A Chiral HPLC-MS/MS Method for Simultaneous Quantification of Warfarin Enantiomers and its Major Hydroxylation Metabolites of CYP2C9 and CYP3A4 in Human Plasma

Research Article

Austin J Anal Pharm Chem. 2014;1(2): 1010.

A Chiral HPLC-MS/MS Method for Simultaneous Quantification of Warfarin Enantiomers and its Major Hydroxylation Metabolites of CYP2C9 and CYP3A4 in Human Plasma

Ju W1, Peng K1, Yang S1, Sun H2, Sampson M2 and Wang MZ1*

1Department of Pharmaceutical Chemistry, the University of Kansas, USA

2Department of Pharmacy, University of North Carolina at Chapel Hill, USA

*Corresponding author: :Wang MZ, Department of Pharmaceutical Chemistry, The University of Kansas, 2095 Constant Ave, Lawrence, KS 66047, USA.

Received: August 07, 2014; Accepted: August 20, 2014; Published: August 22, 2014


Warfarin is an oral anticoagulant that requires frequent therapeutic drug monitoring due to a narrow therapeutic window, considerable inters individual variability in drug response, and susceptibility to drug-drug and drug-diet interactions. Enantiomeric separation and quantification of warfarin enantiomers and clinically important major hydroxylation metabolites are essential for drug interaction studies and phenotypic characterization of CYP2C9 and CYP3A4, the major Cytochrome P450 (CYP) enzymes involved in warfarin metabolism. Here, we describe the development and validation of a chiral high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/ MS)-based quantification of R-warfarin, S-warfarin, S-7-hydroxywarfarin (the major CYP2C9metabolite) and (9R; 10S)-10-hydroxywarfarin (the CYP3A4 metabolite) in human plasma. Simple protein precipitation-based extraction showed good recovery of analyses (82.9 - 96.9%). The developed method exhibited satisfactory intra-day and inter-day accuracy and precision. The lower limits of detection were 0.25nM (or ~0.08 ng/ml) for the war far in enantiomers and 0.1nM (or ~0.04 ng/mL) for S-7-hydroxywarfarinand (9R; 10S)-10- hydroxywarfarin using only 50μL plasma during extraction. The validated method was successfully applied to analyze plasma samples obtained from a healthy human subject who enrolled in a clinical drug interaction study involving warfarin.

Keywords: Warfarin; Chiral separation; HPLC-MS/MS; Hydroxyl war far in; Protein precipitation extraction


AUC: Area Under The Plasma Concentration-Time Curve; COD: Coefficient of Determination; CYP: Cytochrome P450; CV: Coefficient of Variation; DMSO: Dimethylsulfoxide; HPLC-MS/ MS: High Performance Liquid Chromatography-tandem mass spectrometry; INR: International Normalized Ratio; IS: Internal Standard; LLE: Liquid–Liquid Extraction; LLOQ: Lower Limit Of Quantification; LOD: Limit Of Detection; MRM: Multiple Reaction Monitoring; PPE: Protein Precipitation Extraction; PT: Prothrombin Time; QC: Quality Control; SD: Standard Deviation; SPE: Solid- Phase Extraction; S/N: Signal-to-Noise ratio; ULOQ: Upper Limit Of Quantification


Warfarin is the most commonly prescribed oral anticoagulant in North America and is indicated for the prophylaxis and treatment of a number of serious thromboembolic disorders and complications [1]. It elicits an anticoagulant effect by interfering with the synthesis of vitamin K-dependent clotting factors in the liver via inhibition of the vitamin K epoxies reeducates complex subunit 1 (VKORC1) [2]. Warfarin has a narrow therapeutic index and displays considerable inter individual variability in dose requirements. To achieve and maintain optimal anticoagulant activity: warfarin efficacy is frequently monitored by the pro thrombin time (PT) or the Internationally Normalized Ratio (INR). Despite target INR monitoring and careful dose adjustment: the rate of warfarin-related bleeding events has not diminished [3,4]. Genetic factors including VKORC1: concomitant drugs/diet: and various disease states can impact on the efficacy of warfarin by changing its pharmacokinetics. Thus: sensitive and specific analytical methods to quantify warfarin and its clinically significant metabolites in various matrices: especially human plasma: may help to improve the understanding of warfarin pharmacology and detection of potential warfarin-drug/diet interactions.

