tTwo analytical procedures are presented for the determination of the total content and unbound fractionof both warfarin and warfarin alcohols in human plasma. Chromatographic separation was carried outin 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. Fluores-cence detection was performed at 390 nm (excitation wavelength 310 nm). Neither method showed anydetectable interference or matrix effect. Inter-day recovery of the total warfarin and warfarin alcoholsat a concentration level of 1000 ng/mL was 89 ± 3% and 73 ± 3%, respectively, whereas for their unboundfraction (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 detectionwere 0.4 and 0.2 ng/mL for warfarin and warfarin alcohols, respectively. The methods were successfullyapplied to a pooled plasma sample obtained from 69 patients undergoing warfarin therapy.
Determination of total and unbound warfarin and warfarin alcohols in human plasma by high performance liquid chromatography with fluorescence detection
LOMONACO, TOMMASO;MELAI, BERNARDO;FUOCO, ROGER;DI FRANCESCO, FABIO
2013-01-01
Abstract
tTwo analytical procedures are presented for the determination of the total content and unbound fractionof both warfarin and warfarin alcohols in human plasma. Chromatographic separation was carried outin 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. Fluores-cence detection was performed at 390 nm (excitation wavelength 310 nm). Neither method showed anydetectable interference or matrix effect. Inter-day recovery of the total warfarin and warfarin alcoholsat a concentration level of 1000 ng/mL was 89 ± 3% and 73 ± 3%, respectively, whereas for their unboundfraction (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 detectionwere 0.4 and 0.2 ng/mL for warfarin and warfarin alcohols, respectively. The methods were successfullyapplied to a pooled plasma sample obtained from 69 patients undergoing warfarin therapy.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
