Analytical Quantification of Tofacitinib Citrate in Bulk and Nanostructured Lipidic Formulations by HPLC Using a Quality by Design Approach Integrated with Monte Carlo Simulations

Abstract

A novel, robust, and stability-indicating high-performance liquid chromatography method was developed and validated for the quantification of tofacitinib citrate (TFC) using an integrated quality-by-design approach supported by appropriate chemometric tools. The analytical target profile was defined, and critical analytical attributes (CAAs), including peak area and tailing factor, were identified. Chromatographic separation was achieved on a C18 column with photodiode array detection at 287 nm using an isocratic elution of methanol, acetonitrile, and phosphate buffer (pH 5.0) in a 20 : 20 : 60 (v/v/v) ratio. An Ishikawa fishbone diagram was employed to identify potential sources of variability affecting CAAs. Taguchi design facilitated the screening of critical method parameters, followed by method optimization using a face-centered cubic design. The optimized method complied with the International Council for Harmonization Q2 (R1) guideline and Monte Carlo validation criteria, demonstrating excellent linearity (correlation coefficient = 0.9999), precision (coefficient of variation ≤ 0.04%), accuracy (100.1–101.5% recovery), and sensitivity (limit of detection = 150 ng/mL; limit of quantitation = 460 ng/mL). Robustness was confirmed within the method operable design region. Chemometric evaluation using principal component analysis and variance inflation factor effectively addressed multicollinearity. Forced degradation studies confirmed the stability-indicating capability of the method for TFC in topical nanolipidic formulations, establishing its suitability for routine pharmaceutical analysis.