Development and Validation of the HPLC Method for Quantification of the Innovative Drug DD217, Factor Xa Inhibitor, in Rat Plasma for a Pharmacokinetic Study

Introduction. Nowadays, discovery and development of innovative drugs represent a relevant goal for the pharmaceutical market. One of such drugs is N-(5-chloropyridine-2-yl)-2-({4-[ethanimidoyl(methyl)benzoyl}amino)-5-methylbenzamide hydrochloride (hereinafter DD217), an innovative drug that belongs to the class of anticoagulants, namely factor Xa inhibitors.

Aim. The aim of this study was to develop and validate a method for DD217 determination in rat plasma by means of high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) to carry out pharmacokinetic studies.

Materials and methods. Outbred Wistar rats were used in the study. Rats received a single dose of DD217 substance solution intragastrically at a dose of 5, 15, 30 mg/kg. The validated HPLC-MS/MS method was employed for DD217 determination in rat plasma. Nebivolol was chosen as the internal standard of the method. Chromatographic separation involved the use of Phenomenex Luna С8 column (3 µm, 50 × 4.6 mm) and gradient elution with water-acetonitrile solution containing 0.1 % formic acid. The total run time of each sample was equal to 2.0 min. DD217 and nebivolol were detected in positive electrospray ionization mode, the ion transitions monitored were m/z 436,1 → 119,9 and 406,0 → 151,0, respectively. Pharmacokinetic parameters and descriptive statistics were calculated through model-independent method in IBM SPSS Statistics v27 and Julia v1.6.0 (MixedModels v3.8.0, ClinicalTrialUtilities v0.5.1) software.

Results and discussion. Method was validated by linearity, lower limit of quantification, selectivity, accuracy and precision, dilution integrity, matrix effect, recovery, carry-over, and stability. The lower limit of quantification of DD217 in rat plasma was 2.0 ng/mL. The method was successfully applied for establishing the pharmacokinetic parameters of DD217 substance in rat plasma after intragastric administration at a dose of 5, 15, 30 mg/kg.

Conclusion. HPLC-MS/MS method for DD217 determination in plasma was developed, validated and applied in order to evaluate pharmacokinetic parameters of DD217 substance in rat plasma.

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