Abstract
In equine sport, salicylic acid is prohibited with a threshold level of 750 µg mL−1 in urine; hence, doping control laboratories have to establish quantitative and qualitative methods for its determination. A simple and rapid liquid chromatographic/mass spectrometric method was developed and validated for the quantification and identification of salicylic acid. Urine samples after 900-fold dilution and addition of the internal standard (4-methylsalicylic acid) were directly injected to the liquid chromatography/quadrupole time-of-flight mass spectrometry system. Electrospray ionization in negative mode with full scan acquisition mode and product ion scan mode were chosen for the quantification and identification of salicylic acid, respectively. Run time was 2.0 min. The tested linear range was 2.5–50 µg mL−1 (after 100-fold sample dilution). The relative standard deviations of intra- and inter-assay analysis of salicylic acid in horse urine were lower than 2.5% and 2.8%, respectively. Overall accuracy (relative percentage error) was less than 3.3%. Method was applied to two real samples found to be positive for salicylic acid, demonstrating simplicity, accuracy, and selectivity.
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References
Beaumier PM, Fenwick JD, Stevenson AJ, Weber MP, Young LM (1987) Equine Vet. J. 19:207–213
Schulz R, Hagedorn HW, Böck M (1992) Proceedings of the 9th International Conference of Racing Analysts and Veterinarians, New Orland, pp. 223-227
Lakhani KH, Lambert M, Sluyter F, Devolz R, Maylin G, Higgins AJ (2004) Proceedings of the 15th International Conference of Racing Analysts and Veterinarians, Dubai, United Arab Emirates, pp. 67-77
International Federation of Horse racing Authorities. International agreement on breeding, racing and wagering. Article 6. Available: www.horseracingintfed.com/resources/2007_choose_eng.pdf (Aug. 2007)
Marsh MV, Caldwell J, Smith RL (1981) Xenobiotica 11:655–663
Kees F, Jehnich D, Grobecker H (1996) J Chromatogr B 677:172–177
Bakar SK, Niazi S (1983) J. Pharm. Sci. 72:1020–1023
Peng GW, Gadalla MA, Smith V, Peng A, Chiou WL (1978) J. Pharm. Sci. 67:710–712
O’Kruk RJ, Adams MA, Philp RB (1984) J Chromatogr B 310:343–352
Pirola R, Bareggi SR, De Benedittis G (1998) J Chromatogr B 705:309–315
Battezzati A, Fiorillo G, Spadafranca A, Bertoli S, Testolin G (2006) Anal. Biochem 354:274–278
Judefeind A, Van Rensburg P, Langelaar S, Du Plessis J (2007) J. Chromatogr. B 852:300–307
Gonzátlez G, Ventura R, Smith AK, De La Torre R, Segura J (1996) J. Chromatogr. A 719:251–264
Croubels S, Maes S, Baert K, De Backer P (2005) Analytica Chimica Acta 529:179–187
Wang N, Xu F, Zhang Z, Yang C, Sun X, Li J (2008) Biomed Chromatogr 22:149–156
Bae SK, Seo KA, Jung EJ, Kim HS, Yeo SW, Shon JH, Park KM, Liu KH, Shin JG (2008) Biomed Chromatogr 22:590–595
Segarra G, Jauregui O, Casanova E, Trillas I (2006) Phytochemistry 67:395–401
Wilbert SM, Ericsson LH, Gordon MP (1998) Anal. Biochem 257:186–194
Pirkera R, Hucka CW, Popp M, Bonn GK (2004) J Chromatogr B 809:257–264
Vinci F, Fabbrocino S, Fiori M, Serpe L, Gallo P (2006) Rapid Commun Mass Spectrom 20:3412–3420
Gallo P, Fabbrocino S, Vinci F, Fiori M, Danese V, Serpe L (2008) Rapid Commun Mass Spectrom 22:841–854
Van Hoof N, De Wasch K, Poelmans S, Noppe H, De Brabander H (2004) Rapid Commun Mass Spectrom 18:2823–2829
Vonaparti A, Lyris E, Panderi I, Koupparis M, Georgakopoulos C (2008) J. Mass Spectrom 43:1255–1264
Vonaparti A, Lyris E, Panderi I, Koupparis M, Georgakopoulos C (2009) Rapid Commun Mass Spectrom 23:1020–1028
Association of Official Racing Chemists (2002). AORC guidelines for the minimum criteria for identification by chromatography and mass spectrometry. MS Criteria Working group-19 June 2002 Version. Internal communication to AORC members.
Van Eenoo P, Delbeke FT (2004) Chromatographia 59:S39–S44
Verwaal W, Van Bavel M, Boot A, Bravenboer J, De Goei F, Maas V, Van der Putten A (1996) DeWaren Chemicus 26:106
Annesley TM (2003) Clin. Chem 49:1041–1044
Matuszewski BK, Constazer ML, Chavel-Eng CM (2003) Analytical Chemistry 75:3019–3030
Acknowledgments
The authors sincerely thank Agilent Technologies (Santa Clara, California, USA) for the provision of the LC/QTOF-MS 6520 instrument. A. Vonaparti, PhD student, gratefully acknowledges the State Scholarships Foundation of Athens, Greece.
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Vonaparti, A., Lyris, E., Panderi, I. et al. Direct injection horse urine analysis for the quantification and identification of threshold substances for doping control. III. Determination of salicylic acid by liquid chromatography/quadrupole time-of-flight mass spectrometry. Anal Bioanal Chem 395, 1403–1410 (2009). https://doi.org/10.1007/s00216-009-3047-7
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DOI: https://doi.org/10.1007/s00216-009-3047-7