UPLC MS/MS assay for routine quantification of dabigatran – A direct thrombin inhibitor – In human plasma

doi:10.1016/j.jpba.2011.09.018

Journal of Pharmaceutical and Biomedical Analysis

Volume 58, 25 January 2012, Pages 152-156

https://doi.org/10.1016/j.jpba.2011.09.018 Get rights and content

Abstract

This article described the development and full validation of rapid and accurate liquid chromatography method, coupled with tandem mass spectrometry detection, for quantification of dabigatran in human plasma with [¹³C₆]-dabigatran as internal standard. Plasma pretreatment involved a single step protein precipitation with methanol. Separation was performed by ultra performance reversed-phase chromatography on an Acquity UPLC BEH C8 100 mm × 1 mm × 1.7 μm column using a gradient elution mode. The mobile phase was a mix of distilled water containing 0.1% formic acid and methanol containing 0.1% formic acid. Specific multiple reaction monitoring transitions were recorded in positive electrospray ionization. The method was linear over the concentration range of 2–500 μg/L. The intra- and inter-day precision values were below 11.3% and accuracy was within 93.8% and 108.8% for all QC levels (5, 75 and 400 μg/L). The lower limit of quantification was 2 μg/L. Total analysis time was to 10 min including sample preparation.

Introduction

Dabigatran etexilate is a novel oral direct thrombin inhibitor. In recent studies, dabigatran demonstrated its efficacy for prophylaxis and treatment of thromboembolic event during orthopedic surgery and curative treatment of hypercoagulability in atrial fibrillation [1], [2].

Contrary to vitamin K antagonist (VKA), no laboratory monitoring was advised for this drug because its pharmacokinetic profile is supposed to be more predictable [3], [4]. However EMEA underlined the low bioavailability and very large interindividual variability of dabigatran exposition in case of renal impairment, elderly patients or extreme weights [5]. From that point, some arguments are in favor of a monitoring assay [6]. First, dabigatran is eliminated at 80% by kidney leading to accumulation in renal impaired patients. Second, it is also a substrate of P-glycoprotein (P-gp) and may be subject to drug–drug interactions [7], [8]. P-gp inductors or inhibitors can respectively decrease or increase its bioavailability. In these situations, dabigatran accumulation could increase bleeding risk and may require therapeutic drug monitoring.

Several methods were previously published for quantification of dabigatran in phase II/III studies [1], [3], [7], [8], [9], [10], [11]. None of them presented the complete analytical process used and validation criteria according the FDA guidelines [12]. The purpose of this work was to develop a fully validated and routinely available ultra performance liquid chromatography coupled with tandem mass spectrometry (UPLC MS/MS) method by for quantification of dabigatran in human plasma.

Section snippets

Chemicals and reagents

Dabigatran and [¹³C₆]-dabigatran were purchased from Alsachim (Strasbourg, France). LC MS grade methanol and acetonitrile were obtained from Fisher Scientific GmbH (Schwerte, Germany) and distilled water from Aguettant (Lyon, France). Hydrochloric acid 0.1 N was obtained from Sigma–Aldrich (St. Quentin Fallavier, France). A 0.2 μm polyvinylidene fluoride filter was used for mobile phases and was provided by Interchim (Montluçon, France).

Stock solutions, calibration standards and quality control samples

Stock solutions of dabigatran and internal standard [¹³C₆

Liquid chromatography

Representative chromatograms of drug free, LLOQ and supposed over exposed human plasma samples are shown in Fig. 1. Dabigatran and IS retention times and peak shape did not varied over the whole validation period (data not shown). Retention time was near 1.6 min for dabigatran and IS, total run time of 4.5 min allowed return to initials conditions.

Mass spectrometry

In positive electrospray ionization, quantification was performed by addition of 472.19→324.17 m/z and 472.19→306.13 m/z MRM transitions for [dabigatran

Conclusion

In conclusion, this paper described the development and full validation of a single step preparation, rapid (about 10 min), sensible and accurate ultra performance liquid chromatography method using tandem mass spectrometry detection for dabigatran quantification in human plasma. This method is totally compatible with other UPLC MS/MS routine activities and could be applied to 24/7 clinical toxicology to monitor dabigatran accumulation and therapeutic drug monitoring [13].

