Determination and stability of some international screening limited drugs in equine blood by LC-MS/MS

Abstract

Stability of some international screening limited therapeutic substances for doping control in equine blood has been investigated with a validated method by using LC-MS/MS after chemical hydrolysis with orthophosphoric acid. Whole blood samples were extracted with a WAX cartridge on auto-SPE. Stability test of the drug substances performed at the same time in working solution and matrix at +4°C, -20°C, +20°C in dark and +20°C in light conditions for 1, 2, 3 and 4 weeks. For short-time stability, the effects of 6 h storage at +55°C was also investigated. Repeated data were statistically analyzed with ANOVA. All substances in working solution were stable at -20°C and +4°C. In the blood matrix, higher temperatures (+20°C) in light and dark caused degradation of substances at the end of four weeks and short-term study (P< 0.05). In conclusion, these international screening limited substances in blood matrix could be unstable related to temperature and storage time, although in working solution they could be mostly stable in various temperatures for four weeks.

Keywords

• Doping control,equine blood,LC-MS/MS,stability

References

1. 1. Atanasov VN, Stoykova S, Runiov A, et al (2012): Stability of diazepam in blood samples at different storage conditions and in the presence of alcohol. Forensic Sci Int, 215, 159-163.
2. 2. Clauwaert KM, Van Bocxlaer JF, De Leenheer AP (2001): Stability study of the designer drugs "MDA, MDMA and MDEA" in water, serum, whole blood, and urine under various storage temperatures. Forensic Sci Int, 124, 36-42.
3. 3. Commission Decision (2002): Concerning the performance of analytical methods and the interpretation of results. European Comission, 2002/657/EC.
4. 4. Dugan S, Bogema S, Schwartz RW, et al (1994): Stability of drugs of abuse in urine samples stored at -20°C. J Anal Toxicol, 18, 391-396.
5. 5. Giorgi SN, Meeker JE (1995): A 5-year stability study of common illicit drugs in blood. J Anal Toxicol, 19, 392-8.
6. 6. Hippenstiel MJ, Gerson B (1994): Optimization of storage conditions for cocaine and benzoylecgonine in urine: a review. J Anal Toxicol, 18, 104-109.
7. 7. International Federation of Horseracing Authorities (2019): International agreement on breeding, racing and wagering and appendixes (February 2019).https://www.ifhaonline.org/resources/ifAgreement.pdf (14 February 2019).
8. 8. Jane Ellis M, Livesey JH, Evans MJ (2003): Hormone stability in human whole blood. Clinical Biochem, 36, 109–112.

9. 9. Jiménez C, Ventura R, Segura J, et al (2004): Protocols for stability and homogeneity studies of drugs for its application to doping control. Anal Chim Acta, 515, 323-331.
10. 10. Jiménez C, de la Torre R, Segura J, et al (2006): Stability studies of testosterone and epitestosterone glucuronides in urine. Rapid Commun Mass Spectrom, 20, 858-864.
11. 11. Jiménez C, de la Torre R, Ventura M, et al (2006): Stability studies of amphetamine and ephedrine derivatives in urine. J Chromatogr B Analyt Technol Biomed Life Sci, 843, 84-93.
12. 12. Kita K, Ishii T, Hotta K, et al (2018): A dried blood spot assay with UPLC-MS/MS for the simultaneous determination of E6005, a phosphodiesterase 4 inhibitor, and its metabolite in human blood. J Pharm Biomed Anal, 157, 208-216.
13. 13. Moody DE, Monti KM, Spanbauer AC (1999): Long-term stability of abused drugs and antiabuse chemotherapeutical agents stored at -20°C. J Anal Toxicol, 6, 535-40.
14. 14. Moulard Y, Bailly-Chouriberry L, Boyer S, et al (2011): Use of benchtop exactive high resolution and high mass accuracy orbitrap mass spectrometer for screening in horse doping control. Anal Chim Acta, 700, 126-136.
15. 15. Peris‐Vicente J, Esteve‐Romero J, Carda‐Broch S (2015): Validation of analytical methods based on chromatographic techniques: An overview. 1757-1808. In: LA Jared, A Berthod, VP Estévez, AM Stalcup (Ed), Analytical Separation Science. Wiley-VCH Verlag GmbH & Co. KGaA, Germany.
16. 16. Popot MA, Garcia P, Hubert C, et al. (2014): HPLC/ESI-MS(n) method for non-amino bisphosphonates: application to the detection of tiludronate in equine plasma. J Chromatogr B Analyt Technol Biomed Life Sci, 958, 108-116.
17. 17. Saar E, Gerostamoulos D, Drummer OH, et al (2012): Assessment of the stability of 30 antipsychotic drugs in stored blood specimens. Forensic Sci Int, 215, 152-158.
18. 18. Saudan C, Entenza JM, Baume N, et al (2006): Short-term stability of testosterone and epitestosterone conjugates in urine samples: quantification by liquid chromatography-linear ion trap mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci, 844, 168-174.
19. 19. Schenk I, Machnik M, Guddat S, et al (2008): Stability of a representative selection of drugs in horse blood. 225-235. In: E Houghton, FI Keskin, JF Wade, N Yazıcıoglu (Ed), Proceeding of the 17th International Conference of Racing Analysts and Veterinarians, Antalya, Turkey.
20. 20. Shah VP, Midha KK , Dighe S, et al (1992): Analytical methods validation: Bioavailability, bioequivalence, and pharmacokinetic studies. J Pharm Sci, 81, 309-312.
21. 21. Tevis DS, Jarrett JM, Jones DR, et al. (2018): Assessing the stability of Cd, Mn, Pb, Se, and total Hg in whole human blood by ICP-DRC-MS as a function of temperature and time. Clin Chim Acta, 485, 1-6.
22. 22. Van der Merwe PJ, Pieterse JW (1994): Stability of zeranol, nandrolone and trenbolone in bovine urine. Analyst, 119, 2651-2653.
23. 23. Van der Merwe PJ, Grobbelaar E (2003): Stability of 19-norandrosterone and 19-noretiocholanolone glucuronide in urine under various storage conditions. SA Sports Medicine, 15, 14-17.
24. 24. Van Eenoo P, Lootens L, Spaerkeer A, et al (2007): Results of stability studies with doping agents in urine. J Anal Toxicol, 31, 543-548.
25. 25. Ventura R, Jiménez C, Closas N, et al (2003): Stability studies of selected doping agents in urine: caffeine. J Chromatogr B Analyt Technol Biomed Life Sci, 795, 167-177.
26. 26. Wong JK, Wan TS (2014): Doping control analyses in horseracing: a clinician's guide. Vet J, 200, 8-16.