Skip to main content

Quantification of total and free concentrations of R- and S-warfarin in human plasma by ultrafiltration and LC-MS/MS


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.

LC-MS/MS chromatogram of free R- and S-warfarin in human ultrafiltrate

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2


  1. 1.

    Dollery C (ed) (1999) Therapeutic drugs, 2nd edn. Churchill Livingstone, Edinburgh

    Google Scholar 

  2. 2.

    Huang C, Yang J, Du Y, Miao L (2008) Measurement of free concentrations of highly protein-bound warfarin in plasma by ultra performance liquid chromatography-tandem mass spectrometry and its correlation with the international normalized ratio. Clin Chim Acta 393(2):85–89

    Article  CAS  Google Scholar 

  3. 3.

    Jones DR, Boysen G, Miller GP (2011) Novel multi-mode ultra performance liquid chromatography-tandem mass spectrometry assay for profiling enantiomeric hydroxywarfarins and warfarin in human plasma. J Chrom B 879(15–16):1056–1062

    Article  CAS  Google Scholar 

  4. 4.

    Zuo Z, Wo SK, Lo CMY, Zhou L, Cheng G, You JHS (2010) Simultaneous measurement of S-warfarin, R-warfarin, S-7-hydroxywarfarin and R-7-hydroxywarfarin in human plasma by liquid chromatography-tandem mass spectrometry. J Pharm Biomed Anal 52(2):305–310

    Article  CAS  Google Scholar 

  5. 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):507–514

    Article  CAS  Google Scholar 

  6. 6.

    Naidong W, Ring PR, Midtlien C, Jiang X (2001) Development and validation of a sensitive and robust LC-tandem MS method for the analysis of warfarin enantiomers in human plasma. J Pharm Biomed Anal 25(2):219–226

    Article  CAS  Google Scholar 

  7. 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. 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):71–80

    Article  CAS  Google Scholar 

  9. 9.

    US Department of Health and Human Services (2001) Guidance for industry: bioanalytical method validation. US FDA Center for Drug Development and Research, Silver Spring

    Google Scholar 

  10. 10.

    Uno T, Niioka T, Hayakari M, Sugawara K, Tateishi T (2007) Simultaneous determination of warfarin enantiomers and its metabolite in human plasma by column-switching high-performance liquid chromatography with chiral separation. Ther Drug Monit 29(3):333–339

    Article  CAS  Google Scholar 

  11. 11.

    Miura M, Okuyama S, Kato S, Kagaya H, Murata A, Komatsuda A, Wakui H, Sawada K (2011) Simultaneous determination of warfarin and 7-hydroxywarfarin enantiomers by high-performance liquid chromatography with ultraviolet detection. Ther Drug Monit 33(1):108–114

    Article  CAS  Google Scholar 

  12. 12.

    Yacobi A, Levy G (1977) Protein binding of warfarin enantiomers in serum of humans and rats. J Pharmacokinet Biopharm 5(2):123–131

    Article  CAS  Google Scholar 

  13. 13.

    Takahashi H, Kashima T, Nomizo Y, Muramoto N, Shimizu T, Nasu K, Kubota T, Kimura S, Echizen H (1998) Metabolism of warfarin enantiomers in Japanese patients with heart disease having different CYP2C9 and CYP2C19 genotypes. Clin Pharmacol Ther 63(5):519–528

    Article  CAS  Google Scholar 

Download references


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.

Author information



Corresponding author

Correspondence to Berit Packert Jensen.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Jensen, B.P., Chin, P.K.L. & Begg, E.J. Quantification of total and free concentrations of R- and S-warfarin in human plasma by ultrafiltration and LC-MS/MS. Anal Bioanal Chem 401, 2187 (2011).

Download citation


  • Warfarin
  • Enantiomer
  • Free
  • Unbound
  • Ultrafiltration
  • Plasma