References (13)

B.I. Eriksson et al.
A new oral direct thrombin inhibitor, dabigatran etexilate, compared with enoxaparin for prevention of thromboembolic events following total hip or knee replacement: the BISTRO II randomized trial
Journal of Thrombosis and Haemostasis
(2005)
H. Bounameaux et al.
New oral antithrombotics: a need for laboratory monitoring. Against
Journal of Thrombosis and Haemostasis
(2010)
P. Mismetti et al.
New oral antithrombotics: a need for laboratory monitoring
Journal of Thrombosis and Haemostasis
(2010)
X. Delavenne et al.
HPLC MS/MS method for quantification of meprobamate in human plasma: application to 24/7 clinical toxicology
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
(2011)
S.J. Connolly et al.
Randomized evaluation of long-term anticoagulation therapy, newly identified events in the RE-LY trial
The New England Journal of Medicine
(2010)
J. Stangier et al.
The pharmacokinetics, pharmacodynamics and tolerability of dabigatran etexilate, a new oral direct thrombin inhibitor, in healthy male subjects
British Journal of Clinical Pharmacology
(2007)

There are more references available in the full text version of this article.

Cited by (62)

A chemometric comparison of different models in fluorescence analysis of dabigatran etexilate and dabigatran
2021, Spectrochimica Acta - Part A: Molecular and Biomolecular Spectroscopy
Citation 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.
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.
Gradient RP-HPLC method for the determination of potential impurities in dabigatran etexilate in bulk drug and capsule formulations
2019, Arabian Journal of Chemistry
Citation Excerpt :
Dabigatran etexilate converted to a major active metabolite known as dabigatran by non-specific plasma and liver esterase (Blech et al., 2008). Several research papers have been reported in the literature for the determination of DAB (Bernardi et al., 2013a,b; Damle and Bagwe, 2014; Geetharam et al., 2014; Pani Kumar et al., 2015; Khan et al., 2014; Reddy and Rao, 2014) and these papers were limited to the assay of alone DAB from its degradants and concurrently DAB in human plasma and other biological stuff, based on UPLC MS/MS (Delavenne et al., 2012), HPLC and LC/MS/MS (Bernardi et al., 2015; Nouman et al., 2015; Laizure et al., 2014; Li et al., 2014; Härtter et al., 2012). Monitoring and control of by-products or impurities at each stage of synthesis is required to get a final pure form of DAB as per predefined specifications.
A selective RP-HPLC method for separation and determination of potential related impurities (starting materials and by-products of synthesis, and degradants) of dabigatran etexilate (DAB) drug substance has been developed and validated. The separation was accomplished on a Inertsil ODS 3V (250 mm × 4.6 mm, 5 μm) column connected to a photodiode array (PDA) detector using 20 mM ammonium formate with 0.1% of triethylamine (pH: 5.0 adjusted with formic acid) as mobile phase-A and acetonitrile as mobile phase-B, under gradient elution. Two unknown impurities found in dabigatran etexilate industrial batch stability condition at levels more than 0.1% in HPLC analysis were characterized preliminarily by ESI-MS/MS studies. The major unknown (unknown-1) was enriched and isolated by preparative LC and structure was evidenced by ¹H and ¹³C NMR spectroscopy, mass spectrometry and FT-IR. Another unknown (unknown-2) found as secondary degradant and structure was proposed by ESI-MS/MS study. This method can be used for the quality control of both drug substance and drug product. The performance of the method was validated according to the ICH guide lines for specificity, limit of detection, limit of quantification, linearity, accuracy, precision, ruggedness and robustness.
An improved extraction protocol for therapeutic dabigatran monitoring using HPLC-MS/MS
2019, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Citation Excerpt :
However, the INR does not provide an accurate assessment of anticoagulant activity of new oral anticoagulants such as dabigatran, so other tests are required to assess or to adjust dosing in emergency situations. Gas-liquid chromatography [3], chromogenic tests [4–6], high-performance liquid chromatography combined with mass-selective spectrometry detection [7–9], have been used for the quantification of dabigatran in plasma. The HPLC-MS/MS method provides a highly selective and direct determination of the dabigatran in human plasma over a wide analytical concentration range.
