DoE-based HPLC-PDA method development and validation for diverse nature multi-components in plants: AQbD approach for...

Chromatographic methods are widely used in plant drug analysis to estimate single or multiple phytochemicals alone are unable to ensure the authenticity or adulteration of different plant species of the same genus. Additionally, for the compliance to ISO17025:2027, to ensure the accuracy of the results degree of uncertainty of the validated analytical method is also lacking. Design of Experiment and Derringer's Desirability Function were used to optimize the multiple dependent variables and their responses. Thus, the present work demonstrates the analytical quality by design (AQbD) concept in developing an HPLC-PDA method for the simultaneous quantification of eight specialised phytochemicals of Sida complex (PSC) i.e., alkaloids (vasicine, vasicinone, cryptolepine, quindolinone, cryptolepinone); steroids (20-hydroxyecdysone); and flavonoids (rutin, kaempferol) in four Sida species, viz., S. acuta, S. rhombifolia, S. cordifolia, and S. cordata. First ultrasonic-assisted extraction was optimized for optimum recovery of targeted PSCs. Then, the optimum resolution/separation in the sample matrix was optimized for C₁₈ (250 × 4.6 mm, 5 μm) column using 1.0 mM phosphate buffer-acetonitrile as the mobile phases in linear gradient elution. The elution was monitored in the range of 200-400 nm and the detector was set at 254 nm for simultaneous quantitation. A systematic risk analysis was conducted to identify potential critical method variables (CMVs) that affected critical method attributes (CMAs). A definitive screening design was employed to investigate the quantitative relationship and variability among different Sida spp. to address the challenges of un-intentional mixing and adulteration. After the models were built, the method operable design region (MODR) was calculated and validated. The DOE-driven method was optimized to fulfil the criteria of ICH Q2(R2) validation and measurement of uncertainty for ISO-17025:2017 compliance. PSC had a ranges of linearity (15.6-500.00 μg/mL), sensitivities (LOD: 8.15-24.63 μg/mL and LOQ: 27.16-82.11 μg/mL), and recoveries (92.23-97.28 %). The greenness score of the developed method evaluated by three different tools has supported its eco-friendliness and safety. The method was applied to analyze the PSC variability in different parts of Sida spp. aerial parts and possible admixing of phenotypically similar other species in the derived products. The present method could be applied for regular quality analysis of Sida species and their derived therapeutic products.