Separation and determination of warfarin enantiomers in human plasma using a novel polymeric surfactant for micellar electrokinetic chromatography-mass spectrometry

Abstract

Warfarin is a widely used oral anticoagulant which is mostly administrated as a racemic mixture containing equal amount of R- and S-enantiomers. The two enantiomers are shown to exhibit significant differences in pharmacokinetics and pharmacodynamics. In this study, a new chiral micellar electrokinetic chromatography-mass spectrometry (MEKC-MS) method has been developed using a polymeric chiral surfactant, polysodium N-undecenoyl-L,L-leucyl-valinate (poly-L,L-SULV), as a pseudostationary phase for the chiral separation of (+/-)-warfarin (WAR) and (+/-)-coumachlor (COU, internal standard). Under optimum MEKC-MS conditions, the enantio-separation of both (+/-)-WAR and (+/-)-COU was achieved within 23 min. Calibration curves were linear (R=0.995 for (R)-WAR and R=0.989 for (S)-WAR) over the concentration range 0.25-5.0 microg/mL. The MS detection was found to be superior over the commonly used UV detection in terms of selectivity and sensitivity with LOD as low as 0.1 microg/mL in human plasma. The method was successfully applied to determine WAR enantiomeric ratio in patients' plasma undergoing warfarin therapy.

Fig. 1

Chemical structures of (±)-warfarin (WAR), (±)-coumachlor (COU) and polymeric surfactants as pseudostationary phase in MEKC-ESI-MS. The pKa and log P were calculated using Advanced Chemistry Development (ACD/Labs) Software, Version 8.14 for Solaris.(1194–2006, ACD Labs)

Fig. 2

Enantioselectivity of different polymeric surfactants for separation of (±)-warfarin (WAR) and (±)-coumachlor (COU). MEKC-ESI-MS conditions: 120 cm long (50μm, I. D.) fused-silica capillary (UV detection window at 60cm). Applied voltage, +30 kV. UV detection, 254 nm (bandwide,10 nm). Capillary temperature, 20°C. Injection: 5 mbar, 2 sec. Running buffer, 25 mM NH₄OAc/25mM polymeric surfactant at pH=5.5. Spray chamber parameters, nebulizing gas pressure at 4 psi (275.8 mbar); drying gas temperature at 200°C; drying gas flow rate at 6L/min; capillary voltage at −3000V. Sheath liquid composition, MeOH/H₂O (80/20, v/v) containing 5 mM NH₄OAc at pH=6.8; sheath liquid flow rate at 5μL/min. SIM negative ion mode. Sample concentration: 0.1 mg/mL for (±)-WAR and (±)-COU.

Fig. 3

Simultaneous UV and MS detection of (±)-WAR and (±)-COU at the LOD (5μg/mL) of MEKC-UV.

Fig. 4

Determination of (R)-WAR and (S)-WAR concentration and enantiomeric ratio in human plasma samples. The conditions are the same as in Fig. 2. (A) Blank plasma spiked with racemic warfarin (2 μg/mL) standard and coumachlor (2 μg/mL, I. S.); (B) and (C) Normal patient plasma spiked with 2 μg/mL of coumachlor [ B: (R)-WAR = 0.80 μg/mL; (S)-WAR = 0.35 μg/mL; R/S = 2.24 and C: (R)-WAR = 2.30 μg/mL; (S)-WAR = 0.38 μg/mL; R/S = 6.20 ]; (D) Mutant gene (CYP2C9*2 or*3) patient plasma spiked with 2 μg/mL of coumachlor [(R)-WAR = 0.67μg/mL; (S)-WAR= 2.5 μg/mL; R/S= 0.27)].