AJAC  Vol.5 No.17 , December 2014
Doping Control Analysis of 16 Non-Steroidal Anti-Inflammatory Drugs in Equine Plasma Using Liquid Chromatography-Tandem Mass Spectrometry
Abstract: Non-steroidal anti-inflammatory drugs (NSAIDs) are classified as Class 4 agents by the Association of Racing Commissioners International and are banned in racehorses during competition in Pennsylvania (PA). To control the abuse of these agents in racehorses competing in PA, a forensic method for screening and confirmation of the presence of these agents is needed. Equine plasma (0.5 mL) was acidified with 75 μL 1M H3PO4 to increase recovery of the analytes by liquid-liquid extraction using methyl tert-butyl ether (MTBE). Extracted analytes were separated by reversed-phase liquid chromatography using a C8 column under gradient condition. All 16 analytes were detected, quantified and confirmed using a triple quadrupole tandem mass spectrometry with selected reaction monitoring (SRM) in both negative and positive electrospray ionization modes. The limit of detection, quantification and confirmation of the analytes were 1.0 - 5.0 ng/mL, 1.0 - 5.0 ng/mL and 1.0 - 20 ng/mL, respectively. The linear dynamic range of quantification was 5.0 - 200 ng/mL. The method is routinely used in anti-doping analysis to control the abuse of NSAIDs in racehorses competing in PA.
Cite this paper: You, Y. , Uboh, C. , Guan, F. and Soma, L. (2014) Doping Control Analysis of 16 Non-Steroidal Anti-Inflammatory Drugs in Equine Plasma Using Liquid Chromatography-Tandem Mass Spectrometry. American Journal of Analytical Chemistry, 5, 1184-1199. doi: 10.4236/ajac.2014.517126.

[1]   Russeva, V. and Mihailova, D. (1995) Binding of Phenylbutazone to Human Serum Albumin-Characterization and Identification of Binding Sites. Arzneimittel Forschung/Drug Research, 49, 255-258.

[2]   Rahim, S. and Anne-Francoise, A. (1995) Location of Binding Sites on Immobilized Human Serum Albumin for Some Nonsteroidal Anti-inflammatory Drugs. Journal of Pharmaceutical Sciences, 84, 949-952.

[3]   Gerrig, E.L., Lees, P. and Taylor, J.B. (1981) Pharmacokinetics of Phenylbutazone and Its Metabolites in the Horse. Equine Veterinary Journal, 13, 152-157.

[4]   Van Eenoo, P., Delbeke, F.T., Roels, K. and Baert, K. (2003) Detection and Disposition of Tolmetin in the Horse. Journal of Pharmaceutical and Biomedical Analysis, 31, 723-730.

[5]   You, Y.W., Uboh, C.E., Soma, L.R., Guan, F.Y., Li, X.Q., Rudy, J.A. and Chen, J.W. (2008) Screening, Quantification, and Confirmation of Phenylbutazone and Oxyphenbutazone in Equine Plasma by Liquid Chromatography-Tandem Mass Spectrometry. J. Anal. Toxicol. 33, 41-50.

[6]   Dirikolu, L., Woods, W.E., Boyles, J., Lehner, A.J., Harkins, J.D., Fisher, M., Schaeffer, D.J. and Tobin, T. (2009) Nonsteroidal Anti-Inflammatory Agents and Musculoskeletal Injuries in Thoroughbred Racehorses in Kentucky. Journal of Veterinary Pharmacology and Therapeutics, 32, 271-279.

[7]   Jones, E.W. and Hamm, D. (1978) Comparative Efficacy of Phenylbutazone and Naproxen in Induced Equine Myositis. Journal of Equine Medicine and Surgery, 2, 341-347.

[8]   Soma, L.R., Uboh, C.E. and Maylin, G.A. (2012) The Use of Phenylbutazone in The Horse. Journal of Veterinary Pharmacology and Therapeutics, 35, 1-12.

[9]   Insel, P.A. (1990) Analgesic-Antipyretics and Antiinflammatory Agents; Drugs Employed in The Treatment of Rheumatoid Arthritis and Gout. In: Goodman Gilman, A., Rail, T., Nies, A.S. and Taylor, P., Eds., The Pharmacological Basis of Therapeutics, Pergamon Press Inc., New York, 638-681.

[10]   Kazemifard, A.G. and Moore, D.E. (1990) Liquid Chromatography with Amperometric Detection for The Determination of Non-Steroidal Anti-Inflammatory Drugs in Plasma. Journal of Chromatography A, 533, 125-132.

[11]   Sun, Y., Takaba, K., Kido, H., Nakashima, M.N. and Nakashima, K. (2003) Simultaneous Determination of Arylpropionic Acidic Non-Steroidal Anti-Inflammatory Drugs in Pharmaceuitical Formulations and Human Plasma by HPLC with UV Detection. Journal of Pharmaceutical and Biomedical Analysis, 30, 1611-1619.

