Electrochemical chromatographic determinations of morphine antagonists in biological fluids, with applications

The morphine antagonists naltrexone and naloxone were extracted from plasma and urine, separated on a chromatographic column, and assayed by electrochemical detection. Optimum oxidation potentials were 0.65 V for morphine and 0.75 V for naloxone and naltrexone. Assay sensitivities were 2-5 ng/mL for plasma and 10 ng/mL for urine. The assays were applied to determine red blood cell partition coefficients of 1.83 +/- 0.15 (SD) for naltrexone and 1.49 +/- 0.27 (SD) for naloxone in a concentration range of 10-3500 ng/mL. No significant time dependence for the partitioning could be observed. Plasma protein binding in the same concentration range, determined by ultracentrifugation, was 27.7% +/- 2.5% (SD) for naltrexone and 30.1% +/- 5.1% (SD) for naloxone. The degree of protein binding did not change in the presence of morphine for morphine-antagonist ratios between 1:10 and 10:1. No concentration dependencies of red blood cell partitioning or protein binding were observed.