Journal of Chromatographic Science, 2016, Vol. 54, No. 4, 554–560 doi: 10.1093/chromsci/bmv186 Advance Access Publication Date: 11 December 2015 Article

Article

Validation and Application of a Simple UHPLC–MS-MS Method for the Enantiospecific Determination of Warfarin in Human Urine Osama Y. Alshogran1,2, Andrew J. Ocque1,3, François A. Leblond4, Vincent Pichette4,5, and Thomas D. Nolin1,3,* 1

Center for Clinical Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA, USA, Department of Pharmaceutical Sciences, School of Pharmacy, University of Pittsburgh, Pittsburgh, PA, USA, 3 Department of Pharmacy and Therapeutics, School of Pharmacy, University of Pittsburgh, 208 Salk Pavillion, 335 Sutherland Drive, Pittsburgh, PA 15260, USA, 4Service de Néphrologie et Centre de Recherche, Hôpital Maisonneuve-Rosemont, Montréal, QC, Canada, and 5Département de Pharmacologie, Université de Montréal, Montréal, QC, Canada 2

*Author to whom correspondence should be addressed. Email: [email protected] Received 26 February 2015; Revised 20 September 2015

Abstract A simple and rapid liquid chromatographic–tandem mass spectrometric method has been developed and validated for the enantiospecific determination of R- and S-warfarin in human urine. Warfarin enantiomers were extracted from urine using methyl tert-butyl ether. Chromatographic separation of warfarin enantiomers and the internal standard d5-warfarin was achieved using a Astec Chirobiotic V column with gradient mobile phase at a flow rate of 400 µL/min over 10 min. Detection was performed on a TSQ Quantum Ultra triple quadrupole mass spectrometer equipped with a heated electrospray ionization source. Analytes were detected in negative ionization mode using selected reaction monitoring. Calibration curves were linear with a correlation coefficient of ≥0.996 for both enantiomers over a concentration range of 5–500 ng/mL. The intra- and interday accuracy and precision for both analytes were within ±9.0%. Excellent extraction efficiency and negligible matrix effects were observed. The applicability of the method was demonstrated by successful measurement of warfarin enantiomers in urine of patients with kidney disease. The method is simple, accurate and reproducible and is currently being used to support warfarin pharmacokinetic studies.

Introduction Warfarin is the most commonly used oral anticoagulant for the treatment and prevention of thromboembolic disorders (1). The drug acts by inhibiting vitamin K epoxide reductase (VKOR), the enzyme involved in the activation of clotting factors II, VII, IX and X (1). It is administered as a racemic mixture consisting of both R- and S-enantiomers and shows regioselective and stereoselective metabolism (2). S-warfarin is three to five times more potent than R-warfarin, is more rapidly cleared by hepatic metabolism than R-warfarin and is almost exclusively (∼90%) metabolized by cytochrome P450 2C9 (CYP2C9) (2, 3).

Treatment with warfarin is challenging due to its narrow therapeutic window, large inter-individual variability and high likelihood of food and drug interactions (4). As such, individualized dosing and regular monitoring of warfarin is needed to achieve safe and effective clinical outcomes. This requires consideration of patient demographic factors, variations in the genes encoding VKOR and CYP2C9 and comorbid conditions such as liver disease (5, 6). Recently, kidney function has been shown to be an important determinant of S-warfarin metabolic clearance (7), supporting previous findings of a 50% increase in the S-/R-warfarin ratio in end-stage renal disease patients (8), as well as recent observations that patients with advanced kidney

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Enantiospecific Determination of Warfarin in Human Urine disease require lower warfarin dosages (9, 10). Previous warfarin pharmacokinetic studies also indicate that renal excretion of unchanged enantiomers can be modulated (11, 12). To mechanistically evaluate these phenomena, there is a need for robust and fully validated methods capable of quantitative enantiospecific determination of warfarin in urine. Several analytical methods have been reported for enantiospecific quantification of warfarin in plasma. These include conventional HPLC methods with UV (13–18) or fluorescence detection (19), capillary electrophoresis with UV detection (20) and numerous chromatographic techniques [e.g. liquid chromatography (21–26), supercritical fluid chromatography (27), chiral capillary electrochromatography (28) and micellar electrokinetic chromatography (29, 30)] with mass spectrometric (MS) detection. However, to our knowledge, only one other method, which is limited by its complex dual-phase approach, 20 min run-time, and relatively few validation parameters assessed, has been reported for determination of warfarin enantiomers in urine (23). Thus, the goal of this work was to develop and fully validate a simple and fast liquid chromatography–tandem mass spectrometry (LC–MS-MS) assay for determination of R- and S-warfarin in human urine.

Experimental Chemicals and reagents R- and S-warfarin were purchased from Cayman Chemical (Ann Arbor, MI, USA). Racemic phenyl-d5-warfarin (used as internal standard) was purchased from Toronto Research Chemical (North York, ON, Canada). Ammonium acetate and formic acid were obtained from Sigma (St. Louis, MO, USA). Methyl tert-butyl ether (MTBE), methanol, OPTIMA LC–MS grade water and acetonitrile were purchased from Fisher Scientific (Pittsburgh, PA, USA). All chemicals were LC–MS grade or of the highest purity available. Nitrogen gas (ultra-pure, >99.9%) was produced by a Parker Balston nitrogen generator (Haverhill, MA, USA). Argon gas (ultra-pure, >99.9%) was provided by Valley (Wheeling, WV, USA). Blank human urine was obtained from in-house laboratory personnel.

