Identification of hydroxyropivacaine glucuronide in equine urine by ESI⁺/MS/MS

Ropivacaine is a local anesthetic that has a high potential for abuse in racing horses. It can be recovered from urine collected after administration as a hydroxylated metabolite following β-glucuronidase treatment of the urine. Based on these findings, it has been inferred that ropivacaine is present in equine urine as a glucuronide metabolite; however, these metabolites have never been directly identified. Using ESI⁺/MS/MS, the presence of a [M+H]⁺ molecular ion of m/z 467 was demonstrated in urine corresponding to the calculated mass of a hydroxyropivacaine glucuronide + 1. The abundance of this ion diminished after glucuronidase treatment with concomitant appearance of a m/z 291 peak, which is consistent with its hydrolysis to hydroxyropivacaine. In further work, the m/z 467 material was fragmented in the MS/MS system, yielding fragments interpretable as hydroxyropivacaine glucuronide. These data are consistent with the presence of a hydroxyropivacaine glucuronide in equine urine and constitute the first direct demonstration of a specific glucuronide metabolite in equine urine.