A novel ultra high performance liquid chromatography method development strategy was ameliorated by applying quality by design approach. The developed systematic approach was divided into five steps (i) Analytical Target Profile, (ii) Critical Quality Attributes, (iii) Risk Assessments of Critical parameters using design of experiments (screening and optimization phases), (iv) Generation of design space, and (v) Process Capability Analysis (Cp) for robustness study using Monte Carlo simulation. The complete quality-by-design-based method development was made automated and expedited by employing sub-2 μm particles column with an ultra high performance liquid chromatography system. Successful chromatographic separation of the Coenzyme Q10 from its biotechnological process related impurities was achieved on a Waters Acquity phenyl hexyl (100 mm × 2.1 mm, 1.7 μm) column with gradient elution of 10 mM ammonium acetate buffer (pH 4.0) and a mixture of acetonitrile/2-propanol (1:1) as the mobile phase. Through this study, fast and organized method development workflow was developed and robustness of the method was also demonstrated. The method was validated for specificity, linearity, accuracy, precision, and robustness in compliance to the International Conference on Harmonization, Q2 (R1) guidelines. The impurities were identified by atmospheric pressure chemical ionization-mass spectrometry technique. Further, the in silico toxicity of impurities was analyzed using TOPKAT and DEREK software.

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topkat and derek software biotechnological process related qualitybydesignbased method process capability analysis chromatographic separation design space critical parameters harmonization q2 r1 target specificity linearity accuracy precision risk assessments ultra high performance liquid chromatography method development strategy monte carlo systematic approach silico toxicity atmospheric pressure chemical ionizationmass spectrometry of experiments coenzyme q10 robustness m particles column

Shireesha Dharavath,Murali V.N. Kumar Talluri, Naeem Shaikh, Nageswara Rao Ramisetti, Pradipbhai D. Kalariya, Prabha Garg, Srinivas Ragampeta,.Automated statistical experimental design approach for rapid separation of coenzyme Q10 and identification of its biotechnological process related impurities using UHPLC and UHPLC–APCI-MS. 39 (18),3535-3528.

Shireesha Dharavath, et al."Automated statistical experimental design approach for rapid separation of coenzyme Q10 and identification of its biotechnological process related impurities using UHPLC and UHPLC–APCI-MS" 39

Shireesha Dharavath,Murali V.N. Kumar Talluri, Naeem Shaikh, Nageswara Rao Ramisetti, Pradipbhai D. Kalariya, Prabha Garg, Srinivas Ragampeta,"Automated statistical experimental design approach for rapid separation of coenzyme Q10 and identification of its biotechnological process related impurities using UHPLC and UHPLC–APCI-MS"