Warfarin is a racemic mixture of R- and S-enantiomers. S-warfarin is more potent (3-5 times) pharmacologically than R-warfarin [5]: however S-warfarin clearance is greater than that of R-warfarin. Both enantiomers undergo extensive cytochrome P450 (CYP)-mediated metabolism in the liver. CYP2C9 catalyzes the 6- and 7-hydroxylation of S-warfarin: with S-7-hydroxywarfarin (S-7-OH-warfarin) being the predominant metabolite: and the 4’-hydroxylation of R-warfarin [6]. CYP3A4 regio- and stereo selectively catalyzes the 10-hydroxylation of R-warfarin to form (9R; 10S)-10-hydroxywarfarin [(9R; 10S)-10- OH-warfarin] [7]. Inhibition and induction of CYP2C9- and/or CYP3A4-mediated warfarin metabolism by co-administered drugs/diets the main culprit for many clinically important drug interactions [1]. Therefore: specific quantification of S-7-OH-warfarin (or S-6- OH-warfarin) and (9R; 10S)-10-OH-warfarin in plasma will enable phenotypic characterization of CYP2C9 and CYP3A4 catalytic activities during clinical drug-drug and drug-diet interaction studies.

Several analytical methods have been reported to achieve Enantiomeric separation of R-and S-warfarin and/or the stereoisomer’s of clinically important warfarin metabolites. Separation and quantification of R-and S-warfarin in human plasma has been demonstrated using various chiral High Performance Liquid Chromatography (HPLC) columns coupled with detection by UV [8- 12] or tandem mass spectrometry (MS/MS) [13]. However: chiral separation and quantification of hydroxylated warfarin metabolites has mainly focused on 7-OH-warfarinusing a chiral HPLC column coupled with UV [14]: fluorescence [15] or MS/MS detection [16]: or using capillary zone electrophoresis coupled with UV detection [17]. Although specific quantification of the CYP3A4-selective warfarin metabolite: (9R; 10S)-10-OH-warfarin: in human plasma using MS/ MS detection has been reported [18]: analyses were separated by combining phenyl based reverse phase column and chiral column with a complicated sample preparation and a long analytical time (17 min). In addition: previously reported analytical methods for the warfarin enantiomers in plasma involve either extraction using solid phase extraction (SPE) cartridges [8;16;18-20] or phase separation via liquid-liquid extraction (LLE) [9-11,13-15]. Extraction of war far in by Protein Precipitation (PPE) has only been used for microsomal metabolism studies: where protein concentrations are typically much lower (< 2 mg/mL) than in plasma [21,22]. In this study: we aimed to 1) develop a chiral HPLC-MS/MS-based method to quantify the warfarin enantiomers and two stereo isomeric metabolites: S-7- OH-warfarin and (9R; 10S)-10-OH-warfarin: in human plasma for CYP2C9 and CYP3A4 phenotyping with a short chromatographic procedure and 2) determine whether PPE could be used as an alternative extraction method for Enantiomeric analysis of warfarin and its major hydroxylated metabolites in human plasma.

Materials and Methods


R-warfarin: S-warfarin: racemic warfarin: warfarin metabolites (4’-: 6-: 7-: 8- and 10-OH-warfarin): and warfarin-d5 (deuterated phenyl ring; used as internal standard [IS]) were purchased from Toronto Research Chemicals Inc. (North York: ON: Canada). Ammonium acetate and dimethylsulfoxide (DMSO) were obtained from Sigma-Aldrich (St. Louis: MO: USA). Optima-grade water: acetonitrile: methanol: and acetic acid was obtained from Fisher Scientific (Pittsburgh: PA: USA). Blank human plasma (collected in K2-EDTA tubes) was purchased from Innovative Research (Novi: MI: USA). Recombinant human CYP3A4 and CYP2C9 Supersomes™: microsomes prepared from baculovirus-infected insect cells expressing human CYP enzymes and NADPH-cytochrome P450 reductase: were purchased from BD Gentlest (Woburn: MA: USA).

Incubation with recombinant CYP3A4 and CYP2C9 enzymes

The metabolism of R- and S-warfarin by recombinant CYP3A4 and CYP2C9 was conducted according to a previously published protocol [22] with modifications. Briefly: incubation mixtures (50 μL) contained R- or S-warfarin (10 μM final concentration): recombinant CYP enzymes individually (50 pmol/mL): and 100 mM phosphate buffer (pH 7.4) containing 3.3mM MgCl2. Reactions were initiated by the addition of NADPH (1 mM final concentration) and allowed to proceed for 30 min at 37°C. Control incubations were conducted in the absence of NADPH. The reactions were stopped with 400 μL ice-cold methanol-water (7:1: v/v) and vortexes. Following centrifugation to pellet precipitated proteins: the supernatants were dried under nitrogen at 50°C for 45 misusing a 96-well micro plate evaporator (Model SPE Dry 96; Biotage: LLC: Charlotte: NC: USA) and the dried sample reconstituted with 250 μL methanol-water (15:85: v/v) prior to chiral HPLC-MS/MS analysis.

