A novel green QbD-optimized RP-HPLC bioanalytical method for the simultaneous determination of doxorubicin and rifampicin in human plasma

Doxorubicin hydrochloride (DOX) remains a cornerstone in the treatment of numerous malignancies. Recent research has focused on improving the efficacy of chemotherapeutic regimens through synergistic combinations. Rifampicin (RFP), in addition to its well-known antimicrobial properties, exhibits chemosensitizing potential, making it a promising adjuvant to DOX therapy. In this study, a robust and eco-friendly HPLC method was developed and optimized for the simultaneous determination of DOX and RFP. Chromatographic separation was achieved on a C18 column (150 × 4.6 mm, 5 μm particle size) at 25 °C, using an isocratic mobile phase composed of acetonitrile and phosphate buffer (0.02 M, pH 5.43) in a 34.85:65.15 (v/v) ratio, with a flow rate of 0.8 mL/min and UV detection at 254 nm. The method was systematically optimized using a Quality-by-Design (QbD) approach with a full factorial design to assess the influence of key variables on chromatographic performance. The optimized chromatographic separation was achieved within 7 min, with retention times (Rts) of 3.26 min of DOX and 6.62 min for RFP. Excellent linearity was obtained over the ranges of 1.0-40.0 µg/mL for DOX and 1.0-30.0 µg/mL for RFP, with high determination coefficients (r² ≥ 0.999) and lower detection limits of 0.44 µg/mL and 0.39 µg/mL and quantitation limits of 1.35 µg/mL and 1.18 µg/mL, respectively. Greenness evaluation using the Analytical Greenness (AGREE) and Analytical Green Star Area (AGSA) metrics confirmed the method's high environmental sustainability. When applied to spiked human plasma samples, recovery values ranged from 92.38% to 107.20% with low %RSD, demonstrating strong accuracy, precision, and bioanalytical applicability. Overall, the developed method represents the first simple, sensitive, and environmentally benign analytical approach for the simultaneous quantification of doxorubicin HCl and rifampicin in biological matrices, making it highly suitable for routine therapeutic drug monitoring (TDM) in clinical and pharmacokinetic studies.