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

Malli D, Gikas E, Vavagiannis A, Kazanis M, Daniilides K, Gennimata D, Panderi I

University of Athens, School of Pharmacy, Division of Pharmaceutical Chemistry, Panepistimiopolis, Zografou, GR-157 71 Athens, Greece.

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 x 2.1 mm i.d., particle size 5 microm) 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 microg mL(-1) for nateglinide with a limit of quantitation of 0.05 microg mL(-1). Quality control samples (0.05, 4.50 and 16.00 microg 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.

Published 3 September 2007 in Anal Chim Acta, 599(1): 143-50.
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