Development and validation of HPLC-MS/MS method of determination of a new valproic acid derivative and 1,3,4-thiadiazole in rat brain

Introduction. To assess the pharmacokinetic indices of new drugs, it is necessary to develop analytical methods for determining their concentration in biological objects. Purpose of the study. Development and validation of HPLC-mass spectrometric methods for the determination of a new valproic acid derivative and 1,3,4-thiadiazole in rat brain. Methods. The object of the study was a new anticonvulsant N- (5-ethyl-1,3,4-thiadiazol-2-yl)-2-propylpentanamide (valprazolamide). Determination of valprazolamide was carried out by the HPLC-mass spectrometric method. Selectivity, sample transfer, linearity, accuracy, precision, matrix effect, the degree of extraction of valprazolamide were evaluated. Results. A method of HPLC-mass spectrometric determination of valprazolamide in rat brain homogenates was developed (Zorbax Eclipsi plus C18 analytical column - 4 μm 2,0*150 mm, temperature - 30°C; the mobile phase is acetonitrile and deionized water with the following gradient profile: 0-1 minute 10% acetonitrile; 1-5 minutes a linear increase in the concentration of acetonitrile to 90%; 5-6 minutes isocratic plot with a concentration of acetonitrile 90%; 6-10 minutes column conditioning with 10% acetonitrile; the flow rate of the mobile phase is of 0.6 ml/min; the volume of the injected sample accounts for 10 μl; total elution time - 10 minutes, mass detection with negative polarization). The MRM transition of valprazolamide amounted to m/z 254,0 → m/z 167,1. Conclusion. The analytical limits of the method are in the range from 1 to 1000 ng/ml. The developed method is selective, accurate, precise and linear, it meets the requirements for the validation of bioanalytical methods in all respects.

HPLC-MS/MS, valproic acid, 1,3,4-thiadiazole, antiepileptic drugs, mass spectrometry