Determination of warfarin alcohols by ultra-high performance liquid chromatography–tandem mass spectrometry: Application to in vitro enzyme kinetic studies

doi:10.1016/j.jchromb.2013.11.014

Journal of Chromatography B

Volume 944, 1 January 2014, Pages 63-68

https://doi.org/10.1016/j.jchromb.2013.11.014 Get rights and content

Highlights

•
A sensitive UHPLC–MS/MS method for the determination of warfarin alcohol metabolites.
•
The method is validated in Tris–HCl incubation buffer with high accuracy and precision.
•
The method is simple with excellent extraction recovery and short run-time of 5 min.
•
The method is successfully applied to in vitro enzyme kinetic studies of warfarin.

Abstract

A sensitive, accurate, and reproducible ultra-high performance liquid chromatography–tandem mass spectrometry method was developed and validated for determination of warfarin and its alcohol metabolites (RS/SR- and RR/SS-warfarin alcohol) in 10 mM Tris–HCl incubation buffer (pH 7.4). Sample preparation involved acidification with 4% formic acid, followed by liquid–liquid extraction using methyl tert-butyl ether. Chromatographic separation was achieved using a Hypersil Gold C18 (2.1 mm × 100 mm, 1.9 μm) analytical column with gradient elution of solvent A (water containing 0.01% formic acid) and solvent B (acetonitrile containing 0.1% formic acid). The flow rate was 0.4 mL/min and the total run time was 5 min. Detection of analytes was performed using heated electrospray ionization (negative mode) and selected reaction monitoring. Excellent linearity was observed for all analytes over the standard curve concentration ranges of 100–10,000 ng/mL for warfarin, and 0.5–250 ng/mL for warfarin alcohols. The intra- and inter-day accuracy and precision for analytes were within ±10.0%. Excellent recovery and negligible matrix effects were observed. The method is robust, sensitive, accurate and reproducible, and was successfully applied to in vitro enzyme kinetic studies of warfarin.

Introduction

Warfarin is the most commonly used oral anticoagulant for the treatment and prevention of thromboembolic disorders [1]. It is a technically difficult medication to use in clinical practice, due to its notoriously narrow therapeutic window, large inter-individual variability, potential for food and drug interactions, and significant risk if suboptimally dosed [2]. Warfarin is administered clinically as a racemic mixture of both R- and S-enantiomers, and is highly metabolized, exhibiting regioselective and stereoselective metabolism [3], [4]. It undergoes Phase I oxidation mediated mainly by cytochrome P450 (CYP) enzymes, CYP2C9 in particular, producing hydroxy metabolites that can be further metabolized by Phase II conjugation [3], [4]. The effect of various factors (e.g., disease, inflammation, genetic polymorphisms) on CYP2C9 function [5] and their subsequent impact on warfarin dose requirements has been extensively studied [6], [7], and this information is now used to optimize warfarin therapy [8], [9].

Another important metabolic pathway for warfarin elimination is reduction. Warfarin undergoes reduction by hepatic reductases, which generate warfarin alcohols of two diastereoisomers, namely RS/SR-warfarin alcohol (alcohol 1), and RR/SS-warfarin alcohol (alcohol 2) [10], [11]. Warfarin reduction is catalyzed predominantly in the cytosol, producing warfarin alcohol 1 as the major metabolite [12], [13]. Although reduction accounts for up to 20% of warfarin metabolism [11] and produces pharmacologically active alcohol metabolites [14], the effect of altered reductase function on the disposition of warfarin and its alcohol metabolites has not been studied to date. In order to do so, a robust and validated analytical method for the quantitative determination of each analyte is required.

Several assays have been developed for measuring the enantiomers of hydroxywarfarin metabolites in biological samples including HPLC with UV [15], combination of UV/fluorescence and circular dichroism [16] or MS detection [17], [18], [19], capillary zone electrophoresis with UV detection [20], and micellar electrokinetic chromatography with MS detection [21]. However, few analytical methods have been reported for the determination of warfarin alcohol metabolites. These include conventional HPLC methods with UV or fluorescence detection [22], [23], [24], [25], and gas chromatography with mass spectrometric (GC–MS) detection [26]. In general, HPLC with UV or fluorescence detection methods are not as sensitive and selective as MS techniques and require larger sample volume to achieve high sensitivity. The GC–MS method is complex, and requires extensive sample preparation including a derivatization step [26]. In addition, each of the reported methods include minimal or no validation parameters for warfarin alcohols. To date, use of contemporary LC–MS techniques for the measurement of warfarin alcohols has not been reported. Thus, the goal of this work was to develop and validate a simple, rapid and robust ultra-high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) assay for determination of warfarin and its alcohol metabolites. The method was used to examine the enzyme kinetics of warfarin in cytosolic fractions of rat liver tissue.

