Determination of total and unbound warfarin and warfarin alcohols in human plasma by high performance liquid chromatography with fluorescence detection

• Analytical methods for the determination of warfarin and warfarin alcohols.
• Liquid–liquid extraction of total plasmatic warfarin and warfarin alcohols.
• Ultrafiltration of plasma for the unbound fraction of warfarin and warfarin alcohols.
• Limits of detection of warfarin and warfarin alcohols at 0.4 ng/mL level.
• Suitable methods for routine therapy monitoring and pharmacokinetic studies.

Two analytical procedures are presented for the determination of the total content and unbound fraction of both warfarin and warfarin alcohols in human plasma. Chromatographic separation was carried out in isocratic conditions at 25 °C on a C-18 reversed-phase column with a mobile phase consisting of a 70% buffer phosphate 25 mM at pH = 7, 25% methanol and 5% acetonitrile at a flow rate of 1.2 mL/min. Fluorescence detection was performed at 390 nm (excitation wavelength 310 nm). Neither method showed any detectable interference or matrix effect. Inter-day recovery of the total warfarin and warfarin alcohols at a concentration level of 1000 ng/mL was 89 ± 3% and 73 ± 3%, respectively, whereas for their unbound fraction (at a concentration level of 10 ng/mL) was 66 ± 8% and 90 ± 7%, respectively. The intra- and inter-day precision (assessed as relative standard deviation) was <10% for both methods. The limits of detection were 0.4 and 0.2 ng/mL for warfarin and warfarin alcohols, respectively. The methods were successfully applied to a pooled plasma sample obtained from 69 patients undergoing warfarin therapy.