Determination of total and unbound warfarin and warfarin alcohols in human plasma by high performance liquid chromatography with fluorescence detection.
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 25mM at pH=7, 25% methanol and 5% acetonitrile at a flow rate of 1.2mL/min. Fluorescence detection was performed at 390nm (excitation wavelength 310nm). Neither method showed any detectable interference or matrix effect. Inter-day recovery of the total warfarin and warfarin alcohols at a concentration level of 1000ng/mL was 89±3% and 73±3%, respectively, whereas for their unbound fraction (at a concentration level of 10ng/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 0.2ng/mL for warfarin and warfarin alcohols, respectively. The methods were successfully applied to a pooled plasma sample obtained from 69 patients undergoing warfarin therapy.
Department of Chemistry and Industrial Chemistry, University of Pisa, Pisa, Italy.
Tommaso
T
Lomonaco
Silvia
S
Ghimenti
Isabella
I
Piga
Massimo
M
Onor
Bernardo
B
Melai
Roger
R
Fuoco
Fabio
F
Di Francesco
D000438:Alcohols / Q000097:blood*; D000925:Anticoagulants / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D059625:Liquid-Liquid Extraction; D009682:Magnetic Resonance Spectroscopy; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014859:Warfarin / Q000097:blood*
D000438:Alcohols; D000925:Anticoagulants; D014859:Warfarin
High performance liquid chromatography; Sample preparation techniques; Warfarin; Warfarin alcohols