Direct MS-MS identification of isoxsuprine-glucuronide in post-administration equine urine

Isoxsuprine is routinely recovered from enzymatically-hydrolyzed, post-admi nistration urine samples as parent isoxsuprine in equine forensic science. However, the specific identity of the material in horse urine from which is oxsuprine is recovered has never been established, although it has long bee n assumed to be a glucuronide conjugate (or conjugates) of isoxsuprine. Usi ng ESI/MS/MS positive mode as an analytical tool, urine samples collected 4 -8 h after isoxsuprine administration yielded a major peak at m/z 554 that was absent from control samples and resisted fragmentation to daughter ions . Titration of this material with increasing concentrations of sodium aceta te yielded m/z peaks consistent with the presence of monosodium and disodiu m isoxsuprine-glucuronide complexes, suggesting that the starting material was a dipotassium-isoxsuprine-glucuronide complex. Electrospray ionization mass spectrometry negative mode disclosed the presence of a m/z 476 peak th at declined following enzymatic hydrolysis and resulted in the concomitant appearance of peaks at m/z 300 and 175. The resulting peaks were consistent with the presence of isoxsuprine (m/z 300) and a glucuronic acid residue ( m/z 175). Examination of the daughter ion spectrum of this putative isoxsup rine-glucuronide mit 476 peak showed overlap of many peaks with those of si milar spectra of authentic morphine-3- and morphine-6-glucuronides, suggest ing they were derived from glucoronic acid conjugation. These data suggest that isoxsuprine occurs in post-administration urine samples as an isoxsupr ine-glucuronide conjugate and also, under some circumstances, as an isoxsup rine-glucuronide-dipotassium complex.