[12]   Arcelloni, C., Lanzi, R., Pedercini, S., Molteni, G., Fermo, I., Pontiroli, A. and Paroni, R. (2001) High-Performance Liquid Chromatographic Determination of Diclofenac in Human Plasma after Solid-Phase Extraction. Journal of Chromatography B, 763, 195-200.

[13]   Mikami, E., Goto, T., Ohno, T., Matsumoto, H., Inagaki, K., Ishihara, H. and Nishida, M. (2000) Simultaneous Analysis of Anthranilic Acid Derivatives in Pharmaceuticals and Human Urine by High-Performance Liquid Chromatography with Isocratic Elution. Journal of Chromatography B, 744, 81-89.

[14]   Hirai, T., Matsumoto, S. and Kishi, I. (1997) Simultaneous Analysis of Several Non-Steroidal Anti-Inflammatory Drugs in Human Urine by High-Performance Liquid Chromatography with Normal Solid-Phase Extraction. Journal of Chromatography B, 692, 375-388.

[15]   Battista, H.J., Wehinger, G. and Henn, R. (1985) Separation and Identification of Non-Steroidal Antirheumatic Drugs Containing a Free Carboxyl Function Using High-Performance Liquid Chromatography. Journal of Chromatography, 345, 77-89.

[16]   Heitmeier, S. and Blaschke, G. (1999) Direct Assay of Nonopioid Analgesics and Their Metabolites in Human Urine by Capillary Electrophoresis and Capillary Electrophoresis-Mass Spectrometry. Journal of Chromatography B, 721, 109-125.

[17]   El Haj, B.M., Al Ainri, A.M., Hassan, M.H., Bin Khadem, R.K. and Marzouq, M.S. (1999) The GC/MS Analysis of Some Commonly Used Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) in Pharmaceutical Dosage Forms and in Urine. Forensic Science International, 105, 141-153.

[18]   Maure, H.H., Tauvel, F.X. and Kraemer, T. (2001) Screening Procedure for Detection of Non-Steroidal Anti-Inflammatory Drugs and Their Metabolites in Urine as Part of a Systematic Toxicological Analysis Procedure for Acidic Drugs and Poisons by Gas Chromatography-Mass Spectrometry after Extractive Methylation. Journal of Analytical Toxicology, 25, 237-244.

[19]   Hines, S., Pearce, C., Bright, J. and Teale, P. (2004) Development and Validation of a Quantitative Gas Chromatography Mass Spectrometry Confirmatory Method for Phenylbutazone in Equine Plasma. Chromatographia, 59, 109-114.

[20]   Clark, S.B., Turnipseed, S.B., Nandea, G.J. and Madson, M.R. (2004) Confirmation of Phenylbutazone Residues in Bovine Kidney by Liquid Chromatography/Mass Spectrometry. Journal of AOAC International, 85, 1009-1014.

[21]   Hoof, N.V., Wasch, K.D., Poelmans, S., Noppe, H. and Brabander, H.D. (2004) Multi-Residue Liquid Chromatography/ Tandem Mass Spectrometry Method for the Detection of Non-Steroidal Anti-Inflammatory Drugs in Bovine Muscle: Optimization of Ion Trap Parameters. Rapid Communications in Mass Spectrometry, 18, 2823-2829.

[22]   Vinci, F., Fabbrocine, S., Fiori, M., Serpe, L. and Gallo, P. (2006) Determination of Fourteen Non-Steroidal Anti-Inflammatory Drugs in Animal Serum and Plasma by Liquid Chromatography/Mass Spectrometry. Rapid Communications in Mass Spectrometry, 20, 3412-3420.

[23]   Suenami, K., Lim, L.W., Takeuchi, T., Sasajima, Y., Sato, K., Takekoshi, Y. and Kanno, S. (2007) Online Sample Extraction and Enrichment of Non-Steroidal Anti-Inflammatory Drugs by Pre-Column in Capillary Liquid Chromatography Mass Spectrometry. Journal of Chromatography B, 846, 176-183.

[24]   Heffron, B., Taddei, L., Benoit, M. and Negrusz, A. (2013) Quantification of Several Acidic Drugs in Equine Serum Using LC-MS-MS. Journal of Analytical Toxicology, 37, 600-604.

[25]   Bethem, R., Boison, J., Gale, J., Heller, D., Lehotay, S., Loo, J., Musser, S., Price, P. and Stein, S. (2003) Establishing the Fitness for Purpose of Mass Spectrometric Methods. Journal of the American Society for Mass Spectrometry, 14, 528-541.