Determination of nalbuphine by high-performance liquid chromatography with electrochemical detection: application to...

A rapid, selective and reproducible high-performance liquid chromatographic assay with electrochemical detection was developed for the determination of nalbuphine in human plasma. The method involves extraction with chloroform-isopropanol at pH 9.4, back-extraction into dilute phosphoric acid and reversed-phase chromatography on a microBondapak phenyl column. The recovery of nalbuphine and naltrexone (internal standard) was greater than 90%. Calibration curves were linear over a concentration range of 3-36 ng/ml with coefficients of variation, within-day or between-day, not exceeding 8% at any level. Although the limit of detection was 0.3 ng/ml based on a signal-to-noise ratio of 3, the reliable limit of quantitation was 1 ng/ml (coefficient of variation 12%) using 1 ml of plasma. The dual-electrode detector was operated in the screening mode of oxidation (electrode 1, 0.3 V and electrode 2, 0.6 V), providing a greater specificity and reducing background noise. This procedure was applied to a large number of clinical samples in an intravenous dose-range pharmacokinetic study in patients.