Section snippets

Chemicals and reagents

Warfarin (C₁₉H₁₆O₄), d5-7-hydroxywarfarin (C₁₉H₁₁D₅O₅, used as internal standard for warfarin alcohols), formic acid, NADPH, magnesium chloride, and tris(hydroxymethyl)aminomethane (Trizma base) were purchased from Sigma (St. Louis, MO, USA). Warfarin alcohols (C₁₉H₁₈O₄) were synthesized as previously described [10], [27]. Racemic d5-warfarin (C₁₉H₁₁D₅O₄, used as internal standard for warfarin) was purchased from Toronto Research Chemical (North York, Ontario, Canada). Hydrochloric acid, methyl

Results and discussion

In order to support in vitro studies exploring warfarin reduction, we aimed to develop and validate a simple and robust UHPLC–MS/MS method for determination of warfarin and warfarin alcohol metabolites. To our knowledge, this is the first LC–MS/MS method developed for this purpose. The method was validated according to the U.S. Food and Drug Administration (FDA) guidelines for bioanalytical method validation [29]. The method is simple, accurate and precise, and is currently being used to

Conclusions

A simple, robust, rapid, accurate, and precise UHPLC–MS/MS method for the determination of warfarin and warfarin alcohols has been developed and validated. The short run-time and high sensitivity and specificity associated with LC–MS/MS renders this method valuable for studies relating to warfarin alcohols.

References (29)

J. Hirsh et al.
Chest
(2001)
L.S. Kaminsky et al.
Pharmacol. Ther.
(1997)
U.M. Zanger et al.
Pharmacol. Ther.
(2013)
T.A. Moreland et al.
Biochem. Pharmacol.
(1975)
J.J. Hermans et al.
Biochem. Pharmacol.
(1989)
H. Takahashi et al.
J. Chromatogr. B: Biomed. Sci. Appl.
(1997)
D.R. Jones et al.
J. Chromatogr. B: Anal. Technol. Biomed. Life Sci.
(2011)
Z. Zuo et al.
J. Pharm. Biomed. Anal.
(2010)
X. Wang et al.
J. Chromatogr. A
(2013)
M.J. Fasco et al.
J. Chromatogr.
(1977)

C. Banfield et al.

J. Pharm. Sci.

(1984)

Y.W. Wong et al.

J. Chromatogr.

(1989)

T.A. van der Hoeven et al.

J. Biol. Chem.

(1974)

M.J. Kim et al.

J. Clin. Pharmacol.

(2009)

Cited by (14)

