Beckman Unicel DxC®600, meeting all criteria stipulated in the AORC cross-validation protocol. The method achieved an accuracy of 99.8%, within-run relative standard deviation of 0.22% and interday reproducibility of 0.04 mM, all significant improvements on the authors ISE method. A population study was also conducted to ensure the plasma TCO₂ threshold, established with ISE methodology, did not change with the developed HSGCMS method. The concentrations and standard deviations for the two methods were almost identical, HSGCMS mean 30.62 mM, standard deviation 1.65 mM, and ISE 30.65 and 1.55 mM. The results indicate that the fully automated HSGCMS method is suitable for measurement of equine plasma TCO₂ for regulatory purposes.