FREE SHIPPING over $40
OVER 9000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Home / Equine Research Bank / Biomed Chromatogr / Simultaneous quantification of caffeine and its main metabol...
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.
Get Full Text
Publication Date: 2022-07-15 PubMed ID: 35797186DOI: 10.1002/bmc.5445Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • Journal Article

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

References

This article includes 45 references
  1. Aresta A, Palmisano F, Zambonin CG. Simultaneous determination of caffeine, theobromine, theophylline, paraxanthine and nicotine in human milk by liquid chromatography with diode array UV detection. Food Chemistry 93(1), 177-181.
    doi: 10.1016/j.foodchem.2004.11.013google scholar: lookup
  2. Ashihara H, Suzuki T. Distribution and biosynthesis of caffeine in plants. Frontiers in Bioscience 9(2), 1864-1876.
    doi: 10.2741/1367google scholar: lookup
  3. Benowitz NL, Jacob P III, Mayan H, Denaro C. Sympathomimetic effects of paraxanthine and caffeine in humans. Clinical Pharmacology & Therapeutics 58(6), 684-691.
    doi: 10.1016/0009-9236(95)90025-xgoogle scholar: lookup
  4. Bispo MS, Veloso MCC, Pinheiro HLC, De Oliveira RF, Reis JON, De Andrade JB. Simultaneous determination of caffeine, theobromine, and theophylline by high-performance liquid chromatography. Journal of Chromatographic Science 40(1), 45-48.
    doi: 10.1093/chromsci/40.1.45google scholar: lookup
  5. Budhraja A, Camargo FC, Hughes C, Lehner AF, Stirling K, Brennan N, Dowling M, Tobin T. Caffeine and theobromine identifications in post race urines: threshold levels and regulatory significance of such identifications. AAEP Proc 53, 87-94.
  6. Büyüktuncel E. Simultaneous determination of theobromine, paraxanthine, theophylline, and caffeine in urine by reversed-phase high-performance liquid chromatography with diode array UV detection. Analytical Letters 43(16), 2518-2524.
    doi: 10.1080/00032711003731357google scholar: lookup
  7. Caubet MS, Comte B, Brazier JL. Determination of urinary 13C-caffeine metabolites by liquid chromatography-mass spectrometry: The use of metabolic ratios to assess CYP1A2 activity. Journal of Pharmaceutical and Biomedical Analysis 34(2), 379-389.
    doi: 10.1016/s0731-7085(03)00528-4google scholar: lookup
  8. Caubet MS, Elbast W, Dubuc MC, Brazier JL. Analysis of urinary caffeine metabolites by HPLC-DAD: The use of metabolic ratios to assess CYP1A2 enzyme activity. Journal of Pharmaceutical and Biomedical Analysis 27(1-2), 261-270.
    doi: 10.1016/s0378-4347(98)00016-4google scholar: lookup
  9. Chen F, Hu ZY, Parker RB, Laizure SC. Measurement of caffeine and its three primary metabolites in human plasma by HPLC-ESI-MS/MS and clinical application. Biomedical Chromatography 31(6), e3900.
    doi: 10.1002/bmc.3900google scholar: lookup
  10. Choi EJ, Bae SH, Park JB, Kwon MJ, Jang SM, Zheng YF, Lee YS, Lee S, Bae SK. Simultaneous quantification of caffeine and its three primary metabolites in rat plasma by liquid chromatography-tandem mass spectrometry. Food Chemistry 141(3), 2735-2742.
    doi: 10.1016/j.foodchem.2013.05.069google scholar: lookup
  11. Chou CC, Vickroy TW. Antagonism of adenosine receptors by caffeine and caffeine metabolites in equine forebrain tissues. American Journal of Veterinary Research 64(2), 216-224.
    doi: 10.2460/ajvr.2003.64.216google scholar: lookup
  12. . Commission Decision. 2002/657/EC. Off. J. Eur. Commun. L22 2002;8.
  13. Community Reference Laboratories Residues (CRLs). Guidelines for the validation of screening methods for residues of veterinary medicines (initial validation and transfer). .
