High-performance liquid-chromatographic determination of warfarin enantiomers in plasma with automated on-line sample enrichment.
A sensitive and selective chiral high performance liquid chromatographic method was developed for the direct determination of R- and S-warfarin enantiomers in human plasma. The method involved direct injection of human plasma onto a semipermeable surface (SPS) guard column, washing the proteins from the column with aqueous acetonitrile and back flushing the analytes onto a reversed phase ovomucoid silica HPLC column using switching valves. After separation, the analytes were simultaneously detected and quantitated with a fluorometer. The recoveries of R-warfarin from human plasma at 25 and 2500 ng/ml were 98.9% and 88.1%, respectively. The recoveries of S-warfarin at 25 and 2500 ng/ml were 105.4% and 93.9%, respectively. Using 100 microl of human plasma, the lower limit of quantification for both R- and S-warfarins was 25 ng/ml. Linear responses in analyte/internal standard peak height ratios were observed for analyte concentrations ranging from 25 to 2500 ng/ml for both enantiomers. Fluorescence chromatograms of drug-free human plasma showed no interfering peaks with retention times similar to those for R- and S-warfarins and the internal standard. Results from a 3-day validation study for both enantiomers demonstrated excellent precision (1.7-9.0%) and accuracy (97-109%) across the calibration range.
Boppana
Venkata K
VK
GlaxoSmithkline Pharmaceuticals, Drug Metabolism and Pharmacokinetics, Mail Code UW2712, PO Box 1539, King of Prussia, PA 19406, USA. venkata_k_bopanna@gsk.com
Schaefer
William H
WH
Cyronak
Matthew J
MJ
D001774:Blood Chemical Analysis / Q000295:instrumentation / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D005246:Feedback; D005470:Fluorometry / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D011786:Quality Control; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013237:Stereoisomerism; D014859:Warfarin / Q000032:analysis / Q000097:blood* / Q000737:chemistry / Q000145:classification / Q000302:isolation & purification*
D014859:Warfarin