Development and validation of LC–MSMS assay for the determination of the prodrug dabigatran etexilate and its active metabolites in human plasma

doi:10.1016/j.jchromb.2015.02.042

Journal of Chromatography B

Volume 989, 1 May 2015, Pages 37-45

https://doi.org/10.1016/j.jchromb.2015.02.042 Get rights and content

Highlights

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LC–MSMS is used for the determination of dabigatran etexilate and its active metabolites.
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Free/total dabigatran is quantified after treatment with β-glucuronidase enzyme.
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The assay is applicable for therapeutic drug monitoring and pharmacokinetic studies.

Abstract

Dabigatran etexilate (DABE) is a low-molecular-weight prodrug that is converted after oral administration to dabigatran (DAB)—a directly acting oral anticoagulant. In this study, an LC–MSMS assay was developed and validated for the determination of DABE, free DAB and its equipotent O-glucuronide conjugates in plasma. Owing to the susceptibility of DABE and DAB to chemical hydrolysis, cleavage of the O-glucuronide moiety was carried out using β-glucuronidase enzyme. Free and total plasma concentrations of DAB were determined in incurred plasma samples before and after enzymatic cleavage (50 °C and 3 h), respectively. RP-HPLC separation was carried out using acetonitrile: water (30:70, v/v), adjusted to pH 3.0 using formic acid. Tandem mass spectrometric detection at positive electrospray ionization in the MRM mode was then employed for the determination of DABE and DAB. The analysis was carried out within 5.0 min over a linear concentration range of 1.00–600.00 ng/mL for the prodrug and its active metabolite. Validation was carried out according to FDA guidelines for bioanalytical method. The recoveries were higher than 89.48%, the accuracy was within 98.33–110.12% and the RSD was below 10% for the studied compounds in both incurred plasma and quality control samples. Results of incurred sample re-analysis and incurred sample stability revealed less than 10% variability. This indicated good assay precision and sufficient stability of target analytes in their real matrix at the employed experimental conditions. The applicability of the assay for therapeutic drug monitoring and the determination of the pharmacokinetic parameters were demonstrated.

Introduction

Dabigatran etexilate (DABE) is an oral anticoagulant from the class of direct thrombin inhibitors. It offers an alternative to warfarin that does not require frequent monitoring of the clotting tendency of blood while offering similar results in terms of efficacy [1], [2]. DABE is a prodrug that is metabolized by serum esterase to its active metabolite, dabigatran (DAB) [3], [4], [5]. For many drugs, liver glucuronidation is the major metabolic pathway for detoxification [6], [7], [8], [9]. However, in the case of DAB, conjugation with glucuronic acid yields equipotent conjugates [1], [3], [4]. The detailed glucuronidation pathway has been previously shown [5] and was summarized in Fig. S1.

Chemically, DABE is ethyl N-[(2-{[(4-{N’-[(hexyloxy)carbonyl]carbamimidoyl}phenyl)amino] methyl}-1-methyl-1H-benzimidazol-5-yl)carbonyl]-N-2-pyridinyl-β-alaninate. The structural formulae of DABE and DAB are presented in Fig. 1 [2]. Few analytical methods have been reported for the routine quantitative analysis of DABE in its dosage forms such as HPLC [10], [11], [12], [13] and spectrophotometry [14]. The metabolic pattern of DAB was previously revealed using HPLC with online radioactivity detection [4]. Mass spectrometry has been reported for in vitro metabolic profiling [15], determination of permeability coefficient of DABE and its transcellular transport by intestinal P-glycoprotein [16] and clinical evaluation of therapeutic results [17], [18], [19], [20]. Recently, DABE and DAB along with an intermediate metabolite were simultaneously determined in rat plasma using LC–MSMS [21].

