Skip to Content
Merck
  • Development and validation of LC-MSMS assay for the determination of the prodrug dabigatran etexilate and its active metabolites in human plasma.

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

Journal of chromatography. B, Analytical technologies in the biomedical and life sciences (2015-03-24)
Eman G Nouman, Medhat A Al-Ghobashy, Hayam M Lotfy
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
β-Glucuronidase from Helix pomatia, Type H-2, aqueous solution, ≥85,000 units/mL
Sigma-Aldrich
Sertraline hydrochloride, ≥98% (HPLC)
Sertraline for peak identification, European Pharmacopoeia (EP) Reference Standard
USP
Sertraline hydrochloride, United States Pharmacopeia (USP) Reference Standard
Sertraline hydrochloride, European Pharmacopoeia (EP) Reference Standard
Sertraline for system suitability, European Pharmacopoeia (EP) Reference Standard
Supelco
Residual Solvent - Acetonitrile, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
Acetonitrile, anhydrous, 99.8%
Supelco
Acetonitrile, analytical standard
Sigma-Aldrich
Acetonitrile, electronic grade, 99.999% trace metals basis
Sigma-Aldrich
Acetonitrile, biotech. grade, ≥99.93%
Sigma-Aldrich
Acetonitrile, for HPLC, for UV, ≥99.9% (GC)
Sigma-Aldrich
Acetonitrile, suitable for HPLC, gradient grade, ≥99.9%
Sigma-Aldrich
Acetonitrile, HPLC Plus, ≥99.9%
Sigma-Aldrich
Ultrapure Acetonitrile
Supelco
Acetonitrile, Pharmaceutical Secondary Standard; Certified Reference Material
USP
Residual Solvent Class 2 - Acetonitrile, United States Pharmacopeia (USP) Reference Standard
Sigma-Aldrich
Acetonitrile, ReagentPlus®, 99%
Sigma-Aldrich
Acetonitrile, for preparative HPLC, ≥99.8% (GC)
Sigma-Aldrich
Acetonitrile, suitable for DNA synthesis, ≥99.9% (GC)
Sigma-Aldrich
Acetonitrile, ≥99.5% (GC)
Sigma-Aldrich
Acetonitrile, suitable for HPLC-GC, ≥99.8% (GC)
Sigma-Aldrich
Acetonitrile, suitable for HPLC, gradient grade, ≥99.9%
USP
Sertraline hydrochloride racemic mixture, United States Pharmacopeia (USP) Reference Standard
Sigma-Aldrich
Acetonitrile, ACS reagent, ≥99.5%