Determination of morphine by high-performance liquid chromatography with electrochemical detection: application to human and rabbit pharmacokinetic studies

A rapid, sensitive, precise and accurate high-performance liquid chromatographic assay with coulometric electrochemical detection was developed for the determination of morphine in human, rabbit, pig and dog plasma. It includes a one-step extraction procedure with hexane–isoamyl alcohol (1:1, v/v) at pH 8.9 (adjusted with phosphoric acid) and reversed-phase liquid chromatography on a μPorasil column. The mobile phase was composed of 5 mM sodium acetate buffer (pH 3.75)–acetonitrile (25:75, v/v). A flow-rate of 1.2 ml/min at 20°C was used. The working potentials for the electrochemical detector were +0.20 V for detector cell 1, +0.55 V for detector cell 2 and +0.75 V for the guard cell. The limit of detection of morphine was 100 pg/ml of plasma. Repeatability, precision and accuracy were also determined concomitantly. The calibration graphs were linear in the concentration range 0.25–250 ng/ml with correlation coefficients of 0.998±0.01 and with a minimum intercept of 0.05±0.08. The precision in plasma was acceptable, with coefficients of variation less than 11%. The absolute recoveries of morphine and nalbuphine (internal standard) were between 86 and 89% and independent of morphine concentration. Pharmacokinetics after oral morphine MST Continus™ (morphine sulphate tablets) 30 mg, Bard Pharmaceutical, Cambridge, UK] in humans revealed a one-compartment first-order absorption model with one absorption phase and one elimination phase. The absorption and elimination half-lives were 2.46 and 1.80 h, respectively. Pharmacokinetics after intravenous morphine (3 mg/kg) in rabbits showed a linear two-compartment open model with one distribution phase and one elimination phase. The distribution and elimination half-lives were 0.5 and 33.8 h, respectively.