Abstract
The purpose of this work was to demonstrate the use of the AQbD with the DOE approach to the methodical step-by-step development of a UHPLC method for the quantitative determination of the impurity profile of new CPL409116 substance (JAK/ROCK inhibitor) on the preclinical and clinical step of drug discovery studies. The critical method parameters (CMPs) have been tested extensively: the kind of stationary phase (8 different columns), pH of the aqueous mobile phase (2.6, 3.2, 4.0, 6.8), and start (20-25%) and stop (85-90%) percentage of organic mobile phase (ACN). The critical method attributes (CMAs) are the resolution between the peaks (≥2.0) and peak symmetry of analytes (≥0.8 and ≤1.8). In the screening step, the effects of different levels of CMPs on the CMAs were evaluated based on a full fractional design 2(2). The robustness tests were established from the knowledge space of the screening step and performed by application fractional factorial design 2((4-1)). Method operable design region (MODR) was generated. The probability of meeting the specifications for the CMAs was calculated by Monte-Carlo simulations. In relation to literature such a complete AQbD approach including screening, optimization, and validation steps for the development of a new method for the quantitative determination of the full profile of nine impurities of an innovative pharmaceutical substance with the structure-based pre-development pointed out the novelty of our work. The final working conditions were as follows: column Zorbax Eclipse Plus C18, aqueous mobile phase 10 mM ± 1 mM aqueous solution of HCOOH, pH 2.6, 20% ± 1% of ACN at the start and 85% ± 1% of ACN at the end of the gradient, and column temperature 30 °C ± 2 °C. The method was validated in compliance with ICH guideline Q2(R1). The optimized method is specified, linear, precise, and robust. LOQ is on the reporting threshold level of 0.05% and LOD at 0.02% for all impurities.