Development and validation of LC–MSMS assay for the determination of the prodrug dabigatran etexilate and its active metabolites in human plasma
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In the FDA draft guidance, measurement of the concentration of free and total DAB has been requested in order to demonstrate the bioequivalence of DABE generic formulations to the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 Cmax 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 ConfederationCitation 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].
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 SciencesCitation 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.
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 MaterialsCitation Excerpt :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.
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 AnalysisA chemometric comparison of different models in fluorescence analysis of dabigatran etexilate and dabigatran
2021, Spectrochimica Acta - Part A: Molecular and Biomolecular SpectroscopyCitation Excerpt :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.