Simultaneous chromatographic quantification of upadacitinib, nilotinib, and tryptophan decoding myeloid leukemia and liver cancer integrated with ELISA and Flow cytometry.

For the first time, an integrated dual analytical-therapeutic platform is presented for concurrent investigation of hematological (chronic myeloid leukemia, CML) and solid (hepatocellular carcinoma, HCC) malignancies. A six-dimensional analytical framework combining RP-HPLC-DAD, UV-Vis spectrophotometry, ELISA, MTT assay, single-cell fluorescence imaging, and flow cytometry enabled comprehensive biochemical profiling. A novel QbD-driven RP-HPLC-DAD method was developed for the simultaneous quantification of Nilotinib (NIL, tyrosine kinase inhibitor, TKI), Upadacitinib (UPA, Janus kinase inhibitor, JAKI) and tryptophan (TRP) as a critical metabolic biomarker, successfully applied in serum matrices. Because CML and immune-mediated inflammatory diseases (IMIDs) share aberrant JAK-STAT signaling, and both NIL and UPA are metabolized via CYP3A4, continuous quantification is essential for safe co-therapy. Chromatographic separation was achieved on a carefully selected fluorinated Pursuit PFP column (150 × 4.6 mm, 3 μm), a key factor enabling efficient separation of the fluorinated drugs, using methanol-water (75,25, v/v) at 1.0 m/min and 230 nm detection, within a QbD 23 full factorial framework, exhibiting excellent linearity (1.0-50.0 μg/mL). Fluorescence-based determination using flow cytometry with ELISA revealed a pioneer significant pro-apoptotic effect of Upadacitinib in HCC cells via modulation of the Bax/Bcl-2 axis with combination therapy showing superior anticancer effect compared to standard chemotherapy of Doxorubicin (DOX). Sustainability evaluation using AGREE (greenness) and BAGI (blueness) with RGB12 algorithm (whiteness) and spider-diagram visualization, in addition to the recently launched Multi-Color Assessment (MA) tool to confirm the method's multidimensional eco-efficiency in strong alignment with the United Nations Sustainable Development Goals (SDGs). This study further harnesses the Need-Quality-Sustainability (NQS) index and Koel's pyramid principles for holistic evaluation and benchmarking against reported approaches toward sustainable analytical oncology with personalized medicine.