Quantification of total and free concentrations of R- and S-warfarin in human plasma by ultrafiltration and LC-MS/MS
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A sensitive LC-MS/MS assay for quantification of total and free concentrations of R- and S-warfarin in plasma was required to support clinical studies on warfarin enantiomers. Several ultrafiltration devices were evaluated for separation of free warfarin from plasma proteins. The highest precision and lowest non-specific binding was obtained for Centrifree ultrafiltration devices. R- and S-warfarin were extracted from plasma (total) and ultrafiltrate (free) by liquid–liquid extraction with methyl tert-butyl ether using d6-warfarin as internal standard. Mean extraction recovery was 68 ± 4%. The enantiomers were separated on a Chirobiotic V column with isocratic elution using 40% methanol and 0.03% acetic acid in water. Negative mode electrospray ionisation was used for MS/MS detection, monitoring the ion transition m/z 307/161. Calibration curves (quadratic, weighted 1/x) were fitted over the range of 20–2,000 ng/ml (r 2 ≥ 0.995) in plasma and 0.5–20 ng/ml (r 2 ≥ 0.998) in ultrafiltrate. The lower limit of quantification for R- and S-warfarin was 0.5 ng/ml in ultrafiltrate. Intra- and interday precision (% RSD) and bias were within 10% in all cases, and matrix effects were negligible. The assay was applied successfully to analysis of samples from clinical studies.
KeywordsWarfarin Enantiomer Free Unbound Ultrafiltration Plasma
The authors wish to acknowledge the New Zealand Lottery Grants Board and Health Research Council of New Zealand (ref. no. 08/322) for financial support of this study.
- 1.Dollery C (ed) (1999) Therapeutic drugs, 2nd edn. Churchill Livingstone, EdinburghGoogle Scholar
- 5.Vecchione G, Casetta B, Tomaiuolo M, Grandone E, Margaglione M (2007) A rapid method for the quantification of the enantiomers of warfarin, phenprocoumon and acenocoumarol by two-dimensional-enantioselective liquid chromatography/electrospray tandem mass spectrometry. J Chrom B 850(1–2):507–514CrossRefGoogle Scholar
- 7.Radwan MA, Bawazeer GA, Aloudah NM, AlQuadeib BT, Aboul-Enein HY (2011) Determination of free and total warfarin concentrations in plasma using UPLC MS/MS and its application to a patient samples. Biomed Chrom. doi: 10.1002/bmc.1616
- 8.Takahashi H, Kashima T, Kimura S, Muramoto N, Nakahata H, Kubo S, Shimoyama Y, Kajiwara M, Echizen H (1997) Determination of unbound warfarin enantiomers in human plasma and 7-hydroxywarfarin in human urine by chiral stationary-phase liquid chromatography with ultraviolet or fluorescence and on-line circular dichroism detection. J Chrom B 701(1):71–80CrossRefGoogle Scholar
- 9.US Department of Health and Human Services (2001) Guidance for industry: bioanalytical method validation. US FDA Center for Drug Development and Research, Silver SpringGoogle Scholar