A method for the simultaneous determination of warfarin and its active metabolites (warfarin alcohols) in dried blood spots samples was developed and validated. The procedure consisted of the collection of blood sample spots on Whatman 903 filter paper and the liquid extraction of the analytes by a 3:1 v/v methanol/acetonitrile mixture from 6 mm diameter disks punched out from dried blood spots. Extracted samples were analyzed by ultra-high performance liquid chromatography coupled to electrospray positive ionization and tandem mass spectrometry. Chromatographic separation was carried out in isocratic condition at 25 °C on a Poroshell 120 EC-C18 reversed phase column with a mobile phase consisting of 65% water containing 0.1% (v/v) formic acid and 35% acetonitrile containing 0.1% (v/v) formic acid at a flow rate of 0.5 mL min− 1. The method did not show any detectable interference or matrix effect. The limits of detection were 0.007, 0.006 and 0.004 ng/mL for RR/SS-warfarin alcohols, RS/SR-warfarin alcohols and warfarin, respectively. Warfarin and warfarin alcohols recovery from dried blood spots sample was almost quantitative (range 96–103%). The intra- and inter-day precision (expressed as relative standard deviation) was always below 10%. Time stability studies highlighted that the concentrations of warfarin and both diastereoisomers of warfarin alcohols in a pooled blood sample were stable for at least 1 month at − 20 °C and after 4 cycles of thaw–freeze. Moreover, warfarin, RR/SS- and RS/SR-warfarin alcohols concentrations in the dried blood spots were stable for at least 7 days at 25 °C. Hematocrit value had a minor influence on the analytical result compared to blood drop volume. The method was successfully applied to the analysis of 15 blood samples from patients undergoing warfarin therapy and demonstrated its suitability as an alternative for warfarin and warfarin alcohols measurement in plasma. Good correlations between concentrations in plasma and dried blood spots were demonstrated for all three analytes, with correlation coefficients r ≥ 0.95. Warfarin concentration in dried blood spot correlated well with warfarin dosage (r = 0.70, p < 0.01) but not with international normalized ratio (r = 0.47, p = 0.0784) when all the enrolled patients (n = 15) were considered. However, this correlation increased (r = 0.80, p = 0.0302) if a single patient was monitored over time. These preliminary results highlighted that the developed method can provide useful information for the therapeutic drug monitoring of warfarin and its active metabolites, although further studies in a larger clinical trial are needed.
Determination of warfarin and warfarin alcohols in dried blood spots by ultra-high performance liquid chromatography coupled to electrospray ionization-tandem mass spectrometry (UHPLC-ESI-MS/MS)
GHIMENTI, SILVIA;LOMONACO, TOMMASO;BIAGINI, DENISE;BELLAGAMBI, FRANCESCA;DI FRANCESCO, FABIO;FUOCO, ROGER
2017-01-01
Abstract
A method for the simultaneous determination of warfarin and its active metabolites (warfarin alcohols) in dried blood spots samples was developed and validated. The procedure consisted of the collection of blood sample spots on Whatman 903 filter paper and the liquid extraction of the analytes by a 3:1 v/v methanol/acetonitrile mixture from 6 mm diameter disks punched out from dried blood spots. Extracted samples were analyzed by ultra-high performance liquid chromatography coupled to electrospray positive ionization and tandem mass spectrometry. Chromatographic separation was carried out in isocratic condition at 25 °C on a Poroshell 120 EC-C18 reversed phase column with a mobile phase consisting of 65% water containing 0.1% (v/v) formic acid and 35% acetonitrile containing 0.1% (v/v) formic acid at a flow rate of 0.5 mL min− 1. The method did not show any detectable interference or matrix effect. The limits of detection were 0.007, 0.006 and 0.004 ng/mL for RR/SS-warfarin alcohols, RS/SR-warfarin alcohols and warfarin, respectively. Warfarin and warfarin alcohols recovery from dried blood spots sample was almost quantitative (range 96–103%). The intra- and inter-day precision (expressed as relative standard deviation) was always below 10%. Time stability studies highlighted that the concentrations of warfarin and both diastereoisomers of warfarin alcohols in a pooled blood sample were stable for at least 1 month at − 20 °C and after 4 cycles of thaw–freeze. Moreover, warfarin, RR/SS- and RS/SR-warfarin alcohols concentrations in the dried blood spots were stable for at least 7 days at 25 °C. Hematocrit value had a minor influence on the analytical result compared to blood drop volume. The method was successfully applied to the analysis of 15 blood samples from patients undergoing warfarin therapy and demonstrated its suitability as an alternative for warfarin and warfarin alcohols measurement in plasma. Good correlations between concentrations in plasma and dried blood spots were demonstrated for all three analytes, with correlation coefficients r ≥ 0.95. Warfarin concentration in dried blood spot correlated well with warfarin dosage (r = 0.70, p < 0.01) but not with international normalized ratio (r = 0.47, p = 0.0784) when all the enrolled patients (n = 15) were considered. However, this correlation increased (r = 0.80, p = 0.0302) if a single patient was monitored over time. These preliminary results highlighted that the developed method can provide useful information for the therapeutic drug monitoring of warfarin and its active metabolites, although further studies in a larger clinical trial are needed.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
