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

The research investigates the presence of isoxsuprine and its specific compound structure in equine urine following administration, identifying the primary form as isoxsuprine-glucuronide or, in some cases, an isoxsuprine-glucuronide-dipotassium complex.

Overview of Study Methodology

• The study utilized positive mode electrospray ionization mass spectrometry (ESI/MS/MS) as the primary tool for analysis. This techinque allows for the identification and quantification of specific molecular structures in a sample.
• Equine urine samples were collected 4-8 hours after the administration of isoxsuprine, as the recovery of isoxsuprine is common in equine forensic science but the exact identity of the material had not previously been established.

Detection and Identification of Isoxsuprine Compounds

• A major peak at m/z 554 was identified in the urine samples, absent from control samples. This reading resisted fragmentation into daughter ions, indicating a stable compound.
• The study then evaluated the behaviour of this material under varying concentrations of sodium acetate. This revealed peaks consistent with the presence of monosodium and disodium isoxsuprine-glucuronide complexes, suggesting the originally detected compound was a dipotassium-isoxsuprine-glucuronide complex.
• The use of negative mode electrospray ionization mass spectrometry further confirmed the presence of a m/z 476 peak, which diminished after enzymatic hydrolysis and resulted in peaks at m/z 300 and 175. These peaks align with the presence of isoxsuprine (m/z 300) and a glucuronic acid residue (m/z 175), further confirming the presence of the isoxsuprine-glucuronide compound.

Confirmation of Findings

• The overlapping spectral readings between the identified compound and authentic morphine-3 and morphine-6-glucuronides suggest that the detected compound also resulted from glucuronic acid conjugation.
• Overall, the results suggest that isoxsuprine occurs primarily in post-administration equine urine samples as an isoxsuprine-glucuronide conjugate or, in certain circumstances, as an isoxsuprine-glucuronide-dipotassium complex.