Simple and highly sensitive determination of morphine in rat plasma by liquid chromatography with fluorescence detection

Abstract

A simple and highly sensitive liquid chromatographic method for the determination of morphine in rat plasma is described. This method is based on the selective reaction of morphine with benzylamine in neutral media (borate buffer, pH 7.0) in the presence of potassium hexacyanoferrate(III) to give a highly fluorescent derivative. The derivative was determined by isocratic elution using acetonitrile–25 mM phosphate buffer (pH 8.5) on a YMC-Pack polymer C₁₈ reversed-phase column. The detection limit (signal-to-noise ratio=3) for morphine was 5.7 pg (20 fmol) in a 20 μl injection volume (1.4 ng ml⁻¹ plasma), which allowed precise determination of morphine in plasma at very low concentrations with a quite small amount of plasma (50 μl) without prior extraction. The calibration graph was linear from 3 ng ml⁻¹ to 1 μg ml⁻¹ in plasma. The method was applied to monitoring the drug concentrations in rat plasma samples obtained after an intravenous administration of morphine hydrochloride (5.0 mg kg⁻¹).