title: Quantification of Warfarin in Dried Rat Plasma Spots by High-Performance Liquid Chromatography with Tandem Mass Spectrometry.
abstract: This paper presents the development and validation of a novel method for quantification of the oral anticoagulant drug warfarin in dried plasma spots (DPS) by high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). Blood plasma was chosen as a biological fluid to preclude the influence of the hematocrit on the results of the analysis. A 30μL sample of rat plasma was placed onto Whatman 903 Protein Saver Card and was allowed to dry. A single DPS is sufficient for preparing eight 3.2 mm discs, each containing approximately 1.5-1.6μL of plasma. Warfarin extraction from one 3.2 mm disc was carried out by adding 200μL of the acetonitrile : water mixture (1 : 1, v/v) containing 10 mM NH4COOH (pH 4.0), with incubation on a shaker at 1000 rpm for 1 h at 25°C. After chromatographic separation, warfarin and coumachlor (an internal standard) were measured using negative-ion multiple-reaction monitoring with ion transitionsm/z307 → 161 for warfarin andm/z341 → 161 for the internal standard. The working range of this method is 10-10,000 ng/mL. Within this range, intra- and interday variability of precision and accuracy was <13% and recovery was 82-99%. The results indicate that the new method requires only small plasma samples and may be useful for pharmacokinetic research on warfarin.
authors.affiliation: Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, Academician Lavrentiev Avenue 8, Novosibirsk 630090, Russia.
authors.forename: Alexander
authors.identifier: 0000-0001-8362-2443
authors.initials: A
authors.lastname: Chernonosov