Quantitation of plasma warfarin concentrations by high performance liquid chormatography.

A rapid, sensitive, and specific high performance liquid chromatographic (HPLC) method for the quantitative analysis of warfarin in plasma is described. The method involves an extraction from acidified plasma, removal of basic substances, and reextraction into ether. The method is sensitive (0.06-9.0 microgram/ml) and precise (coefficient of variation less than 2%) and makes use of a Bondapak C-18 column. Several methods have been reported for the analysis of warfarin; each has some disadvantage in terms of specificity, sensitivity, reproducibility, or convenience. A fluorometric method (1) was rapid, but in our hands was neither precise nor sensitive and yielded values consistently higher than the proposed method. An HPLC method (2) that incorporated a Permaphase column and dioxane as the mobile phase did not perform at all with a Bondapak C-18 column. Gas-liquid chromatography involved derivatization (3), which did not afford reproducibility in our laboratory. Two other published HPLC methods (4,5) showed interference from diazepam. The method described in this paper is specific, accurate, and convenient. It has sufficient sensitivity to measure low warfarin levels, is linear to 9 microgram/ml, and is free of interference by common drugs. The clinical utility of this method is illustrated with three case reports.