Synthesis of nickel-doped ceria nanospheres for in situ profiling of Warfarin sodium in biological media
2022, Bioelectrochemistry
Citation Excerpt :
Thus, it is highly needed to develop a cost-effective and high sensitive method for the detection of WFS in biological samples. As yet, there are various methods for detecting WFS such as liquid–liquid micro extraction [31,32], phosphorescence [33], supercritical fluid chromatography-tandem mass spectrometry [34], capillary zone electrophoresis [35–37], high-performance liquid chromatography [38,39], and micellar electro kinetic chromatography-electrospray ionization mass spectrometry [40]. However, the electrochemical sensor has become an effective technique for the rapid detection of WFS owing to its low-cost, simple and fast analysis, excellent sensitivity and selectivity.
Venous thromboembolism is one of the major disorders, which is significantly increased the mortality and morbidity rate. Warfarin sodium (WFS) is the most extensively prescribed drug for the prevention of thromboembolic diseases however, it has a narrow therapeutic index. Recently, many methods for detecting and monitoring the level of WFS have been proposed. However, the electrochemical method has gained more interest than the other traditional method due to its ease of operation. This article describes the hydrothermal synthesis of nickel-doped cerium oxide (CeO₂@Ni) nanospheres for the selective electrochemical determination of WFS. Various spectroscopic techniques have been used to analyze the chemical composition, and surface morphology of CeO₂@Ni nanospheres. Further, the prepared CeO₂@Ni nanospheres modified electrode demonstrated excellent electrocatalytic behavior for WFS detection, with an ultralow detection limit of 6.3 × 10^-9 M, a linear range of 1.0 × 10^-8 M to 1.51 × 10^-4 M and 1.51 × 10^-4 M to 9.51 × 10^-4 M, and a higher sensitivity of 2.9986 µA µM⁻¹ cm². Therefore, we believe that the CeO₂@Ni nanosphere electrocatalyst can serve as a potential electrode catalyst for the sensing of WFS in real-time applications.
Sensitive warfarin sensor based on cobalt oxide nanoparticles electrodeposited at multi-walled carbon nanotubes modified glassy carbon electrode (Co<inf>x</inf>O<inf>y</inf>NPs/MWCNTs/GCE)
2017, Electrochimica Acta
Citation Excerpt :
Because of very narrow therapeutic window of warfarin, precise control of its amount in the blood is needed [6]. Several analytical methods such as high performance liquid chromatography [7,8], micellar electrokinetic chromatography-mass spectrometry (MEKC-MS) [9], capillary zone electrophoresis [10], supercritical fluid chromatography–tandem mass spectrometry (SCF-MS-MS) [11], liquid chromatography (LC) [12,13], multi-mode UPLC (ultra performance liquid chromatography)-MS-MS [14,15], spectrofluorimetry [16,17], sensitized fluorescence [18], solid-phase room-temperature transmitted phosphorescence [19] and electrochemical methods [20–23] have been suggested for the determination and monitoring of warfarin. However, some of them are highly costly and time-consuming, while others suffer from low selectivity and sensitivity.
In this work, cobalt oxide nanoparticles were electrodeposited on multi-walled carbon nanotubes modified glassy carbon electrode (MWCNTs/GCE) to develop a new sensor for warfarin determination. The modified electrodes were characterized by cyclic voltammetry, scanning electron microscopy (SEM) along with energy dispersive x-ray spectroscopy (EDS), and electrochemical impedance spectroscopy (EIS). The presence of cobalt oxide nanoparticles on the electrode surface enhanced the warfarin accumulation and its result was the improvement in the electrochemical response. The effect of various parameters such as pH, scan rate, accumulation potential, accumulation time and pulse amplitude on the sensor response were investigated. Under optimal conditions, the differential pulse adsorptive anodic stripping voltammetric (DPASV) response of the modified electrode was linear in the ranges of 8 nM to 50 μM and 50 μM to 800 μM with correlation coefficients greater than 0.998. The limit of detection of the proposed method was 3.3 nM. The proposed sensor was applied to determine warfarin in urine and plasma samples.
Synthesis of conductive polymeric ionic liquid/Ni nanocomposite and its application to construct a nanostructure based electrochemical sensor for determination of warfarin in the presence of tramadol
2017, Talanta
Citation Excerpt :
Recent data recommend that warfarin drug interactions might be the main cause of the bleeding effects in patients receiving anticoagulant therapy. Numerous methods such as high-performance liquid chromatography [15,16], capillary electrophoresis [17], liquid-liquid micro-extraction [18,19] and electrochemistry methods [20–24] have been described for the measurement of warfarin. Tramadol ((±)-trans-2-[(dimethyl-amino) methyl]-1-(3-methoxyphenyl) cyclohexanol) which is a synthetic 4-phenyl-piperidine analog of codeine is a centrally acting analgesic with potency and efficiency ranging between weak opioids and morphine [25].
