This study was designed to develop and validate a simple and efficient high performance liquid chromatography (HPLC) method to determine flunixin concentrations in Asian elephant’s (Elephas maximus) plasma. Flunixin was administered orally at a dose of 0.8 mg/kg, and blood samples were collected. Flunixin extraction was performed by adding an equal amount of acetonitrile to plasma and centrifuging at 4500 rpm for 25 minutes. The supernatant was removed, and flunixin was analyzed using HPLC-UV detection.
Two methods were developed and tested utilizing two different mobile phases either with or without adding methanol (ACN: H2O vs. ACN: H2O: MeOH). Both methods showed excellent linearity and reproducibility. The limit of detection was 0.05 ug/ml and limit of quantification was 0.1 ug/ml. the efficiency of flunixin recovery was maximized by the addition of methanol to mobile phase (ACN: H2O: MeOH as 50:30:20) at 95% in comparison to 23% without methanol.
In conclusion, adding methanol to HPLC methods for extraction of flunixin from elephants’ plasma yielded higher recovery rate than without methanol.
TheUnited States Pharmacopeia. The national formulary. Rockville, MD: The United States Pharmacopeial Convention Inc, USP 32nd revision, NF 27th edition 2009, pp. 2396–2398.
2.
LeesP. and HigginsA.J., Flunixin inhibits prostaglandin E2 production in equine inflammation, Res Vet Sci37 (1984), 347–349.
3.
HardieE.M., RawlingsC.A., ShottsE.B., et al., Escherichia coliinduced lung and liver dysfunction in dogs: effects of flunixin meglumine treatment, Am J Vet Res48 (1987), 56–62.
4.
DowS.W., RosychukR.A., McChesneyA.E., et al., Effects of flunixin and flunixin plus prednisone on the gastrointestinal tract of dogs, Am J Vet Res51 (1990), 1131–1138.
5.
ReedS.K., MesserN.T., TessmanR.K. and KeeganK.G., Effects of phenylbutazone alone or in combination with flunixin meglumine on blood protein concentrations in horses, Am J Vet Res67 (2006), 398–402.
6.
KurtF., MarK.U. and GaraiM., Giants in chains: History, biology and preservation Asian elephants in Asia. In: WemmerC and ChristenCA (eds.). Elephants and Ethics: Toward a Morality of Coexistence, The John Hopkins University Press, Maryland, (2006), pp. 327–345.
7.
OlsenD., North American Region Studbook for the African Elephant (Loxodonta africana). Indianapolis Zoo, (2011), pp. 144.
8.
LewisK.D., ShepherdsonD.J., OwensT.M. and KelleM., A survey of elephant husbandry and foot healthy in North American zoos, Zoo Biol29 (2011), 221–236.
9.
MikotaS.K., SargentE.L., RanglackG.S., Medical management of the elephant. Bloomfield,MI, Indira Publishing House, (1994), pp. 298.
10.
CsutiB., SargentE., BechertU., The Elephant’s Foot: Prevention and Care of Foot Conditions in Captive Asian and African Elephants, Iowa State University Press, (2011), pp. 186.
11.
MortensonJ., SierraS., Determining dosages for anti-inflammatory agents in elephants, Proc Am Assoc Zoo Vet (1998), pp. 477–479.
12.
AnhaltP., Comparative pharmacokinetics of trimethoprim– sulfamethoxazole administered intravenously and orally to captive elephants, J Zoo Wildlife Med22 (1994), 409–416.
13.
CarwardineP.C., Plasma metronidazole levels in an Indian elephant (Elephas maximus) after rectal administration, Vet Rec12 (1987), 120:440.
14.
PittsN.I. and MitchellG., Pharmacokinetics and effects of succinylcholine in African elephant (Loxodonta africana) and impala (Aepyceros melampus), Eur J Pharm Sci15 (2002), 251–260.
15.
RosinE., SchultzD., PerryB. and TeareJ.A., Pharmacokinetics of ampicillin administered orally in Asian elephants, Elephas Maximus24 (1993), 515–518.
16.
OdensvikK. and JohanssonM.I., High Performance liquid chromatography method for determination of flunixin in bovine plasma and pharmacokinetics after single and repeated doses of drug, Am J Vet Res56 (1994), 489–495.
17.
JaussaudP.H., CourtotD. and GuyotJ.J., Identification of a flunixin metabolite in the horse by gas chromatography±mass spectrometry, J Chromat B423 (1987), 123–130.
18.
HardeeG.E., SmithJ.A. and HarrisS.J., Pharmacokinetics of flunixin meglumine in the cow, Res Vet Sci39 (1985), 110–112.
19.
JedziniakP., Determination of FLUNIXIN and 5-HYDROXYFLUNIXIN in Bovine Plasma with HPLC-UV-Method Development, Validation and Verification, Bull Vet Inst Pulawy51 (2007), 261–266.
20.
MiyazakiY., HoriiY., IkenagaN., ShimodaM. and KokueP., Possible active transport mechanism in pharmacokinetics of flunixin-meglumin in rabbits, J Vet Med Sci63 (2001), 885–888.
21.
Wasfi, Pharmacokinetics, metabolism and urinary detection time of flunixin after intravenous administration in camels, J Vet Pharmacol Therap21 (1998), 203–208.
22.
NavarreC.B., RavisW. R., NagillaR.A., SimpkinsS.H. and PughD.G., Pharmacokinetics of phenylbutazone in llamas following single intravenous and oral doses, J Vet Pharmacol Therap24 (2001), 227–231.
23.
SamsR.A., GerkenD.F. and ShcraftA., Detection and identification of flunixin after multiple intravenous and intramuscular doses to horses, J Anal Toxicol23 (1999), 372–378.
24.
BelalF., SawsanA.R., ManalF. and FatmaF., Micellar high performance liquid chromatographic determination of flunixine meglumine in bulk, Anal Chem Res3 (2015), 63–69.
25.
BechertU., ChristensenM., NguyenC., NeelkantR. and BendasE., Pharmacokinetics of orally administered phenylbutazone in African and Asian elephants (Loxodonta africana and Elephas maximus), J Zoo Wildlife Med39 (2008), 188–200.
26.
BechertU. and ChristensenM., Pharmacokinetics of orally administered ibuprofen in African and Asian elephants (Loxodonta africana and Elephas maximus), J Zoo Wildlife Med38 (2007), 258–268.
