title: Bupivacaine in the horse: relationship of local anaesthetic responses and urinary concentrations of 3-hydroxybupivacaine.
abstract: Bupivacaine is a potent local anaesthetic used in equine medicine. It is also classified as a Class 2 foreign substance by the Association of Racing Commissioners International (ARCI). The identification of residues in postrace urine samples may cause regulators to impose significant penalties. Therefore, an analytical/pharmacological database was developed for this medication. The highest no-effect dose (HNED) for the local anaesthetic effect of bupivacaine was determined to be 0.25 mg by using an abaxial sesamoid local anaesthetic model. Administration of the HNED of bupivacaine to eight horses yielded a peak urine concentration of apparent bupivacaine of 23.3 ng/mL 2 h after injection as determined with enzyme-linked immunosorbent assay (ELISA) screening. The major metabolite recovered from beta-glucuronidase-treated equine urine after dosing with bupivacaine is a hydroxybupivacaine, either 3-hydroxybupivacaine, 4-hydroxybupivacaine, or a mixture of the two. To determine which positional isomer occurs in the horse, 4-hydroxybupivacaine was obtained from Maxxam Analytics, Inc., and 3-hydroxybupivacaine was synthesized, purified, and characterized. Furthermore, a quantitative mass spectrometric method was developed for the metabolite as recovered from horse urine. Following subcutaneous injection of the HNED of bupivacaine, the concentration of the hydroxybupivacaine recovered from horse urine reached a peak of 27.4 ng/mL at 4 h after administration as measured by gas chromatography/mass spectrometry (GC/MS). It was also unequivocally demonstrated with ion chromatography that the hydroxybupivacaine metabolite found in horse urine is exclusively 3-hydroxybupivacaine and not 4-hydroxybupivacaine. The mean pH of the 4-h urine samples was 7.21; the mean urine creatinine was 209.5 mg/dL; and the mean urine specific gravity was 1.028. There was no apparent effect of pH, urine creatinine concentration, or specific gravity on the concentration of 3-hydroxybupivacaine recovered. The concentration of bupivacaine or its metabolites after administration of a HNED dose are detectable by mass spectrometric techniques. This study also suggests that recovery of concentrations less than approximately 30 ng/mL of 3-hydroxybupivacaine from postrace urine samples is unlikely to be associated with a recent local anaesthetic effect of bupivacaine.
authors.affiliation: Maxwell H. Gluck Equine Research Center and the Department of Veterinary Science, University of Kentucky, Lexington 40506-0099, USA.
authors.forename: J D
authors.initials: JD
authors.lastname: Harkins
authors.forename: A
authors.initials: A
authors.lastname: Lehner
authors.forename: W
authors.initials: W
authors.lastname: Karpiesiuk
authors.forename: W E
authors.initials: WE
authors.lastname: Woods
authors.forename: L
authors.initials: L
authors.lastname: Dirikolu
authors.forename: J
authors.initials: J
authors.lastname: Boyles
authors.forename: W G
authors.initials: WG
authors.lastname: Carter
authors.forename: T
authors.initials: T
authors.lastname: Tobin
mesh_terms: D000779:Anesthetics, Local / Q000008:administration & dosage / Q000493:pharmacokinetics* / Q000652:urine; D000818:Animals; D002045:Bupivacaine / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000737:chemistry / Q000493:pharmacokinetics* / Q000652:urine; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D008401:Gas Chromatography-Mass Spectrometry / Q000662:veterinary; D006736:Horses / Q000378:metabolism* / Q000652:urine; D007279:Injections, Subcutaneous / Q000662:veterinary
chemical_list: C121551:3-hydroxybupivacaine; D000779:Anesthetics, Local; D002045:Bupivacaine