In the present study the bioavailability and pharmacokinetics properties of pantoprazole (proton pump inhibitor)/amitriptyline (tricyclic antidepressant) in novel formulated effervescent granules was estimated in rabbit plasma using a validated, selective and rapid LC-MS/MS method. Separation and detection of pantoprazole, amitriptyline and internal standards namely omeprazole and dothiepin, respectively, were achieved at ambient column temperature on C 18 . Acetonitrile: 4mM ammonium acetate solution (comprising 0.05 % formic acid) (40:60, v/v) was used as mobile phase and the flow rate of 0.6 mLmin -1 was applied. Liquid-liquid extraction technique with diethyl ether: dichloromethane (70:30, v/v) was used to extract the cited drugs from rabbit plasma. Multiple reactions monitoring (MRM) in the positive ionization mode was carried out for quantification. The method was validated over linear concentration range of 0.01-4μgmL −1 and 0.001–0.1 μgmL −1 for Pan and Ami respectively, with regression coefficient (r 2 ) ≥ 0.9961. The intra- and inter-run precisions (%CV) were ≤4.03. The extraction recoveries were in the range of 95.92%–100.24 %. Pan and Ami were stable during three freeze-thaw cycle and post-preparative stability. The work also aimed to formulate immediate release novel effervescent granules by melt granulation technique. Nine formulae were assessed by validated dissolution test for their micrometric properties and dissolution profile. Experimental design was applied to select formula that fulfilled the desired criteria of optimum release of pantoprazole and amitriptyline with optimum micrometric properties for the study. A single period randomized open-label parallel design was applied on Chancellor's rabbit. The selected formula showed superior pharmacokinetic parameters for pantoprazole and amitriptyline than that of marketed products.

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Background: Design of experiment (DOE) is considered the most powerful tool to identify factors that affect variation and improve the response by tuning these factors. In the present work, DOE was applied to establish an innovative, sensitive and precise HPLC method for the simultaneous determination of Escitalopram oxalate, Paroxetine hydrochloride hemihydrate and Clonazepam in presence of their related compounds in drug substance and drug products. Methods: Buffer molarity, % organic modifier (acetonitrile content) at the beginning/end of gradient, flow rate at the beginning/end of gradient, pH of mobile phase and column temperature were screened using Plackett-Burman design (PBD) model. The main effect plot showed that % organic modifier at the beginning/end of gradient and flow rate at the beginning of gradient were statistically significant variables influencing peaks resolution (p<0.05). Box-Behnken design (BBD) was then used as optimization model in order to achieve the highest possible resolution with the least possible experimental trials through studying interaction and quadratic effects of these three factors. Finally, the optimum condition for predicated peak resolution could be achieved by desirability function. Results: After optimization, chromatographic separation was attained on Agilent Zorbax SB C18 (4.6×250mm, 5µm) column using gradient elution of mobile phase: (A) potassium dihydrogen phosphate (pH 2.7; 0.025M) and (B) acetonitrile at ambient column temperature with the last eluted compound at less than 17 min. The flow rate was maintained at 1/2.3 mLmin-1 with UV detection at 245/210 nm using time programming. Conclusion: The optimized chromatographic method was used for stability indicating assay of the cited drugs in presence of their related compounds according to ICH Q2R1 guidelines.