A new sample extraction protocol was developed for pharmacokinetic studies of dabigatran with high-performance liquid chromatography separation - electrospray ionization time-of-flight mass spectrometry analysis. After protein precipitation with acetonitrile, free dabigatran and its metabolites are separated into water phase by water-dichloromethane liquid-liquid extraction to purify the sample from proteins and endogenous lipophilic compounds. Chromatographic separation was achieved on an Agilent Zorbax SB-CN column (150 × 4.6 mm, 5 µm)) using 0.1% aqueous solution of formic acid and acetonitrile (80:20) as the mobile phase. Agilent Zorbax SB-CN column was selected to improve sample resolution and to avoided early elution of dabigatran previously seen when using a C18 column. The extended calibration curve was constructed from 5 to 1000 ng/L while precision and accuracy were assessed at four levels across the linear dynamic ranges. Within-run precision was <5.6% and the between-run precision was <3.9%. The method accuracy ranged from 89.8% to 104.4%. The developed method was successfully applied to 30 patient samples to evaluate antithrombotic efficacy and anticoagulant activity of dabigatran following knee endoprosthesis surgery.
Measurement of apixaban, dabigatran, edoxaban and rivaroxaban in human plasma using automated online solid-phase extraction combined with ultra-performance liquid chromatography-tandem mass spectrometry and its comparison with coagulation assays
2018, Clinica Chimica Acta
Citation Excerpt :
Accuracy, intra- and inter-assay precision of our online SPE-UPLC-MS/MS assay for all four DOACs were high, as presented in Table 3. These assay parameters of our method are generally comparable with those of previously described LC-MS/MS assays of DOACs, albeit with most of these assays being limited to measurement of only one, two, or three DOACs [11,13–21,39,40], and only three of them measuring all four DOACs [14,20,21], however, none of which used automated online solid-phase extraction for sample preparation to obtain high-purity samples which prevent mass spectrometer contaminations. The recovery of our assay was good for dabigatran (>90%), edoxaban (>85%), rivaroxaban (>90%), and moderate for apixaban (>60%) (Table 3).
Measurement of direct oral anticoagulants (DOACs) concentration in patient blood is essential in special clinical circumstances.
We developed a fast, selective and sensitive method for simultaneous measurement of DOACs in human plasma consisting of an automated online solid-phase extraction method coupled with ultra-performance liquid chromatography electrospray ionization-tandem mass spectrometry (online SPE-UPLC-MS/MS).
The calibration curves of all DOACs were linear over the working range (apixaban: 0.25–760 μg/L, r > 0.99; dabigatran: 0.5–900 μg/L, r > 0.99; edoxaban: 0.6–800 μg/L, r > 0.99; rivaroxaban: 0.5–900 μg/L, r > 0.99). Limits of detection in the plasma matrix were < 0.2 μg/L, whereas the lower limits of quantification were < 0.6 μg/L for all DOACs. The intraassay and interassay CV for all DOACs were < 6%. Mean recoveries were between 61.4% and 91.6%. Method comparison between our online SPE-UPLC-MS/MS assay and commercially available functional based coagulation assays using patient samples showed a high degree of correlation for all investigated DOACs.
We developed and validated the first online SPE-UPLC-MS/MS method for fast, sensitive, specific, and reliable measurement of the new generation of DOACs and compared this method with commercial available coagulation assays.
Microdosing Cocktail Assay Development for Drug–Drug Interaction Studies
2018, Journal of Pharmaceutical Sciences
Citation Excerpt :
The interconversion between the 2 forms depends on pH, enzyme activity, sample processing temperature, and time.29 Methods for the determination of dabigatran in human plasma have also been reported,30-34 but these methods lack the sensitivity required to support microdosing studies. Several methods exist for midazolam,35-37 one assay was sensitive and was utilized for a microdose study; however, sample preparation was expensive and complex.36