Equipment and LC–MS-MS conditions Liquid chromatography was performed with an Accela series UHPLC system (Thermo Scientific, San Jose, CA, USA) including an autosampler and ultra-high performance binary pump. Chromatographic separation of the samples was achieved with an Astec Chirobiotic V column (150 × 2.1 mm ID, 5 μm) and an Astec Chirobiotic V guard column (10 × 2.1 mm ID; 5 μm) each supplied by Supelco (Bellefonte, PA, USA). The flow rate was 400 µL/min and composed of solvent A [10 mM ammonium acetate ( pH 5.0) and acetonitrile (90 : 10, v/v)] and of solvent B (acetonitrile). The step gradient was as follows: 100% of solvent A for 0–0.5 min; a linear gradient to 15% B from 0.5 to 5.0 min; followed by a step to 50% B for 2 min. The column was then re-equilibrated to initial conditions for 3 min. The total runtime was 10 min. The autosampler was kept at 10°C, and the column temperature was held at 40°C. MS–MS detection was performed on a TSQ Quantum Ultra triple quadrupole mass spectrometer (Thermo Scientific, San Jose, CA, USA) equipped with a heated electrospray ionization (ESI) source. Analytes were detected in negative ionization mode using selected reaction monitoring. The spray voltage was set to 3,000 V, and the vaporizer temperature was set to 360°C. The sheath gas and auxiliary gas were set to 65 and 55 (arbitrary units), respectively. The ion transfer tube was set

to 350°C. Collision gas (argon) pressure was set at 1.5 mTorr. The scan width was set to 0.01 m/z, scan time was set to 0.5 s and full width at half maximum was set to 0.7 for both quadrupole one (Q1) and three (Q3). The ion transitions were m/z 307.1 → 161.1 for warfarin (collision energy = 21 V) and m/z 312.2 → 255.3 for d5-warfarin (collision energy = 26 V). Signal output was captured and processed with Xcaliber software v2.2 (Thermo Scientific, San Jose, CA, USA).

Preparation of calibration standard and quality control samples Each analyte (R- and S-warfarin) was dissolved in methanol to obtain a 1.0 mg/mL stock solution. These stock solutions were mixed with methanol to prepare an intermediate stock solution that was spiked into blank human urine to create calibration standards at concentrations of 5, 25, 50, 125, 250 and 500 ng/mL for both enantiomers. Three quality control (QC) samples (LQC, MQC and HQC) containing both analytes were made by spiking a separate 1.0 mg/mL stock solution into blank urine at concentrations of 15, 75 and 200 ng/mL for both analytes. The selection of QC levels was based on the current US Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines for bioanalytical method validation (31, 32). All stock solutions, standards and QC samples were stored at −80°C to simulate the storage conditions of study samples.

Sample preparation A 500 µL aliquot of urine samples was mixed with 20 µL internal standard d5-warfarin working solution (5 µg/mL) dissolved in methanol. Samples were then acidified by addition of 4% (v/v) formic acid in water (250 µL) and briefly vortex-mixed. The analytes were extracted by MTBE (2 mL), where they were continuously mixed for 20 min at room temperature on an Eberbach shaker. The upper organic layer was removed and evaporated to dryness at 37°C for 10 min under a stream of nitrogen. The residue was reconstituted with 100 µL of acetonitrile–water (25 : 75, v/v). Samples were loaded into autosampler vials, and 15 µL was injected into the LC–MS-MS system.

Assay validation Calibration and linearity Calibration curves were constructed over a linear range of 5–500 ng/mL using six concentrations of R- and S-enantiomers in blank urine. Each calibration level was run in duplicate except for the lower limit of quantification (LLOQ) which was run in triplicate. For each curve, the absolute peak-area ratios of the analyte to the internal standard were calculated and plotted against the nominal analyte concentration. Calibration curves were generated by weighted (1/y) linear regression analysis. Accuracy and precision Accuracy and precision were determined by the analysis of QC samples spiked at three concentrations. Six replicate QC samples at each concentration were analyzed daily for 2 days, followed by analysis of 12 replicate QC samples at each concentration on the third day, for a total of n = 24 QC samples at each concentration. Intraday accuracy and precision were determined from the 12 replicate QC samples on Day 3, and interday accuracy and precision were calculated from all 24 QC samples. The calculated mean concentration relative to the nominal concentration was used to express accuracy (% bias). Means, standard deviations and relative standard deviations (% RSD) were

556 calculated from the QC values and used to estimate the inter- and intraday precision. A dilution analysis was also performed. Samples were spiked to twice the concentration of the highest standard (i.e. 1,000 ng/mL), then diluted 1 : 4 or 1 : 8 with blank urine before analysis. Each dilution level was processed in triplicate and back calculated against the standard curves.

Extraction efficiency and matrix effect The extraction efficiency of both enantiomers at QC levels of urine samples were evaluated by measuring R- and S-warfarin as described above, and comparing the response with those achieved after direct injection of neat solutions of the analytes spiked in mobile phase. The matrix effect of urine at QC levels was assessed according to the method of Vecchione et al. (25). The detector response of the analytes extracted in urine was compared with that obtained from samples extracted in water, representing 100% (no matrix effect). The matrix effect was considered to be negligible if the measured concentration in urine deviated from water by