High-performance liquid chromatographic determination of the conjugate metabolites of moxisylyte in human plasma and urine

Sensitive and specific high-performance liquid chromatographic methods with fluorescence detection are described for the determination of the metabolites of mox sylyte (4-(2-dimethylaminoethoxy)-5-isopropyl-2-methylphenyl acetate) in human plasma and urine. Deacetylmoxisylyte glucuroconjugate (DAM-G) was hydrolysed enzymatically using β-glucuronidase and quantified as the difference between the DAM concentrations determined after and before hydrolysis. The two sulphate derivatives (deacetylmoxisy;yte sulphoconjugate, DAM-S and monomethyldeacetylmoxisylyte sulphoconjugate, MDAM-S), were analysed without prior hydrolysis. Their extraction from plasma and urine, as well as that of DAM from plasma, involved the use of C₁₈ cartridges adapted on a Benchmate workstation. DAM in urine was quantified after liquid-liquid extraction. The two methods were validated for specificity, linearity, intra- and inter-day precision and accuracy. Precision was generally ≤15% and accuracy ≤12%. In plasma, the limits of quantification were 2.5 ng/ml for DAM and 2.8 ng/ml for the two sulphates; in urine, they were 40 ng/ml for DAM and 200 ng/ml for the sulphates. These methods were used for pharmacokinetic studies in healthy subjects.