Development of a validated, sensitive hplc method for determination of 2- amino-7-hydroxy-4-(4-methoxy phenyl)-4-hydrogen-chromene-3-carbonitrile in plasma as a new compound with antiplatelet aggregation activity.

Platelet aggregation inhibitors are a group of cardiovascular therapeutic agents which are widely used in primary and secondary prevention of thrombotic cerebrovascular or cardiovascular diseases [1].

Anti-Platelet drugs which are currently in use suffer from various side effects and inefficiency in some patients [2].

Therefore, new anti-platelet agents are on demand. 2-amino-7-hydroxy-4-(4-methoxy phenyl)-4-hydrogen- chromene-3-carbonitrile, which is called DEMOK-10 from now on, has been recently introduced as a potent platelet aggregation inhibitor with IC50 value of 7.6 µM [3].

To provide valuable information for subsequent preclinical evaluation, the present study was aimed to study the pharmacokinetic (PK) of this drug candidate following IV administration into rats. The separation was achieved on a LiChrospher® RP-8 (5 µm, 250 × 4 mm) column at room temperature, using a mixture of water: acetonitrile: methanol (60:35:5 v/v) as the mobile phase. The analyte was detected with a UV detector set at 237 nm and the flow rate was 1.0 ml/min.

Liquid-liquid extraction with 1 ml of chloroform and isopropyl alcohol (80:20 v/v) was chosen as the most appropriate method for preparation of plasma samples. After validation of the HPLC method, DEMOK-10 was administered via IV injection to rats (n=6) and blood samples were collected at the predetermined time intervals. Plasma samples were separated and stored at -20 ℃ until analysis.

The Retention times of DEMOK-10 and internal standard (IS, carbamazepine) were 11.6 and 8.4 min, respectively. Limit of detection and limit of quantification of DEMOK-10 were 35 ng/ml and 100 ng/ml, respectively. The method was linear over the concentration range of 0.1 to 25 μg/ml and the regression coefficient of the calibration curve was greater than 0.999.

The inter- and intra-day relative standard deviations (RSD) and relative errors (RE) obtained by analysis of QC samples were less than 9%. Analytical recoveries of analyte and IS were greater than 80%. According to the results the mean values for elimination half-life (T 1/2), mean residence time (MRT), total clearance (Cl) and volume of distribution at steady state (V ss) were 1.5 ± 1.1 h, 1.84 ± 1.24 h, 23.28 ± 6.29 ml/h and 37.53 ± 17.25 ml, respectively.

Keywords: HPLC, plasma, DEMOK-10, antiplatelet, validation, pharmacokinetic.


  1. Chiarito, M., Stefanini, G.G.: Antiplatelet therapy for secondary prevention of cardiovascular disease: challenging the certainties. Lancet (London, England). 397, 2443–2444 (2021).
  2. Kim K, Park KI. A Review of Antiplatelet Activity of Traditional Medicinal Herbs on Integrative Medicine Studies. Evid Based Complement Alternat Med. 2019;2019:7125162. Published 2019 Jan 3. doi:10.1155/2019/7125162
  3. Kobarfard, F 2020, 'Synthesis of Cyanodihydropyrone Derivatives from Resorcinol and Evaluation of their Antiplatelet Aggregation Activity', Doctoral thesis, Sahid Beheshti University of Medical Sciences, Tehran.
Year: 2021
City: Shymkent
Category: Medicine