Fully automated analytical method for codeine quantification in human plasma using on-line solid-phase extraction and high-performance liquid chromatography with ultraviolet detection

A simple, sensitive and fully automated analytical method for the analysis of codeine in human plasma is presented. Samples are added with oxycodone, used as internal standard (I.S.), and directly loaded in the autosampler tray. An on-line sample clean-up system based on solid-phase extraction (SPE) cartridges (Bond-Elut C2, 20 mg) and value switching (Prospekt) is used. Isocratic elution improved reproducibility and allowed the recirculation of the mobile phase. A Hypersil BDS C18, 3 microns, 10 x 0.46 cm column was used and detection was done by UV monitoring at 212 nm. Retention times of norcodeine (codeine metabolite), codeine and oxycodone (I.S.) were 5.5, 6.4 and 9.1 min, respectively. Morphine was left to elute in the chromatographic front. Detection limit for codeine was 0.5 microgram l-1 and inter-assay precision (expressed as relative standard deviation) and accuracy (expressed as relative error) measured at 2 micrograms l-1 were 5.03% and 1.82%. Calibration range was 2-140 micrograms l-1.