In the FDA draft guidance, measurement of the concentration of free and total DAB has been requested in order to demonstrate the bioequivalence of DABE generic formulations to the innovator product [22]. In the literature, the pharmacokinetics, pharmacodynamics and tolerability of orally administered DABE were investigated using the results of LC–MSMS in conjunction with various clinical parameters [4], [23], [24]. In the latter studies, the concentration of free and total DAB was determined before and after base-catalysed hydrolysis of the glucuronide moiety. Lack of evidence for the stability of DABE and DAB at the described sample preparation conditions, along with the presence of several reports highlighting the liability of DABE to chemical hydrolysis [10], [11], [12], raised a concern about the validity of the outcomes of these studies.

Therefore, the aim of this study was to develop a reliable and valid LC–MSMS bioanalytical assay for the determination of DABE and free and total DAB in human plasma. The reliability of base-catalysed hydrolysis and enzymatic hydrolysis of DAB-O-glucuronide conjugates into free DAB was compared. The applicability of the proposed assay for therapeutic drug monitoring and determination of the pharmacokinetic parameters of DAB was investigated.

Section snippets

Materials

DABE and DAB reference standards were obtained from US Biological Life Sciences (USA), whereas sertraline hydrochloride (IS) was supplied by Makin Research Center (Egypt). Pradaxa capsules (Boehringer Ingelheim GmbH, Germany) labelled to contain 75 or 150 mg DABE (as mesilate) were obtained from local market. Human blank plasma was obtained from the Holding Company for Biological Products and Vaccines (VACSERA), Egypt. The β-glucuronidase aqueous solution, 197,000 U/mL (Cat. Number G0876), was

Liquid chromatography and mass spectrometric conditions

Coupling of LC with MSMS detection is a highly selective technique that results in minimal interference from endogenous impurities. This could be explained on the basis that in the MRM mode, only the ions derived from the target analytes are monitored [27]. Initially, several trials using different columns (C₁₈ Xterra, Waters and C₁₈ Luna, Phenomenex) and mobile phase compositions using methanol and different ratios of acetonitrile (70, 60, 50 and 40%) were tried in order to optimize the

Conclusion

A fast and accurate LC–MSMS assay was developed and validated for the simultaneous determination of the prodrug DABE, free and total DAB in human plasma. Enzymatic cleavage of DAB-O-glucuronide conjugates followed by simple protein precipitation procedure was employed for sample preparation. Results of the validation studies showed that the developed assay was selective, accurate and precise over a concentration range that covers the C_max of the drug. Results also confirmed appropriate

