An improved extraction protocol for therapeutic dabigatran monitoring using HPLC-MS/MS

J Chromatogr B Analyt Technol Biomed Life Sci. 2019 Nov 1:1130-1131:121808. doi: 10.1016/j.jchromb.2019.121808. Epub 2019 Oct 4.

Abstract

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.

Keywords: Anticoagulant; Dabigatran; Extraction protocol; HPLC-MS/MS; Pharmacokinetics.

MeSH terms

  • Chromatography, High Pressure Liquid / methods*
  • Dabigatran / blood*
  • Dabigatran / isolation & purification*
  • Dabigatran / pharmacokinetics
  • Drug Monitoring
  • Humans
  • Limit of Detection
  • Linear Models
  • Reproducibility of Results
  • Tandem Mass Spectrometry / methods*

Substances

  • Dabigatran