Direct injection high-performance liquid chromatographic assay of morphine with electrochemical detection, a polymeric column and an alkaline eluent.

A simple method is described for the direct determination of morphine in untreated plasma or serum, using reversed-phase high-performance liquid chromatography with amperometric electrochemical detection. A basic eluent [0.05 mol/l phosphate buffer-isopropanol-tetrahydrofuran (88:10:2) (pH 9.5)] allows both reversed-phase chromatography of morphine under ionization control conditions and its detectability at an unprotected thin-layer glassy carbon electrode at a potential of 350 mV (vs. an Ag/AgCl reference electrode). In addition, the alkaline mobile phase promotes the ionization of serum proteins, which, being poorly retained by the hydrophobic column packing [poly(styrene-divinylbenzene) copolymer], elute early in the chromatogram, leaving a clean baseline. Up to 50 microliters of simply filtered plasma can be injected. The absolute limit of detection is 0.75 ng on-column. No interferences were observed from more than 80 opiate and non-opiate drugs. The intra- and inter-assay relative standard deviations (n = 5) were 3.2 and 6.6%, respectively, at a morphine concentration of 100 ng/ml in plasma and 0.09 and 4.2%, respectively, at the level of 500 ng/ml.