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New disposable ion-selective sensors for the determination of dabigatran etexilate: The oral anticoagulant of choice in patients with non-valvular atrial fibrillation and COVID-19 infection
2022, Measurement: Journal of the International Measurement Confederation
Citation Excerpt :
Used to prevent systemic and stroke embolism among patients with venous thromboembolism and atrial fibrillation [1,3]. Some analytical techniques are reported for the determination of dabigatran etexilate, including spectroflourimetric [4], voltammetric [4,5], HPLC [7–11], UHPLC [12], and liquid chromatography mass spectrometry (LC-MS/MS) [13]. Ion-selective electrodes (ISE) have often been used in potentiometric determination of various pharmaceutical preparations [14–16].
Selective, sensitive, and reproducible ion-selective electrodes containing dabigatran etexilate- phosphotungstate ion pair as a modifier have been fabricated to determine dabigatran etexilate in pure and pharmaceutical dosage form. The modified electrodes ion selective carbon paste electrode and ion selective pencil graphite electrode showed fast and linear response within a concentration range of 1.0 × 10⁻⁵ to 1.0 × 10⁻² M and 1.0 × 10⁻⁶ to 1.0 × 10⁻² M with a detection limit of 4.36 × 10⁻⁶ M and 4.26 × 10⁻⁷ M by ISCPE and ISGPE, respectively. Electrodes are selective to dabigatran etexilate over common excipients. The present study demonstrated the determination of dabigatran etexilate in pure and pharmaceutical dosage forms with high accuracy and precision.
Development and validation of an ultra-high performance liquid chromatography with tandem mass spectrometry method for the simultaneous quantification of direct oral anticoagulants in human plasma
2021, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Citation Excerpt :
After precipitation, the clear supernatant was diluted with water to attenuate possible matrix effects. In recent studies, diazepam, sertraline, and risperidone were reported as ISs in HPLC-UV methods [23-26]. However, owing to their different chemical structures and elution behaviors compared to DOACs, they cannot efficiently compensate for the error during UHPLC-MS analysis.
Direct oral anticoagulants are widely used to treat and prevent thromboembolic disorders. With rising clinical application, monitoring concentrations of direct oral anticoagulants are necessary in certain clinical conditions. A rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry method was developed for the simultaneous determination of dabigatran etexilate, dabigatran, rivaroxaban, edoxaban, and apixaban, in human plasma. Protein precipitation with methanol was performed for sample preparation. The direct oral anticoagulants and internal standards were separated under gradient conditions using a C18 column, at an analytical run time of 8 min. The mobile phase was composed of 0.1% (v/v) formic acid in water (solvent A) and 0.1% (v/v) formic acid in acetonitrile (solvent B) at a flow rate of 0.3 mL/min. Mass detection was performed in multiple reaction monitoring using positive ionization mode. The method was validated over a range of 1.0–500 ng/mL for dabigatran etexilate, 0.1–500 ng/mL for dabigatran, and 0.5–500 ng/mL for edoxaban, rivaroxaban, and apixaban. The method detection limits of five analytes were in the range of 0.05–0.5 ng/mL. The lower limits of quantification of five analytes ranged from 0.1 to 1 ng/mL. The linearity (r² values) was higher than 0.997. The accuracy of the low, medium, and high quality control samples were between 85.9 and 114%, and intra- and inter-day precision were below 9.47%. This validated method was successfully used to determine the plasma concentrations of rivaroxaban in 32 patients, and of dabigatran etexilate and dabigatran in 1 patient.
Comparative study of boron-doped diamond, basal-plane pyrolytic graphite, and graphite flake paste electrodes for the voltammetric determination of rivaroxaban and dabigatran etexilate in pharmaceuticals and urine samples
2021, Diamond and Related Materials
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DAB has been approved in Europe for the prevention and treatment of deep vein thrombosis in patients undergoing knee or hip replacement surgery [33], prophylaxis of pulmonary embolism, and prevention of stroke and venous thromboembolism in patients with acute coronary syndrome and atrial fibrillation [44]. The literature describes various procedures for the determination of RIV, for example, by using high-performance liquid chromatography (HPLC) [32,34,45–48], micellar liquid chromatography (MLC) [36], thin-layer chromatography (TLC) [47], and ultra-high-performance liquid chromatography (UHPLC) [49,50], whereas DAB is determined by using HPLC [32,34,44,46], UHPLC [49], or liquid chromatography (LC) [51,52]. However, the aforementioned methods are time-consuming and are limited by the need for expensive instrumentation.
As anticoagulants, rivaroxaban (RIV) and dabigatran etexilate (DAB) are high-risk drugs that can cause life-threatening bleeding. They are a group of drugs that require optimal dosing, therefore their monitoring in the patient is of great importance, especially in emergencies. In this study, we developed a rapid, sensitive square-wave voltammetric (SWV) method by using boron-doped diamond, graphite flake paste, and basal-plane pyrolytic graphite electrodes (BDDE, GFPE, and BPPGE, respectively) to determine the presence of anticoagulants, namely, RIV and DAB in pharmaceutical and urine samples. The supporting electrolyte composition and SWV parameters were investigated. The sensitivity, stability, recovery, calibration curve, and limit of detection (LOD) of the developed method were evaluated. Under the optimal experimental condition, a wider linear response was achieved using BDDE in the detection of both RIV and DAB. The calibration curve for RIV was in the range of 0.5–30.0 μmol L⁻¹ for BDDE and 1.0–10.0 μmol L⁻¹ for GFPE. The calibration curve for DAB was in the range of 0.01–0.7 μmol L⁻¹ for BDDE and 0.07–1.0 μmol L⁻¹ for BPPGE, which shows a linear relationship between the concentration of DAB and the anodic current. LOD was equal to 0.145 μmol L⁻¹ (for RIV at BDDE), 0.297 μmol L⁻¹ (for RIV at GFPE), 2.78 nmol L⁻¹ (for DAB at BDDE), and 16.7 nmol L⁻¹ (for DAB at BPPGE). Finally, BDDE, GFPE, and BPPGE were applied to detect the presence of RIV and DAB in pharmaceuticals and spiked urine samples with satisfactory results (98.2–100.5% for RIV and 99.22–102.8% for DAB). Cyclic voltammetry (CV) was applied to understand the electrode mechanism of RIV and DAB oxidation. The character of RIV and DAB signals was irreversible and diffusion-controlled. In addition, the roughness factor (RF) of BDDE, GFPE, and BPPGE was investigated, which were equal to 0.367, 0.558, and 0.422, respectively.