Methodology for analysis of a microdosing drug cocktail designed to evaluate the contribution of drug transporters and drug metabolizing enzymes to disposition was developed using liquid chromatography–mass spectrometry–based detection. Fast and sensitive methods were developed and qualified for the quantification of statins (pitavastatin, pitavastain lactone, rosuvastatin, atorvastatin, 2-hydroxy, and 4-hydroxy atorvastatin), midazolam, and dabigatran in human plasma. Chromatographic separation was accomplished using reversed-phase liquid chromatography or hydrophilic interaction liquid chromatography with gradient elution and detection by tandem mass spectrometry in the positive ionization mode using electrospray ionization. The lower limit of quantitation (LLOQ) for the statins assay was 1 pg/mL for the 6 analytes with a linear range from 1 to 1000 pg/mL processing 250 μL plasma sample. The midazolam assay LLOQ was 0.5 pg/mL with a linear range of 0.5 to 1000 pg/mL. For the dabigatran assay, the LLOQ was 10 pg/mL with a linear range of 10 to 5000 pg/mL processing 100 μL plasma sample. The intraday and interday precision and accuracy of the assays were within acceptable ranges, and the assays were successfully applied to support a study where a microdose cocktail was dosed to healthy human subjects for simultaneous assessment of clinical drug-drug interactions mediated by major drug transporters and CYP3A.
Evaluation of dabigatran, rivaroxaban and apixaban target-specific assays in a multicenter French study
2017, Thrombosis Research
Dabigatran etexilate, rivaroxaban and apixaban (DOACs) are widely used and measurement of their concentration is desirable in certain clinical situations. Target-specific assays are available but limited information exists on their performance especially in their ability to accurately measure low and high concentrations.
To define, in a multicenter study, the precision and accuracy of DOAC measurements in daily practice.
15 plasma samples (kindly provided by Hyphen-Biomed) spiked with 5 blinded concentrations of dabigatran, rivaroxaban or apixaban (targeted 0–40–100–250–500 ng/mL, actual concentrations measured by HPLC-MS/MS), were sent to 30 haemostasis laboratories. DOAC concentration, PT and aPTT were measured once in each sample using local reagents. Interlaboratory precision was determined by its coefficient of variation (CV) and accuracy by its bias.
464 DOAC measurements were performed in the 30 laboratories using 4 dabigatran and 5 rivaroxaban/apixaban calibrated assays on 3 analysers. Inter-laboratory CVs were below 18% for concentrations ≥ 100 ng/mL, and higher for concentrations ~ 40 ng/mL; biases were below 8% for all drugs and concentrations. In DOAC-free samples, concentrations were all below the lower limit of quantification except for one value (dabigatran: 35 ng/mL). Depending on the concentrations, significant differences were found between reagents in rivaroxaban and apixaban concentration values. PT and aPTT ratios displayed a low sensitivity to apixaban.
Our results suggest that calibrated DOAC assays allow the reliable measurement of a wide range of drug concentrations, even though improvement of their performances is necessary, especially for measuring low concentrations.

View all citing articles on Scopus

View full text

Short communicationUPLC MS/MS assay for routine quantification of dabigatran – A direct thrombin inhibitor – In human plasma

Abstract

Introduction

Section snippets

Chemicals and reagents

Stock solutions, calibration standards and quality control samples

Liquid chromatography

Mass spectrometry

Conclusion

Journal of Thrombosis and Haemostasis

Journal of Thrombosis and Haemostasis

Journal of Thrombosis and Haemostasis

Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences

Randomized evaluation of long-term anticoagulation therapy, newly identified events in the RE-LY trial

The New England Journal of Medicine

The pharmacokinetics, pharmacodynamics and tolerability of dabigatran etexilate, a new oral direct thrombin inhibitor, in healthy male subjects

British Journal of Clinical Pharmacology

Short communication
UPLC MS/MS assay for routine quantification of dabigatran – A direct thrombin inhibitor – In human plasma