The current work integrates design of experiment (DoE) techniques for developing, validating, and quantifying Cefotaxime sodium through RP-HPLC. Four independent factors were considered: flow rate, mobile phase ratio, oven temperature, and injection volume. A full factorial design was applied to optimize the parameters and determine the significant variables. Using the DoE approach, a more environmentally friendly separation and quantification were achieved with the help of the Venusil XBP. C8 column (5 um × 4.6 × 250 mm), a flow rate of 0.8 ml/minute, and a mobile phase ratio consisting of a binary combination of A:B (15:65). The organic phase (A) consisted of acetonitrile, while the aqueous phase (B) consisted of ammonium acetate, and the pH was adjusted to 6.1 with the aid of glacial acetic acid. The injection volume was set at 20 μl, and the elution was performed at a wavelength of 235 nm using a UV detector. The method was validated according to International Conference on Harmonisation guidelines, and it successfully determined the amount of Cefotaxime in Cefotaxime-loaded nanosponges. Tools such as Analytical Greenness metric (AGREE), “National Environmental Method Index” (NEMI), and “Green Analytical Procedure Index” (GAPI) were used to assess the greenness of the proposed method, along with a comparison of the proposed method with other reported HPLC methods. The proposed method proved to be eco-friendly, with better qualitative and quantitative results, as well as greener NEMI and GAPI quadrants. It also achieved a higher AGREE score (0.68) than the reported method (0.44).
Key words: Cefotaxime sodium, DoE, QbD, AGREE, GAPI, NEMI, Green analytical chemistry (GAC), ICH guidelines
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