Determination of nateglinide in human plasma by high-performance liquid chromatography with pre-column derivatization using a coumarin-type fluorescent reagent Original Research Article

A sensitive and selective high-performance liquid chromatographic method has been developed and validated for the determination of nateglinide in human plasma. Nateglinide and the internal standard, undecylenic acid, were extracted from plasma by liquid–liquid extraction using a mixture of ethyl acetate–diethyl ether, 50:50 (v/v). Pre-column derivatization reaction was performed using a coumarin-type fluorescent reagent, N-(7-methoxy-4-methyl-2-oxo-2H-6-chromenyl)-2-bromoacetamide. The derivatization proceeded in acetone in the presence of potassium carbonate and catalyzed by 18-crown-6 ether. The fluorescent derivatives were separated under isocratic conditions on a Hypersil BDS-C8 analytical column (250.0 mm × 2.1 mm i.d., particle size 5 μm) with a mobile phase that consisted of 65% acetonitrile in water and pumped at a flow rate of 0.50 mL min−1. The excitation and emission wavelengths were set at 345 and 435 nm, respectively. The assay was linear over a concentration range of 0.05–16.00 μg mL−1 for nateglinide with a limit of quantitation of 0.05 μg mL−1. Quality control samples (0.05, 4.50 and 16.00 μg mL−1) in five replicates from five different runs of analysis demonstrated intra-assay precision (%coefficient of variation <6.8%), inter-assay precision (%coefficient of variation <1.6%) and an overall accuracy (%relative error) less than −3.4%. The method can be used to quantify nateglinide in human plasma covering a variety of pharmacokinetic or bioequivalence studies.