Analysis of new potential anticonvulsant compounds in mice brain tissue by SPE/HPLC/DAD

This paper describes a novel reversed-phase high performance liquid chromatography (RP-HPLC) with photo-diode array detection (DAD) method for the determination of three new derivatives of 4-alkyl-5-(3-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione with different antiepileptic activity in the brains of mice treated with the doses of 300 mg kg−1 of body weight. Samples were prepared by solid-phase extraction (SPE) method using BAKERBOND™ spe Octadecyl (C18) and analyzed by the use of an isocratic elution mode over an Zorbax Extend-C18 column (150 mm × 4.6 mm I.D., 5-μm, Agilent Technologies). The mobile phase consisted of 80% methanol (for compound TP-315) and 85% acetonitrile (for compound TP-321) for 80% 2-propanol (for TP-323) at a flow rate of 1.0 mL min−1 and 0.5 mL min−1 in the last case. Gradient elution mode was also proposed for all examined analytes in mixture with common antiepileptic drugs: carbamazepine, phenobarbital and phenytoin in view of possible synergistic activity. Photodiode-array investigations of the peaks after degradation studies indicate the stability of the compounds under conditions proposed for sample preparation procedure. Linear coefficients of correlation (r2) were >0.995 for all analytes. The proposed strategy gives extraction yields higher than 95% with the intra- and inter-day relative standard deviation lower than 3% and 5%, respectively. This method was applied to the analysis of brain tissue of mice treated with investigated compounds. Obtained results enable to explain the differences in their pharmacological activity.

► Separation method in sample together with standard antiepileptic drugs taking into account observed their synergic action. ► Quantitative determination of new ant-epileptic compounds in mouse brain tissue by HPLC–DAD after sample clean-up step by the use of SPE procedure. ► Explanation of the different anti-epileptic activity.