  14. Ferraz GC, Teixeira-Neto AR, Mataqueiro MI, Lacerda-Neto JC, Queiroz-Neto A. Effects of intravenous administration of caffeine on physiologic variables in exercising horses. American Journal of Veterinary Research 69(12), 1670-1675.
    doi: 10.2460/ajvr.69.12.1670google scholar: lookup
  15. Guerreiro S, Toulorge D, Hirsch E, Marien M, Sokoloff P, Michel PP. Paraxanthine, the primary metabolite of caffeine, provides protection against dopaminergic cell death via stimulation of ryanodine receptor channels. Molecular Pharmacology 74(4), 980-989.
    doi: 10.1124/mol.108.048207google scholar: lookup
  16. Harkins JD, Rees WA, Mundy GD, Stanley SD, Tobin T. An overview of the methylxanthines and their regulation in the horse. Equine Pratice 20(1), 10-16.
  17. Hertzsch R, Richter A. Systematic analysis to assess the scientific validity of the international residue limits for caffeine and theophylline in horse-racing. Veterinary Record 185(8), 230.
    doi: 10.1136/vr.105404google scholar: lookup
  18. Hetzler RK, Knowlton RG, Somani SM, Brown DD, Perkins RM 3rd. Effect of paraxanthine on FFA mobilization after intravenous caffeine administration in humans. Journal of Applied Physiology 68(1), 44-47.
    doi: 10.1152/jappl.1990.68.1.44google scholar: lookup
  19. Houghton E. Deuteromethylation of dimethylxanthines: A gas chromatographic mass spectrometric method for confirmatory analysis in horse urine extracts. Biomedical Mass Spectrometry 9(3), 103-107.
    doi: 10.1002/bms.1200090305google scholar: lookup
  20. Huang M, Gao JY, Zhai ZG, Liang QL, Wang YM, Bai YQ, Luo GA. An HPLC-ESI-MS method for simultaneous determination of fourteen metabolites of promethazine and caffeine and its application to pharmacokinetic study of the combination therapy against motion sickness. Journal of Pharmaceutical and Biomedical Analysis 62, 119-128.
    doi: 10.1016/j.jpba.2011.12.033google scholar: lookup
  21. IFHA. Recommendations for Control of Feed Contaminants and Environmental Substances 2021. .
  22. Jiang Z, Gao X, Liang J, Ni S. Simultaneous quantitation of serum caffeine and its metabolites by ultra-high-performance liquid chromatography-tandem mass spectrometry for CYP1A2 activity prediction in premature infants. Biomedical Chromatography 35(9), e5141.
    doi: 10.1002/bmc.5141google scholar: lookup
  23. Jodynis-Liebert J, Flieger J, Matuszewska A, Juszczyk J. Serum metabolite/caffeine ratios as a test for liver function. The Journal of Clinical Pharmacology 44(4), 338-347.
    doi: 10.1177/0091270004263468google scholar: lookup
  24. Kaiser S. Untersuchung zur Pharmakokinetik der Methylxanthine Coffein und Theobromin hinsichtlich der Dopingrelevanz beim Pferd. Doctoral dissertation (Vol. 2006) Hannover, Tierärztliche Hochsch..
  25. Kertys M, Žideková N, Pršo K, Maráková K, Kmeťová K, Mokrý J. Simultaneous determination of caffeine and its metabolites in rat plasma by UHPLC-MS/MS. Journal of Separation Science 44(23), 4274-4283.
    doi: 10.1002/jssc.202100604google scholar: lookup
  26. Kowblansky M, Scheinthal BM, Cravello GD, Chafetz L. Specific gas chromatographic determination of xanthines and barbiturates by flash-heater N-butylation. Journal of Chromatography a 76(2), 467-470.
    doi: 10.1016/s0021-9673(01)96932-8google scholar: lookup
  27. Kress M, Meissner D, Kaiser P, Hanke R, Wood WG. The measurement of theophylline in human serum or plasma using gas chromatography and isotope dilution-mass spectrometry (GC-IDMS) taking other substituted xanthines into consideration. Clinical Laboratory 48(9-10), 535-540.
  28. Krul C, Hageman G. Analysis of urinary caffeine metabolites to assess biotransformation enzyme activities by reversed-phase high-performance liquid chromatography. Journal of Chromatography B: Biomedical Sciences and Applications 709(1), 27-34.
    doi: 10.1016/s0378-4347(98)00016-4google scholar: lookup
  29. Lambert MBT, Miller J, Kelly R, Evans JA. Urinary theobromine levels after adding cocoa husks to horses feed. Proceedings of the 6th International Conference of Racing Analysis and Veterinarians (Hong Kong) pp. 137-141.
