Analysis of morphine and its 3- and 6-glucuronides by high performance liquid chromatography with fluorimetric...

A rapid, sensitive and simple to operate high performance liquid chromatographic method for the simultaneous determination of morphine, morphine-3-glucuronide and morphine-6-glucuronide in plasma is described. The drug and its metabolites were extracted from plasma using commercially available reversed phase octylsilane bonded silica columns (1 mL Bond Elut C8, 50 mg). Chromatographic separation of morphine and its metabolites was achieved using a mobile phase, consisting of 2 mM sodium dodecyl sulphate in 0.05% phosphoric acid:acetonitrile (71.5:28.5 by volume), at a flow-rate of 1.2 mL/min, in conjunction with a Waters Nova-Pak C18 column (300 x 3.9 mm). The analytical column was used in combination with a Guard-Pak module containing a Nova-Pak C18 Guard-Pak insert. Using fluorescence detection (excitation 245 nm, emission 335 nm), plasma levels in the region of 5-10 micrograms/L for the drug and its metabolites can be detected with only 200 microL of plasma. The method has been applied to studies of the disposition of morphine and its metabolites in premature neonates requiring mechanical ventilation who were receiving the drug intravenously; preliminary findings in patients at steady state are presented.