In the current study, poly(MImEO8BS)-Ni nanocomposite was synthesized and applied to modify a glassy carbon electrode along with conductive polymeric ionic liquids. The electrochemical investigation of the modified electrode as well as its efficiency for voltammetric oxidation of warfarin is elucidated. The electrode was used to study the voltammetric oxidation of warfarin by employing cyclic voltammetry (CV), linear sweep voltammetry (LSV), chronoamperometry, and square wave voltammetry (SWV) as diagnostic techniques. It has been observed that warfarin oxidation at the surface of modified electrode occurs at a potential of about 230 mV which is less positive than that of an unmodified glassy carbon electrode. SWV demonstrated a linear dynamic range from 1.0×10⁻⁶ to 1.0×10⁻⁴ M and a detection limit of 1.5×10⁻⁷ M for warfarin. In addition, this modified electrode was utilized for simultaneous determination of warfarin and tramadol. Finally, the modified electrode was employed for determination of warfarin and tramadol in pharmaceutical compounds.
Enzyme-catalyzed Michael addition for the synthesis of warfarin and its determination via fluorescence quenching of L-tryptophan
2017, Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy
A sensitive fluorescence sensor for warfarin was proposed via quenching the fluorescence of l-tryptophan due to the interaction between warfarin and l-tryptophan. Warfarin, as one of the most effective anticoagulants, was designed and synthesized via lipase from porcine pancreas (PPL) as a biocatalyst to catalyze the Michael addition of 4-hydroxycoumarin to α, β-unsaturated enones in organic medium in the presence of water. Furthermore, the spectrofluorometry was used to detect the concentration of warfarin with a linear range and detection limit (3σ/k) of 0.04–12.0 μmol L^− 1 (R² = 0.994) and 0.01 μmol L^− 1, respectively. Herein, this was the first application of bio-catalytic synthesis and fluorescence for the determination of warfarin. The proposed method was applied to determine warfarin of the drug in tablets with satisfactory results.
Mn-doped ZnS phosphorescent quantum dots: Coumarins optical sensors
2017, Sensors and Actuators, B: Chemical
Citation Excerpt :
Molecular structures of these coumarins are shown in Fig. 1. Several analytical methods for coumarins determination were reported in the literature, mostly chromatographic methods [1–34], whereas little work has been done in relation to electrochemical [35–39], capillary electrophoresis [40–42], spectrophotometric [43] and luminescence [11,30,44–55] determination methods. Generally, these analytical techniques are time-consuming, require sophisticated equipment and, consequently, not routinely applicable in quality control laboratories.
Mn-doped ZnS quantum dots (QDs) have increasingly attracted much attention as luminescent sensors. Mn⁺² doping promotes a strong phosphorescence-like emission, allowing the development of novel and highly selective sensing phases. In this vein, a coumarins phosphorescent sensor was developed based on the room temperature phosphorescence quenching of water-soluble Mn-doped ZnS QDs. Phosphorescence lifetimes were measured in order to clarify the involved quenching mechanism. Warfarin determination in pharmaceutical products was performed, reaching good figures of merit. The developed methodology is an alternative, rapid, simple, sensitive and selective method for coumarins determination in pharmaceutical and toxicological samples.
An electrochemical sensor for warfarin determination based on covalent immobilization of quantum dots onto carboxylated multiwalled carbon nanotubes and chitosan composite film modified electrode
2015, Materials Science and Engineering C
Citation Excerpt :
Therapeutic concentration of WAR is about 2.0–5.0 μg ml− 1 and has a long half-life (20–60 h) [7]. A number of methods have been used for the determination of WAR including high performance liquid chromatography [8–15], liquid chromatography [16,17], capillary electrophoresis [18], phosphorescence [19], fluorescence [20], cloud point extraction [21], and spectrofluorimetric [22]. Electrochemical techniques are alternative methods for the WAR determination because they are simple, fast, sensitive and low cost.
A method is described for the construction of a novel electrochemical warfarin sensor based on covalent immobilization of CdS-quantum dots (CdS-QDs) onto carboxylated multiwalled carbon nanotubes/chitosan (CS) composite film on the surface of a glassy carbon electrode. The CdS-QDs/CS/MWCNTs were characterized by field-emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), Fourier transform infra-red (FTIR) spectroscopy, XRD analysis and electrochemical impedance spectroscopy (EIS). The sensor showed optimum anodic stripping response within 90 s at an accumulation potential of 0.75 V. The modified electrode was used to detect the concentration of warfarin with a wide linear range of 0.05–80 μM and a detection limit (S/N = 3) of 8.5 nM. The proposed sensor has good storage stability, repeatability and reproducibility and was successfully applied for the determination of warfarin in real samples such as urine, serum and milk.

View all citing articles on Scopus

View full text

Short CommunicationDetermination of warfarin alcohols by ultra-high performance liquid chromatography–tandem mass spectrometry: Application to in vitro enzyme kinetic studies

Highlights

Abstract

Introduction

Section snippets

Chemicals and reagents

Results and discussion

Conclusions

Chest

Pharmacol. Ther.

Pharmacol. Ther.

Biochem. Pharmacol.

Biochem. Pharmacol.

J. Chromatogr. B: Biomed. Sci. Appl.

J. Chromatogr. B: Anal. Technol. Biomed. Life Sci.

J. Pharm. Biomed. Anal.

J. Chromatogr. A

J. Chromatogr.

J. Pharm. Sci.

J. Chromatogr.

J. Biol. Chem.

J. Clin. Pharmacol.

Short Communication
Determination of warfarin alcohols by ultra-high performance liquid chromatography–tandem mass spectrometry: Application to in vitro enzyme kinetic studies