Detection and Identification of Flunixin After Multiple Intravenous and Intramuscular Doses to Horses

Abstract

The objectives of the study were to compare various methods to determine flunixin in test samples collected periodically from horses after intramuscular (IM) and intravenous (IV) dosing at the maximum recommended dosage and to document detection times for this drug in test samples. Flunixin, a nonsteroidal anti-inflammatory drug approved for use in horses, was administered to eight mares in five consecutive daily doses of 1.1 mg per kilogram of body weight by the IM or IV route. Flunixin was detected in urine samples collected at various times after drug administration by flunixin enzyme-linked immunosorbent assay (ELISA), thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and gas chromatographic-mass spectrometric (GC-MS) methods. Detection time was defined as the time period over which flunixin was detected and was dependent on the method used. The shortest detection times were 24 to 48 h and were observed when the TLC method was used. On the other hand, detection times were as long as 15 days when HPLC, GC-MS, and flunixin ELISA methods were used. The use of these more sensitive tests to monitor official samples collected from racehorses could result in positive tests for flunixin when it is exerting no detectable clinical effects because it produces clinical effects lasting only 24-36 h in horses.

Quantification and confirmation of flunixin in equine plasma by liquid chromatography–quadrupole time-of-flight tandem mass spectrometry

“…Previous studies in which flunixin concentration in urine was reported using HPLC and/or GC/MS indicated limits of detection of 50 ng/ml by HPLC and of 5 ng/ml by GC/ MS [11,7,24]. From the results obtained, it can be noted that these methods (HPLC and GC/MS) are limited by low sensitivity.…”

“…With a lower dose of 1.1 mg/kg the third compartment could not be well defined due to the limited LOQ of HPLC method of quantification [4]. Administration of flunixin by intramuscular route prolonged its detection in plasma due to the slower absorption [24,25].…”

“…From the results obtained, it can be noted that these methods (HPLC and GC/MS) are limited by low sensitivity. Unlike GC/MS and HPLC, LC/Q-TOF/MS/MS provides enhancement in sensitivity, with LOD of 0.10 ng/ ml that is nearly 50 times more sensitive for flunixin in plasma than that by GC/MS or HPLC [24]. Other investigators [12][13][14] using LC/MS/MS have demonstrated increased sensitivity in the determination of flunixin and 5-hydroxyflunixin in milk and meat products.…”

“…1). Methods available for the quantification of flunixin in plasma include high performance liquid chromatography (HPLC) [1][2][3][4][5][6]24,25], thin-layer chromatographic (TLC) densitometry and gas chromatography-mass spectrometry (GC/MS) [7][8][9][10][11]24,25]. The use of LC/MS/MS in the determination of flunixin and its metabolite in bovine milk and meat was recently reported [12][13][14].…”

Doping control in horses: housing conditions and oral recycling of flunixin by ingestion of contaminated straw

“…Urine was sampled from spontaneously urinating horses. Based upon results obtained by Sams et al. , 1999; urine samples were collected before administration and after from at least 24 h up to 15 days.…”