Development and validation of LC–MS/MS method for simultaneous determination of dabigatran etexilate and its active metabolites in human plasma, and its application in a pharmacokinetic study
2021, Journal of Pharmaceutical and Biomedical Analysis
Dabigatran is a direct thrombin inhibitor widely used for preventing various thrombotic events. Although there are several established LC–MS/MS based methods for quantification of plasma dabigatran etexilate and its active metabolites (dabigatran and dabigatran acylglucuronide), so far, there are no studies for simultaneous quantification of dabigatran etexilate, dabigatran, and dabigatran acylglucuronide in human plasma samples. In the present study, a novel and sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed and validated for assessment of dabigatran pharmacokinetics in human plasma samples according to FDA guidelines. We used the new method to simultaneously quantify dabigatran etexilate, dabigatran, and dabigatran acylglucuronide in human plasma samples. After deproteinization using acetonitrile, the supernatants were evaporated, dissolved in a mobile phase, and finally injected onto an HPLC system with a silica-based C₁₈ reverse-phase column. Mass spectrometer system was operated in multiple reaction monitoring mode (MRM) (dabigatran etexilate: m/z 629.464→290.100; dabigatran: m/z 472.300→289.100, and dabigatran acylglucuronide: m/z 648.382→289.100) and all the components were confirmed using positive electrospray ionization (ESI). Correlation coefficients > 0.999 were achieved for all the calibration curves with linear regression. The intra-day and inter-day accuracies of dabigatran etexilate, dabigatran, and dabigatran acylglucuronide were 95.84–109.44 %, 99.4–103.42 %, and 98.37–104.42 %, respectively, while their corresponding precisions were 3.84–9.79, 1.07–8.76 %, and 2.56–4.51 %, respectively. We successfully applied the new method to determine the pharmacokinetic profiles of dabigatran etexilate, dabigatran, and dabigatran acylglucuronide in humans. Our findings demonstrated the method as robust, reliable, and sensitive.
A chemometric comparison of different models in fluorescence analysis of dabigatran etexilate and dabigatran
2021, Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy
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But these methods are usually inferior in accuracy, sensitivity, selectivity and universality [14]. Some direct determination methods such as liquid chromatography or ultra performance liquid chromatography with tandem mass spectrometry (LC-MS/MS or UPLC-MS/MS) [15–25], high performance liquid chromatography (HPLC) combined with ultraviolet detection (UV) [26–29], diode array detection (DAD) [30,31] or fluorescence detection (FLD) [32] have been also reported to measure DABE or DAB levels in different matrices. However, the instruments for mass spectrometric methods are expensive and require individuals with specialized experience, which makes it difficult for routine clinical applications.
In this paper, a simple, rapid, low-cost and potential method was established for the simultaneous quantitative analysis of dabigatran etexilate (DABE) and dabigatran (DAB) in spiked biological fluids. It combined excitation-emission matrix fluorescence (EEMF) with different second-order calibration methods, including the self-weighted alternating normalized residue fitting (SWANRF) algorithm based on trilinear decomposition model, the multivariate curve resolution - alternating least-squares (MCR-ALS) based on bilinear decomposition model and the unfolded partial least-square coupled with residual bilinearization (U-PLS/RBL) based on latent variables model. The proposed method showed “second-order advantage”, that is, satisfactory quantitative results were successfully obtained even in the presence of unknown interferences and serious spectral overlap. The recoveries of DABE and DAB in spiked biological fluids were 91.7%–101.7% for SWANRF, 95.9%–117.8% for MCR-ALS, 83.0%–109.6% for U-PLS/RBL, respectively. Figures of merit and other statistical parameters were also calculated to assess the performance of the proposed method. Moreover, the modeling procedures and characteristics of three different models in EEMF analysis were discussed and compared.
Optimization by response surface methodology of a dispersive magnetic solid phase extraction exploiting magnetic graphene nanocomposite coupled with UHPLC-PDA for simultaneous determination of new oral anticoagulants (NAOs) in human plasma
2020, Journal of Pharmaceutical and Biomedical Analysis
In this paper a dispersive magnetic-solid phase extraction (MSPE) using a graphene nanocomposite (rG/Fe₃O₄) followed by ultra high performance liquid chromatography with photodiode array detection has been developed for the simultaneous analysis of new class of oral anticoagulants (NOAs) in human plasma. The performance of the nanocomposite graphene@Fe₃O₄ on the magnetic solid phase extraction of apixaban, rivaroxaban and dabigatran has been optimized using a Box-Behnken design of experiment. The amount of graphene nanocomposite, the sample pH and the adsorption time were the investigated parameters as a function of the extraction recovery. The analytical method was fully validated based on linearity, limit of detection (LOD), limit of detection (LOQ), inter- and intra-day precision and trueness, and extraction yield. Under optimal condition, excellent linearity (R² > 0.9987) over the range (0.001–5.0 μg/mL), limit of detection (0.003 μg/mL), precision (0.81–8.97% RSD) and trueness (−5 to 9 % BIAS%) were observed for the target drugs. The average extraction recovery under optimal from plasma samples ranged between 96.6–98.6% for apixaban, rivaroxaban and dabigatran and the internal standard.
The proposed method was developed, validated and successfully applied to the measurement of these NOAs in patients. The new approach offers an attractive alternative for the simultaneous analysis of the selected NOAs from plasma samples, providing several advantages including fewer sample preparation steps, ease of performance, and higher recoveries compared to traditional methodologies.

