Assays - Dextrorphan and 3-Hydroxymorphinan

High-performance liquid-chromatographic determination of Dextrorphan and 3-Hydroxymorphinan in human plasma based on a selective pre-column sample cleanup

Author: H.J. Mascher
Publisher: J. Chromatogr. 420 (1987) 217-222

Dextromethorphan in a widely used non-narcotic antitussive agent. Its major metabolites are dextrorphan and 3-hydroxymorphinan. Since both substances are found in human plasma almost exclusively as glucuronides and sulphates, enzymic hydrolysis is a necessary part of the determination process. Several methods have been described for the determination of dextrorphan in human plasma, and one of these has also been used for the determination of 3-hydroxymorphinan. The assays were based on gas chromatography with flame-ionization detection, high-performance liquid chromatography (HPLC) with fluorescence detection)m HPLC with electrochemical detection and fluorimetry. Some of these procedures are very time-consuming, involving extraction, evaporation and lenghty chromatography; moreover, some of them lack selectivity and sensitivity.
This paper describes a procedure for the determination of dextrorphan and 3-hydroxymorphinan in human plasma, based on pre-column clean-up following enzymic hydrolysis. The first step is a selective sample clean-up on-line on a pre-column, the second step is the separation on a reversed-phase column with fluorescence detection. This procedure is highly sensitive: the limit of detection for dextrorphan and 3-hydroxymorphinan is as low as 0.2 ng/ml plasma. Moreover, this procedure is rapid, because the time-consuming process of extraction and evaporation are eliminated. The total analysis time after enzymatic hydrolysis is ca. 5 minutes, which permits analysis of as many as 80 samples per day.