Development and validation of HPLC-MS/MS method of determination of a new derivative of valproleic acid and 1,3,4-thiadiazole in rabbit blood plasmafor pharmacokinetic study

Objective. To develop and validate the HPLC-mass spectrometric method for determining a new derivative of valproic acid and 1,3,4-thiadiazole in rabbit plasma. Material and methods. The object of the study was N-(5-ethyl-1,3,4-thiadiazol-2-yl) -2-propylpentanamide (valprazolamide) - a new antiepileptic drug from the group of thiadiazole derivatives of valproic acid. Valprazolamide was determined in rabbit plasma using an Agilent 1260 Infinity II high performance liquid chromatograph (Agilent Technologies, Germany). An AB Sciex QTrap 3200 MD triple quadrupole mass spectrometer (AB Sciex, Singapore) with an electrospray ion source (Turbo V with a TurboIonSpray probe) was used as a detector. Selectivity, carry over, linearity, accuracy, precision, matrix effectand recovery were evaluated for the developed method. Results. A method of HPLC-mass spectrometric determination of valprazolamide in rabbit plasma was developed (Phenomenex Synergi C18 analytical column 4 μm 2.0 x 50 mm, gradient elution: 0-1 minute 10% aqueous acetonitrile solution; 1-3 minutes linear increase in concentration acetonitrile in a mixture up to 90%; 3-4 minutes isocratic section with an acetonitrile concentration of 90%; 4-5 minutes conditioning the column with a 10% solution; flow rate of the mobile phase - 0.6 ml / min; volume of sample injected -20 μl; total time gradient elution - 5 min t; mass spectrometric detection). Mass detection conditions: positive polarization, voltage of the electrospray 5500.0 V, declustering potential 41.0; the curtain gas pressure is 20.0 psi, the spray gas is 40.0 psi, the input potential is 3.5 V. The MRM transitions for valprazolamide were m/z 256.1 → m/z 130.1 and m/z 81.0. Acetazolamide (m/z 223.1 → m/z 73.0) was used as an internal standard. Conclusion. The analytical range of the method for determining valprazolamide is 1-1000 ng/ml. The developed method is selective, accurate, precision and linear, it meets the requirements for the validation of bioanalytical methodsin all respects.

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