  30. Löscher W, Ungemach FR, Kroker R. Pharmakotherapie bei Haus-und Nutztieren. Georg Thieme Verlag.
  31. Machnik M, Kaiser S, Koppe S, Kietzmann M, Schenk I, Düe M, Thevis M, Toutain PL. Control of methylxanthines in the competition horse: Pharmacokinetic/pharmacodynamic studies on caffeine, theobromine and theophylline for the assessment of irrelevant concentrations. Drug Testing and Analysis 9(9), 1372-1384.
    doi: 10.1002/dta.2097google scholar: lookup
  32. McLean C, Graham TE. Effects of exercise and thermal stress on caffeine pharmacokinetics in men and eumenorrheic women. Journal of Applied Physiology 93(4), 1471-1478.
    doi: 10.1152/japplphysiol.00762.2000google scholar: lookup
  33. Mendes VM, Coelho M, Tomé AR, Cunha RA, Manadas B. Validation of an LC-MS/MS method for the quantification of caffeine and theobromine using non-matched matrix calibration curve. Molecules 24(16), 2863.
    doi: 10.3390/molecules24162863google scholar: lookup
  34. . OAC-I Peer Verified Methods, Policies and Procedures, 1993. AOAC International 2200 Wilson Blvd., Suite 400, Arlington, Virginia 22201-3301, USA.
  35. Okuro M, Fujiki N, Kotorii N, Ishimaru Y, Sokoloff P, Nishino S. Effects of paraxanthine and caffeine on sleep, locomotor activity, and body temperature in orexin/ataxin-3 transgenic narcoleptic mice. Sleep 33(7), 930-942.
    doi: 10.1093/sleep/33.7.930google scholar: lookup
  36. Ptolemy AS, Tzioumis E, Thomke A, Rifai S, Kellogg M. Quantification of theobromine and caffeine in saliva, plasma and urine via liquid chromatography-tandem mass spectrometry: A single analytical protocol applicable to cocoa intervention studies. Journal of Chromatography B 878(3-4), 409-416.
    doi: 10.1016/j.jchromb.2009.12.019google scholar: lookup
  37. Queiroz-Neto A, Zamur G, Carregaro AB, Mataqueiro MI, Salvadori MC, Azevedo CP, Harkins JD, Tobin T. Effects of caffeine on locomotor activity of horses: Determination of the no-effect threshold. Journal of Applied Toxicology: An International Journal 21(3), 229-234.
    doi: 10.1002/jat.748google scholar: lookup
  38. Reyes-Contreras C, Domínguez C, Bayona JM. Determination of nitrosamines and caffeine metabolites in wastewaters using gas chromatography mass spectrometry and ionic liquid stationary phases. Journal of Chromatography a 1261, 164-170.
    doi: 10.1016/j.chroma.2012.05.082google scholar: lookup
  39. Sniegocki T, Zmudzki J, Posyniak A, Semeniuk S. Gas chromatography-mass spectrometric confirmatory method for the determination of clenbuterol residues in animal urine and liver samples. Bulletin of the Veterinary Institute in Puławy 47(1), 139-144.
  40. Stavric B. Methylxanthines: Toxicity to humans. 3. Theobromine, paraxanthine and the combined effects of methylxanthines. Food and Chemical Toxicology 26(8), 725-733.
    doi: 10.1016/0278-6915(88)90073-7google scholar: lookup
  41. Supelco. Guide to Derivatization Reagents for GC application note. Bulletin 909 A Bellefonte, PA 16823-0048 USA .
  42. Todi F, Mendonca M, Ryan M, Herskovits P. The confirmation and control of metabolic caffeine in standardbred horses after administration of theophylline. Journal of Veterinary Pharmacology and Therapeutics 22(5), 333-342.
    doi: 10.1046/j.1365-2885.1999.00226.xgoogle scholar: lookup
  43. Ventura R, Jiménez C, Closas N, Segura J, De la Torre R. Stability studies of selected doping agents in urine: Caffeine. Journal of Chromatography B 795(2), 167-177.
    doi: 10.1016/s1570-0232(03)00557-9google scholar: lookup
  44. World Anti-Doping Agency. The 2020 Monitoring Program 2021. .
  45. Youden WJ, Steiner EH. Statistical manual of the AOAC-Association of Official Analytical Chemists (p. 35). AOAC-I .

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
Subscribe to our newsletter
Join 99,344 horse owners like you who receive our email updates on horse health and nutrition.