Simultaneous Determination of Warfarin and 7-Hydroxywarfarin Enantiomers by High-Performance Liquid Chromatography With Ultraviolet Detection

Abstract

Warfarin is administered clinically as a racemic mixture of two enantiomers, (R)-warfarin and (S)-warfarin. (S)-Warfarin has more potent anticoagulant activity than (R)-warfarin and is metabolized mainly to (S)-7-hydroxywarfarin by CYP2C9. A simple, rapid, and sensitive high-performance liquid chromatography method with ultraviolet detection was developed for the simultaneous quantitative determination of the (R)- and (S)-enantiomers of warfarin and 7-hydroxywarfarin in human plasma. Analytes and the internal standard (p-chlorowarfarin) were separated using a mobile phase of 0.5% KH2PO4 (pH 3.5)-methanol (41:59, v/v) on a Chiral CD-Ph column at a flow rate of 0.5 mL/min and were detected at a ultraviolet absorbance of 305 nm. Analysis required 200 μL of plasma and involved a simple and rapid solid-phase extraction with an Oasis HLB cartridge, which gave high recovery (greater than 91.8%) and good selectivity for all analytes. The lower limit of quantification for the (R)- and (S)-enantiomers of warfarin and 7-hydroxywarfarin was 2.5 ng/mL for each analyte. Inter- and intraday coefficients of variation for all analytes were less than 14.2% and accuracies were within 6.6% over the linear range. Our results indicate that this method is applicable to the simultaneous monitoring of the enantiomers of warfarin and 7-hydroxywarfarin in human plasma. The S/R-enantiomer ratio of warfarin and the (S)-warfarin/(S)-7-hydroxywarfarin ratio 3 hours after administration in 67 CYP2C9*1/*1 patients ranged from 0.24 to 0.75 and from 1.83 to 19.02, respectively, whereas these ratios in a CYP2C9*3/*3 patient were 1.12 and 17.02, respectively.

Drug interactions between warfarin and lenvatinib in a patient with the CYP2C9 * 1/*3 and VKORC1 ‐1639G/A genotype

“…The median plasma concentration of ( S )‐warfarin required to yield the target INR response is reported to be significantly associated with the VKORC1 − 1639G > A polymorphism (0.68 μg/mL for the G/G genotype, 0.48 μg/mL for the A/G genotype and 0.27 μg/mL for the A/A genotype) . This patient with the VKORC1 −1639G/A genotype showed a higher plasma concentration of ( S )‐warfarin than in a previous report (0.48 μg/mL) or our previous data (0.54 μg/mL) . The plasma concentration of ( S )‐warfarin seemed to be increased by co‐administration of lenvatinib.…”

“…The patient had CYP2C9 * 1/ * 3 and VKORC1 − 1639G/A genotypes. The plasma trough concentration (C 0 ) of each warfarin enantiomer in this patient was monitored using high‐performance liquid chromatography (HPLC) . On day 8 after beginning lenvatinib plus warfarin therapy, the C 0 values of ( R )‐ and ( S )‐warfarin were 1.8 and 0.67 μg/mL, respectively.…”

Review Metode Analisis Warfarin dalam Plasma dengan Berbagai Instrumen

“…Berbagai metode telah banyak dikembangkan untuk menganalisis warfarin secara kuantitatif dalam plasma darah, seperti HPLC dengan detektor Fluoresensi (FLD) yang memberikan nilai batas bawah deteksi mencapai 0 0,4 ng/mL [3], detektor Diode Array (DAD) yang memberikan nilai batas bawah deteksi mencapai 20 ng/mL [4], detektor UV yang memberikan nilai batas bawah deteksi mencapai 1,25 ng/mL [5] maupun detektor MS yang memberikan nilai batas bawah deteksi mencapai 0,08 ng/mL [6]. Selain metode HPLC, digunakan pula metode elektroforesis dengan basis Carbon Paste Electrode [7].…”

“…Nilai pH dan pKa dari senyawa menentukan derajat ionisasi analit serta afinitasnya terhadap fase diam dan fase gerak. Pada uji bioanalisis warfarin, banyak fase gerak yang dibuat bersifat asam dengan penambahan buffer [3,8,11,16,20,[23][24][25][26][27][28] atau asam format [1,[5][6][7]10,14,15,17,[21][22][23]26,[29][30][31][32][33][34][35][36]. Hal ini menyebabkan warfarin akan terdapat dalam bentuk tak ter-ion sehingga bersifat non polar dan akan lebih terjerap pada fase diam, sehingga puncak yang dihasilkan dapat memiliki resolusi yang baik.…”

“…Pada pemisahan warfarin ini, sering digunakan fase gerak asetonitril untuk pelarut bersifat non polar dan air untuk pelarut bersifat polar [1,2,5,7,8,13,18,[29][30][31]33,[37][38][39]. Asetonitril bersifat polar-aprotik, merupakan fase gerak yang umum digunakan dalam sistem HPLC karena berbagai kelebihan yang dimiliki sehingga dapat memenuhi persyaratan pemilihan fase gerak yang baik, diantaranya adalah absorbansi di daerah panjang gelombang sinar UV yang rendah sehingga tidak mengganggu absorbansi sampel, menghasilkan noise yang rendah, dan memberikan bentuk puncak yang baik pada kolom C18.…”