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

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%.