Characterization of bromhexine and ambroxol in equine urine: effect of furosemide on identification and confirmation

The purpose of this study was two-fold: (1) to develop a simple and sensitive screening procedure for identifying and confirming bromhexine and ambroxol and, (2) to determine the effect of furosemide on the detection of bromhexine, ambroxol, or their metabolites in urine. Female horses (450-550 kg) treated with bromhexine or ambroxol (1 g, p.o.) were used. Urine samples were collected up to 48 h post-drug administration and analysed. Blind samples were used in evaluating the sensitivity of these methods and reproducibility of the results. Bromhexine and ambroxol were extensively metabolized in the horse. These agents and their respective metabolites were identified and confirmed using thin-layer chromatography (TLC) and gas chromatography-mass spectrometry (GC-MS), respectively. Hydroxy-bromhexine and desmethyl-bromhexine were major metabolites found to be unique to bromhexine-treated horses. These metabolites selectively absent from ambroxol-treated horse urine provide a chemical means to distinguish bromhexine from ambroxol administration in horses. These specific metabolites were similarly identified and confirmed in "blind" horse urine samples. The concomitant presence of furosemide (300 mg, i.v.) with bromhexine or ambroxol did not mask the presence of these agents or alter their metabolite profile. By application of the methods described in this study, bromhexine and ambroxol metabolites in horse urine can be easily identified and confirmed.