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Development and validation of LC–MSMS assay for the determination of the prodrug dabigatran etexilate and its active metabolites in human plasma

Highlights

Abstract

Introduction

Section snippets

Materials

Liquid chromatography and mass spectrometric conditions

Conclusion

J. Chromatogr. B

J. Thromb. Haemost.

J. Chromatogr. B

J. Chromatogr. B

J. Chromatogr. B

J. Chromatogr. B

Trends Anal. Chem.

Dabigatran etexilate—a novel, reversible, oral direct thrombin inhibitor: interpretation of coagulation assays and reversal of anticoagulant activity

Thromb. Haemost.

Dabigatran acylglucuronide, the major human metabolite of dabigatran: in vitro formation, stability, and pharmacological activity

Drug Metab. Dispos.

The metabolism and disposition of the oral direct thrombin inhibitor, dabigatran, in humans

Drug Metab. Dispos.

Boehringer Ingelheim

Confirmation of diosmetin 3-O-glucuronide as major metabolite of diosmin in humans, using micro-liquid-chromatography–mass spectrometry and ion mobility mass spectrometry

Anal. Bioanal. Chem.

Quantification of glucuronide metabolites in biological matrices by LC–MS/MS

Disposition of the selective cholesterol absorption inhibitor ezetimibe in healthy male subjects

Drug Metab. Dispos.

Validated stability-indicating high performance liquid chromatographic assay method for the determination of dabigatran etexilate mesylate

Res. J. Pharm. Biol. Chem. Sci.