Preparation of calibration standards and quality controls

Individual stock solutions of R-war far in: S-war far in: racemic war far in: warfarin-d5: and each war far in metabolite (4’-: 6-: 7-: 8- and 10-OH-warfarin) were prepared in DMSO. Working standard mixtures were prepared by mixing and diluting the stock solutions of racemes warfarin:7-OH-warfarin: and 10-OH-warfarin.Calibration standards were prepared by spiking 1.0 μL working standard mixture into 49 μL blank human plasma to yield the following final concentrations: 0.25: 0.5: 1.25: 2.5: 5: 12.5: 25: 50: 125: 250: 500: 1250: 2500: and 5000 nM for R- and S-warfarin: and 0.05: 0.1: 0.25: 0.5: 1: 2.5: 5: 10: 25: 50: 100: 250: 500: and 1000 nM for S-7- OH-warfarin and (9R;10S)-10-OH-warfarin.Quality controls (QCs): prepared separately from individual stock solutions: had final plasma concentrations of 1.25: 2.5: 50: and 1250 nM for R- and S-warfarin: and 0.25: 0.5: 10: and 250 nM for S-7-OH-warfarin and (9R;10S)-10- OH-warfarin. Calibration standards were prepared in triplicate: while QCs in quadruplicate; all were processed as plasma samples before chiral HPLC-MS/MS analysis.

Sample preparation and HPLC-MS/MS analysis

Following thawing at room temperature: plasma samples were prepared using PPE rather than SPE or LLE. Specifically: plasma samples (50 μL) were mixed with 400 μl methanol-water (7:1: v/v) containing 30 nM warfarin-d5 as the IS. After overtaxing for 10 s: the samples were centrifuged at 2250 g for 15 min at 4°C to pellet precipitated proteins. The supernatants were evaporating dander nitrogen at 50°Cfor 45 min. The dried samples were reconstituted with 100 μL methanol-water (15:85: v/v) prior to chiral HPLC-MS/ MS analysis.

HPLC-MS/MS quantification of warfarin and its metabolites was performed on a Waters ACQUITY I-Class UPLC (operated at normal HPLC pressure) coupled to a Xevo TQ-S triple quadruple mass spectrometer equipped with an electro spray ionization source (Waters Corporation: Milford: MA: USA). Enantiomeric separation of warfarin and its metabolites was achieved with an Astec CHIROBIOTIC® V Chiral HPLC column (100 mm × 4.6 mm: 5 μm; Supelco: Inc: Bellefonte: PA) protected by an ACQUITY column in-line filter (0.2 μm; Waters Corporation).HPLC mobile phase (A) consisted of 100% (v/v) water with 5mM ammonium acetate (pH4.0: adjusted with acetic acid): while (B) consisted of100% (v/v) acetonitrile. The initial gradient began with 10% B and was held for 0.2 min. Mobile phase B increased linearly to 40% over 5 min and remained at 40% for 1 min. The system was re-equilibrated with 10% B for 2 min prior to the next injection. A flow rate of 0.8 mL/min was used throughout the study. The auto sampler was set at 6°C and the HPLC column heated at 50°C.The sample injection volume was 10 μL.

The mass spectrometer was operated under negative ion mode for multiple reaction monitoring (MRM) of warfarin and its metabolites. Typical instrument conditions were: capillary voltage: 0.50 kV; source offset: 50 V; desolation temperature: 500°C; desolation gas flow: 1000 L/h; cone gas flow: 150 L/h; nebulizer gas flow: 7.0 bar; and collision gas flow: 0.15 ml/min. Analyze-specific instrument parameters (i.e.: collision energy: capillary voltage: cone voltage) were optimized prior to analysis using the Intel Start™ auto-tune with infusion of analyze standards. The specific MRM transitions used for quantification were m/z 307.1 →161.0 for warfarin: m/z 312.2→ 255.1 for warfarin-d5: m/ z323.1→177.0 for 6-: 7- and 8-OH-warfarin: and m/z 323.1→ 250.3 for 10-OH-warfarin. Peak area ratios of analyze vs. IS were used to generate calibration curves and calculate analyze concentrations in plasma samples.

Method validation

Method selectivity was investigated by comparing HPLC-MS/MS chromatograms from blank human plasma and plasma spiked with standards at the Lower Limit of Quantification (LLOQ). To ensure MRM transitions for warfarin and 6-: 7-: 8-: and 10-OH-warfarin did not cross-detect 4’-OH-warfarin: chromatograms from plasma spiked with 4’-OH-warfarin (200 nM final concentration) also were compared.

