Simultaneous determination of codeine and it seven metabolites in plasma and urine by high-performance liquid chromatography with ultraviolet and electrochemical detection

Abstract

A sensitive and selective high-performance liquid chromatography method has been developed for the measurement of codeine and its seven metabolites, norcodeine, morphine, normorphine, codeine-6-glucuronide, morphine-6-glucuronide, morphine-3-glucuronide and norcodeine glucuronide, in plasma and urine. The compounds were recovered from plasma and urine using solid-phase extraction with C 18 cartridges and separated on a reversed-phase C 8 column with a mobile phase consisting of 77% buffer (5 m M sodium phosphate monobasic and 0.70 m M sodium dodecyl sulfate, pH 2.35) and 23% acetonitrile. Codeine, norcodeine, codeine-6-glucuronide, norcodeine glucuronide and morphine-3-glucuronide were detected by ultraviolet detection at 214 nm, with a detection limit of 0.02 nmol/ml for each compound in plasma. Morphine-6-glucuronide, normorphine and morphine were monitored by electrochemical detection at 350 mV, with a detection limit of 0.003 nmol/ml for each compound in plasma. The assay showed good reproducibility and accuracy using external standardization. The recovery and inter-day variation for all compounds in plasma samples were 63.40–77.90% and 3.49–16.77% (R.S.D.) and while in urine were 64.98–90.13% and 2.93–9.96% (R.S.D.), respectively.