Determination of Warfarin in Human Serum and Serum Ultrafiltrate by Column-switching Liquid Chromatographic System Using a Semi-micro Bore Column.

Abstract

A simple and sensitive reversed-phase high-performance liquid chromatographic method was developed for the analysis of warfarin (WF) in both human serum and serum ultrafiltrate. WF and internal standard (I.S.) were extracted from the serum by simple deproteinization and filtration with a membrane filter. The Column-switching system utilized the short polymer-coated mixed-function (PCMF) phase deproteinization column coupled with a C18 semi-micro analytical column using a mobile phase of acetonitrile, water and NH4H2PO4, followed by a post-column photochemical reaction and fluorimetric detection. The extraction recovery was greater than 80% and the WF and I. S. were separated from the endogenous components in the serum and serumultrafiltrate. The calibration curve was linear from 5 to 1000 ng/mL of serum and 1 to 150 ng/ mL of the serum ultrafiltrate. The limit of quantification was 5 and 1 ng/mL for found to be the serum and serum ultrafiltrate, respectively. The inter and intra-day coefficients of variation for the serum and serum ultrafiltrate ranged less than 10%, respectively. WF was found to be the stable in the serum for a 4-month period during storage at -20°C.