Bupivacaine in the horse: relationship of local anaesthetic responses and urinary concentrations of 3-hydroxybupivacaine

Bupivacaine is a potent local anaesthetic used in equine medicine. It is al so classified as a Class 2 foreign substance by the Association of Racing C ommissioners International (ARCI). The identification of residues in postra ce urine samples may cause regulators to impose significant penalties. Ther efore, an analytical/pharmacological database was developed for this medica tion. 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 loca l anaesthetic model. Administration of the HNED of bupivacaine to eight hor ses yielded a peak urine concentration of apparent bupivacaine of 23.3 ng/m L 2 h after injection as determined with enzyme-linked immunosorbent assay (ELISA) screening, The major metabolite re covered from beta-glucuronidase-treated equine urin e after dosing with bupivacaine is a hydroxybupivacaine, either 3-hydroxybu pivacaine, 4-hydroxybupivacaine, or a mixture of the two. To determine whic h positional isomer occurs in the horse, 4-hydroxybupivacaine was obtained from Maxxam Analytics, Inc., and 3-hydroxybupivacaine was synthesized, puri fied, and characterized. Furthermore, a quantitative mass spectrometric met hod was developed for the metabolite as recovered from horse urine, Followi ng 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 spec trometry (GC/MS), It was also unequivocally demonstrated with ion chromatog raphy that the hydroxybupivacaine metabolite found in horse urine is exclus ively 3-hydroxybupivacaine and not 4-hydroxybupivacaine. The mean pH of the 4-h urine samples was 7.21; the mean urine creatinine wa s 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 o f bupivacaine or its metabolites after administration of a HNED dose are de tectable by mass spectrometric techniques, This study also suggests that re covery of concentrations less than approximate to 30 ng/mL of 3-hydroxybupi vacaine from postrace urine samples is unlikely to be associated with a rec ent local anaesthetic effect of bupivacaine.