The intra-day accuracy and precision of the method were determined by replicate analysis (n = 4) of the QC son the same day. The inter-day accuracy and precision were determined by replicate analysis of the QC son three separate days. Accuracy (%) was defined as the closeness of the average QC concentrations determined by the method to the true concentrations. Precision (Coefficient of Variation [CV]; %) was defined as the spread of the individual measures of multiple QC preparations.

The extraction recovery was determined by comparing peak area ratios of analyses vs. IS from QC samples that were spiked with analyze before extraction to ratios from blank plasma extracts spiked with known amounts of pure analyze post-extraction (represents 100% recovery).The extraction recovery was examined at three QC concentrations (2.5: 50: and 1250 nM for R- and S-warfarin; 0.5: 10: and 250 nM for S-7-OH-warfarin and (9R;10S)-10-OH-warfarin) with quadruplicate samples at each concentration.

Matrix effects during MS/MS analysis were evaluated by comparing peak areas of analyze from blank plasma extracts spiked with known amounts of pure analyze to peak areas from samples prepared in the reconstitution solvent (i.e.: methanol-water [15:85: v/v]) with known amounts of pure analyses. The matrix effects also were examined at three QC concentrations (2.5: 50: and 1250 nM for R- and S-warfarin; 0.5: 10: and 250 nM for S-7-OH-warfarin and (9R; 10S)-10-OH-warfarin) with quadruplicate samples at each concentration. Results were expressed as % signal remaining relative to standards in the reconstitution solvent: which represents100% signal remaining.

Freeze-thaw stability was evaluated by exposing QCs to three freezing (-80°C) and thawing (room temperature) cycles before sample preparation. The thermal stability was evaluated by comparing peak areas of standards in methanol-water (15:85: v/v) incubated at 50°Cfor 50 min (temperature and time needed during drying) to those incubated at 4°C for the same period of time. Sample stability in the auto sampler was evaluated by repeat-analysis of the same QCs stored in a thermostatic auto sampler (6°C) for 24 h.

The Limit of Detection (LOD) was defined as the lowest concentration of calibration standard that had a signal-to-noise ratio (S/N) greater than 10. The LLOQ was defined as 5 times the LOD with an accuracy of 80-120%: and an imprecision of <20%.

Analysis of human plasma samples from a clinical study

Blood samples were obtained from a healthy volunteer who participated in a clinical drug-drug interaction study involving warfarin as the victim drug. The human subject received placebo as the perpetrating drug and a single oral dose of 10 mg warfarin the morning of the study following an overnight fast. Venous blood (5 ml) was collected in K2-EDTAVacutainer® tubes (BD Biosciences: Franklin Lakes: NJ: USA) via an intravenous line at 0: 0.5: 1: 1.5: 2: 3: 4: 6: 8: 10: 12: 24: 36: 48: 60: 72: 96: 120: 144: and 168 h post-warfarin administration. Within 1 h following collection: blood samples were centrifuged (3000 rpm at 4°C for 10 min) and the resulting plasma samples transferred to pre-labeled cry tubes for storage at -80°C until analysis. The clinical study (Clinical Trials.gov registry number: NCT01250535) was approved by the Institutional Review Board of the University of North Carolina at Chapel Hill (Chapel Hill: NC: USA). The area under the plasma concentration–time curve (AUC): terminal elimination half-life (t1/2): maximum plasma drug concentration (C max): and time to reach C max (T max) were calculated using the trapezoidal rule–extrapolation method and non compartmental analysis (Phoenix Win Nonlin version 6.3; Pharsight: Mountain View: CA: USA).

Results and Discussion

HPLC-MS/MS analysis and enantiomeric separation

Enantiomeric separation of warfarin and warfarin-d5 (as IS) was achieved using a chiral HPLC column. The extracted ion chromatograms showed that both racemic warfarin and warfarin-d5 standards produced two well-resolved peaks at 4.44 and 4.80 min for warfarin (Figure 1A): and 4.43 and 4.78 min for warfarin-d5 (Figure 1B). These peaks are distinguishable from the hydroxylated warfarin metabolites due to their characteristic MRM transitions. Using synthetic optically pure R- and S-warfarin standards: it was determined that R-war far in eluted at 4.44 min and S-war far in at 4.80 min (data not shown). Due to the stereo chemical similarity between war far in and war far in-d5 (deuterated phenyl ring; Figures 1A and 1B): it can be postulated that R-war far in-d5 eluted at 4.43 min and S-warfarin-d5 at 4.78 min. Subsequently: R-warfarin-d5served as the IS for quantification of R-war far in and (9R; 10S)-10-OH-warfarin: while S-warfarin-d5 was used as the IS for quantification of S-war far in and S-7-OH-war far in.