Cross Validation of HS-GC/MS to Quantify Total Carbon Dioxide in Horse Plasma.
Abstract: The use of alkinizing agents by trainers to counteract the accumulation of lactic acid in racehorses has been well documented. A by-product of this administration is elevated total carbon dioxide (tCO2) concentrations. Most regulatory authorities have set the threshold for tCO2 in plasma at 37 mM. The quantification of tCO2 often presents a challenge to laboratories due to the instrumentation required, which can be expensive to use and maintain. The Beckman DxC 600 (Brea, CA) is currently used in our laboratory for tCO2 quantification. The goal of this research was to determine if another analytical method could be used for this procedure. We report the use of headspace gas chromatography-mass spectrometry (HS-GC/MS) as an acceptable alternative to the use of the Beckman DxC 600. A six-point calibration curve ranging from 10 to 60 mM was analyzed along with controls at 15, 25 and 45 mM. Imprecision was found to be <3% at all concentrations. Inaccuracy was measured at <4% at all concentrations. Measurement of uncertainty was determined using the Simplified GUM approach and was found to be 3% at 99.7% confidence level with eight measurements at a tCO2 concentration of 45 mM. Furthermore, the HS-GC/MS was cross-validated according to international protocols with all parameters being met. During cross validation, a standard at a known concentration was analyzed by both instruments. The average difference using 25 replicates in calculated concentrations was <0.1 mM. Also, plasma samples from four untreated horses were analyzed by both instruments. The difference in calculated concentrations between the two instruments was <0.6 mM for all horses. The results show that the use of HS-GC/MS is an acceptable alternative to the use of the Beckman DxC 600 for the quantification of tCO2 in horse plasma.
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Publication Date: 2016-12-31 PubMed ID: 28035033DOI: 10.1093/jat/bkw133Google Scholar: Lookup
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Summary
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This study explores the use headspace gas chromatography-mass spectrometry (HS-GC/MS) as an effective alternative to the currently used Beckman DxC 600 for quantifying total carbon dioxide (tCO2) in horse plasma, particularly in the context of monitoring alkalizing substances that enhance horse racing performance.
Background and Goal
- Alkalizing agents are sometimes used by horse trainers to reduce lactic acid accumulation in racehorses, resulting in elevated carbon dioxide (tCO2) concentrations. This necessitates tCO2 quantification in labs.
- The threshold for tCO2 in plasma has been set at 37 mM by most regulatory bodies. However, measuring tCO2 presents a challenge due to expensive instrumentation required, such as the Beckman DxC 600 which is currently used.
- The study intended to decipher whether there is a different analytical method that can be used to measure tCO2. The aim was to establish headspace gas chromatography-mass spectrometry (HS-GC/MS) as a reliable alternative.
Methods and Results
- A six-point calibration curve was analyzed along with controls at three different concentrations. The accuracy and precision of the results were found to be satisfactory, with less than 3% and 4% respectively at all concentrations.
- Uncertainty in measurements was calculated using the Simplified GUM approach and it was found to be 3% at 99.7% confidence level with eight measurements at a tCO2 concentration of 45 mM.
- The HS-GC/MS method was evaluated and cross validated according to international protocols. All desired parameters were achieved in the process.
Comparison with Existing Method
- The cross-validation process compared the results obtained from both HS-GC/MS and Beckman DxC 600 using a known standard and plasma samples from four untreated horses.
- The average difference in calculated concentrations from both instruments was found to be insignificant (<0.1 mM and <0.6 mM for the standard and horse samples respectively), indicating the reliability of the HS-GC/MS.
Conclusion
- The final conclusion drawn from this study is that employing HS-GC/MS for measuring tCO2 in horse plasma is an acceptable and alternate method to using the Beckman DxC 600.
Cite This Article
APA
Heffron B, Bash J, Larsen AK.
(2016).
Cross Validation of HS-GC/MS to Quantify Total Carbon Dioxide in Horse Plasma.
J Anal Toxicol, 41(3), 230-235.
https://doi.org/10.1093/jat/bkw133 Publication
Researcher Affiliations
- Analytical Forensic Testing Laboratory, Department of Biopharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, 2242 West Harrison Street, Suite 110, Chicago, IL 60612, USA.
- Analytical Forensic Testing Laboratory, Department of Biopharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, 2242 West Harrison Street, Suite 110, Chicago, IL 60612, USA.
- Analytical Forensic Testing Laboratory, Department of Biopharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, 2242 West Harrison Street, Suite 110, Chicago, IL 60612, USA.
MeSH Terms
- Animals
- Calibration
- Carbon Dioxide / blood
- Doping in Sports
- Gas Chromatography-Mass Spectrometry / methods
- Horses / blood
- Reference Standards
- Reproducibility of Results
Citations
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