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Biomedical chromatography : BMC2022; 36(10); e5445; doi: 10.1002/bmc.5445

Simultaneous quantification of caffeine and its main metabolites by gas chromatography mass spectrometry in horse urine.

Abstract: Caffeine is a naturally occurring alkaloid and it is metabolized to paraxanthine, theophylline and theobromine. Analysis of caffeine and its metabolites is challenging since the metabolites theophylline and paraxanthine generate similar product and precursor ions. In this study, a new method was developed for the simultaneous analysis of caffeine, paraxanthine, theobromine and theophylline in horse urine using gas chromatography-mass spectrometry (GC-MS). Urine samples were treated using solid-phase extraction followed by the elution with dichloromethane-isopropanol (90:10) after the pH was adjusted to 6, and then derivatization with N-methyl-N-trimethylsilyl-trifluoroacetamide-1% trimethylchlorosilane before analysis with GC-MS. Sample preparation and derivatization steps were optimized and the method permitted elution all of these analytes within 13 min. The method was fully validated according to Commission Decision, 2002/657/EC guidelines. The calibration curves were linear with a correlation coefficient of >0.99. Precision and accuracy were well within the 15% acceptance range and the method was robust. The validation results demonstrated that the method is highly reproducible, easily applicable and selective. The method was applied to urine samples collected from racehorses to demonstrate its applicability.
© 2022 John Wiley & Sons Ltd.
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Publication Date: 2022-07-15 PubMed ID: 35797186DOI: 10.1002/bmc.5445Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research presents a novel method for analyzing caffeine and its major metabolites — paraxanthine, theobromine, and theophylline — in horse urine using gas chromatography-mass spectrometry, with applications to test racehorses. This method overcomes challenges in distinguishing amongst these metabolites due to their similar ions and allows for simultaneous analysis of all components.

Methodology

  • The research involved analyzing the urine of horses, which required careful extraction and treatment of the samples to enable precise detection and measurement of caffeine and its metabolites.
  • The process used solid-phase extraction, where compounds are separated based on their physical and chemical characteristics. Subsequent elution methodology employed a mixture of dichloromethane-isopropanol at a ratio of 90:10 after pH adjustment to 6.
  • A derivative step was added using N-methyl-N-trimethylsilyl-trifluoroacetamide-1% trimethylchlorosilane to help increase detection sensitivity before further analysis with gas chromatography-mass spectrometry (GC-MS), a powerful tool known for its capability to analyze and quantify complex mixtures.
  • All steps from preparation to derivatization were optimized so that all analytes could be eluted within 13 minutes, making the process time-efficient.

Validation and Results

  • The research followed the Commission Decision, 2002/657/EC guidelines to ensure full validation of their method.
  • The calibration curves obtained were linear with a maximum correlation coefficient greater than 0.99, implying a strong positive relationship between observed and predicted values of the measures.
  • The precision and accuracy fell well within the 15% acceptance range, showcasing the method’s reliable repeatability and credibility.
  • The method was deemed robust and highly reproducible, making it a potentially universal procedure for such compound analysis.
  • The method’s effectiveness in application was demonstrated by successfully applying it to real cases involving urine samples from racehorses.

Significance

  • This new methodology offers a reliable, efficient, and robust means of simultaneously analyzing caffeine and its main metabolites in horse urine.
  • It overcomes the analytical challenges of distinguishing between similar ions made from the metabolites theophylline and paraxanthine.
  • With applications in testing racehorses, it can be a significant tool for ensuring integrity and fairness in racing sports by monitoring potential misuse of these compounds.

Cite This Article

APA
Göktaş EF, Kabil E, Yatanaslan L, Güneş E, Dirikolu L. (2022). Simultaneous quantification of caffeine and its main metabolites by gas chromatography mass spectrometry in horse urine. Biomed Chromatogr, 36(10), e5445. https://doi.org/10.1002/bmc.5445

Publication

Biomedical chromatography : BMC
ISSN: 1099-0801
NlmUniqueID: 8610241
Country: England
Language: English
Volume: 36
Issue: 10
Pages: e5445

Researcher Affiliations

Göktaş, Eylem Funda
  • Doping Control Laboratory, Istanbul Pendik Veterinary Control Institude, Istanbul, Turkey.
Kabil, Erol
  • Doping Control Laboratory, Istanbul Pendik Veterinary Control Institude, Istanbul, Turkey.
Yatanaslan, Levent
  • Doping Control Laboratory, Istanbul Pendik Veterinary Control Institude, Istanbul, Turkey.
Güneş, Ertuğrul
  • Doping Control Laboratory, Istanbul Pendik Veterinary Control Institude, Istanbul, Turkey.
Dirikolu, Levent
  • Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA, USA.

MeSH Terms

  • Animals
  • Caffeine / analysis
  • Gas Chromatography-Mass Spectrometry / methods
  • Horses
  • Solid Phase Extraction
  • Theobromine / chemistry
  • Theobromine / urine
  • Theophylline / chemistry

Grant Funding

  • 120O917 / The Scientific And Technological Research Council of Turkey

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Citations

This article has been cited 1 times.
  1. Mullin A, Scott M, Vaccaro G, Gittins R, Ferla S, Schifano F, Guirguis A. Handheld Raman Spectroscopy in the First UK Home Office Licensed Pharmacist-Led Community Drug Checking Service. Int J Environ Res Public Health 2023 Mar 8;20(6).
    doi: 10.3390/ijerph20064793pubmed: 36981705google scholar: lookup
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