High-performance liquid-chromatographic determination of warfarin enantiomers in plasma with automated on-line sample enrichment.

Abstract

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.

• Authors: Venkata K Boppana, William H Schaefer, Matthew J Cyronak
• Journal: Journal of biochemical and biophysical methods (J Biochem Biophys Methods) Vol. 54 Issue 1-3 Pg. 315-26 (Dec 31 2002) ISSN: 0165-022X [Print] Netherlands
• PMID: 12543507 (Publication Type: Evaluation Study, Journal Article, Validation Study)
• Copyright: Copyright 2002 Elsevier Science B.V.

Chemical References

• Warfarin

Topics

• Blood Chemical Analysis (instrumentation, methods)
• Chromatography, High Pressure Liquid (instrumentation, methods)
• Feedback
• Fluorometry (instrumentation, methods)
• Humans
• Quality Control
• Reproducibility of Results
• Sensitivity and Specificity
• Stereoisomerism
• Warfarin (analysis, blood, chemistry, classification, isolation & purification)