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An RP-HPLC method for simultaneous determination of 9 NSAIDs (paracetamol, salicylic acid, ibuprofen, naproxen, aceclofenac, diclofenac, ketorolac, etoricoxib, and aspirin) and their commonly prescribed combination drugs (thiocolchicoside, moxifloxacin, clopidogrel, chlorpheniramine maleate, dextromethorphan, and domperidone) was established. The separation was performed on Kromasil C18 (250 × 4.6 mm, 5 m) at 35 ∘ C using 15 mM phosphate buffer pH 3.25 and acetonitrile with gradient elution at a flow rate of 1.1 mL/min. The detection was performed by a diode array detector (DAD) at 230 nm with total run time of 30 min. Calibration curves were linear with correlation coefficients of determination (r 2 ) > 0.999. Limit of detection (LOD) and Limit of quantification (LOQ) ranged from 0.04 to 0.97 g/mL and from 0.64 to 3.24 g/mL, respectively. As an application tool of quality by design, full factorial experimental design was used for the testing of robustness of the method. The prediction profiler correlating various parameters and responses was established from the results of design of experiments (DOE).

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Quality-by-design-based methods hold greater level of confidence for variations and greater success in method transfer. A quality-by-design-based ultra high performance liquid chromatography method was developed for the simultaneous assay of sumatriptan and naproxen along with their related substances. The first screening was performed by fractional factorial design comprising 44 experiments for reversed-phase stationary phases, pH, and organic modifiers. The results of screening design experiments suggested phenyl hexyl column and acetonitrile were the best combination. The method was further optimized for flow rate, temperature, and gradient time by experimental design of 20 experiments and the knowledge space was generated for effect of variable on response (number of peaks ≥ 1.50 - resolution). Proficient design space was generated from knowledge space by applying Monte Carlo simulation to successfully integrate quantitative robustness metrics during optimization stage itself. The final method provided the robust performance which was verified and validated. Final conditions comprised Waters® Acquity phenyl hexyl column with gradient elution using ammonium acetate (pH 4.12, 0.02 M) buffer and acetonitrile at 0.355 mL/min flow rate and 30°C. The developed method separates all 13 analytes within a 15 min run time with fewer experiments compared to the traditional quality-by-testing approach.

A validated stability-indicating HPLC method was established, and comprehensive stress testing of ivabradine, a cardiotonic drug, was carried out as per ICH guidelines. Ivabradine was subjected to acidic, basic and neutral hydrolysis, oxidation, photolysis and thermal stress conditions, and the resulting degradation products were investigated by LC-PDA and LC-HR-MS/MS. The drug was found to degrade in acid and base hydrolysis. An efficient and selective stability assay method was developed on Phenomenex Luna C18 (250 × 4.6 mm, 5.0 µm) column using ammonium formate (10 mM, pH 3.0) and acetonitrile as mobile phase at 30 °C in gradient elution mode. The flow rate was 0.7 ml/min and detection wavelength was 286 nm. A total of five degradation products (I-1 to I-5) were identified and characterized by LC-HR-MS/MS in combination with accurate mass measurements. The drug exhibited different degradation behaviour in HCl and H2SO4 hydrolysis conditions. It is a unique example where two of the five degradation products in HCl hydrolysis were absent in H2SO4 acid hydrolysis. The present study provides guidance to revise the stress test for the determination of inherent stability of drugs containing lactam moiety under hydrolytic conditions. Most probable mechanisms for the formation of degradation products have been proposed on the basis of a comparison of the fragmentation pattern of the drug and its degradation products. In silico toxicity revealed that the degradation products (I-2 to I-5) were found to be severe irritants in case of ocular irritancy. The analytical assay method was validated with respect to specificity, linearity, range, precision, accuracy and robustness.