Development of highly sensitive and specific HPLC assay for plasma morphine using direct injection technique and post-column derivatization.

An extremely simple, rapid and reproducible analytical method was developed for the determination of morphine in human plasma using a high performance liquid chromatography utilizing a column-switching technique and protein-coated precolumn. Morphine from plasma (500 microL) was preconcentrated on the protein-coated pre-column without sample pre-treatment. This column acted at the same time as a clean up device. The drug was transferred on-line to the analytical column followed by post-chromatographic derivatization and fluorimetric detection. Post-column derivatization was based on the oxidative dimerization of morphine to fluorescent pseudomorphine by potassium hexacyanoferrate (III). The average morphine recoveries over a concentration range of 10 to 100 ng/mL ranged from 94.84 to 100.70%, and relative standard deviations ranged from 1.36 to 2.13%.