Stereoselective high-performance liquid chromatographic assay with fluorometric detection of the three isomers of mivacurium and their cis- and trans-alcohol and ester metabolites in human plasma

An improved high-performance liquid chromatography assay for the three stereoisomers of the muscle relaxant mivacurium and its metabolites in plasma is presented. The principal steps in the assay are precipitation of plasma proteins by acetonitrile, lyophilization of the supernatant and ion-exchange chromatography on Spherisorb 5-SCX column, with gradient elution (acetonitrile from 32 to 68% v/v and ionic gradient from 7 to 56 mmol l⁻¹ Na₂SO₄), a flow-rate of 2.0 ml min⁻¹, d-tubocurarine as internal standard and fluorometric detection (excitation wavelength=280 nm, emission wavelength=320 nm). Quantitation limit of cis-cis, cis-trans, trans-trans isomers were 0.003, 0.002 and 0.005 μmol l⁻¹, respectively. Quantitation limits for the monoester_cis metabolite were 0.011 μmol l⁻¹, for the monoester_trans metabolite 0.017 μmol l⁻¹, for the amino-alcohol_trans 0.020 μmol l⁻¹ and for the amino-alcohol_cis 0.021 μmol l⁻¹. None of eight drugs used during anaesthesia interfered with the assay in vitro. Satisfactory performance was demonstrated by the measurement of the isomers and their metabolites in plasma of two patients over a 6-h period after repeated injections of mivacurium.