Determination of nalbuphine in human plasma by automated high-performance liquid chromatography with electrochemical...

A sensitive plasma assay for the agonist/antagonist analgesic nalbuphine utilizing reversed-phase high-performance liquid chromatography (HPLC) with electrochemical detection is described. It involves extracting nalbuphine from plasma at pH 9 into an ethyl acetate/toluene/isopropanol mixture and then back extracting it into aqueous phosphoric acid. The extract is injected onto an octyl column using a solvent mixture of acetonitrile and phosphoric acid. Intra-day assay coefficients of variation (CV) ranged from 0.2 to 4.2% over a concentration range from 0.21 to 42 ng/ml. Nalbuphine extraction recovery exceeded 90% using 1 or 3 ml of plasma. No degradation of the drug in frozen plasma was observed after 18 weeks. The limit of detection is 0.1 ng/ml using 3 ml of plasma.