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Objectives: Analytical methods are necessary for the field of pharmaceuticals to discover the drug, determination of drugs and metabolites in biological matrices. The present study aimed to develop liquid chromatography-tandem mass spectrometry (LC-MS) and validation for quantification of Pimobendan in the formulation. Methods: The mobile phase composition was methanol, acetonitrile and 0.1 M phosphate buffers in the ratio of 45:15:40 (v/v) mixtures with isocratic elution. Prontosil ODS C18 column (250 × 4.6 mm, 5μ) was selected for separation with 264nm of LC UV detector wavelength. Ionization mass spectrophotometer was analyzed at nitrogen gas flow at 300 Psi and 300°C of source temperature. Mass spectrometry analysis of Pimobendan drug fragments was performed by introducing column eluting into equipment for Q1 and MS scan. Results: The drug was eluted at 3.56 min retention and the calibration curve was achieved at 0.5-250 μg/ml standard concentration. Different parameters of validation (linearity, specificity, precision, accuracy, selectivity and robustness) were performed and results confirm that all the parameters are in the acceptable limit. In the electrospray ionization spectra of Pimobendan [M+H], positive ions were observed at an m/z value of 335.42. The product-ions of 335.42 were observed at m/z value of 319.18, 287.19, 277.09, 261.08, 250.16, 223.30, 111.47 and 86.05. Conclusion: The proposed method is successfully achieved the acceptance criteria of all validation parameters and stability studies.

Stability Indicating LC-MS Method Development and Validation for the Study of the Forced Degradation Behavior of Pimobendan Drug