Central composite design-assisted green high-performance thin-layer chromatographic method development for simultaneous analysis of gallic acid and quercetin in Clerodendrum chinense

The increasing global reliance on herbal medicines and nutraceuticals has highlighted the need for scientifically validated and standardised phytopharmaceutical products. However, the chemical complexity and variability of phytoconstituents necessitate reliable and environmentally sustainable analytical methods for quality assessment and phytochemical estimation. This study aimed to develop and validate a robust and sensitive HPTLC densitometric method for the simultaneous quantification of gallic acid and quercetin in polyphenol-enriched leaves of Clerodendrum chinense (Osbeck) Mabb. Chromatographic separation was achieved on TLC plates by the use of a mobile phase consisting of ethyl acetate: toluene: methanol: formic acid (3:4:1:0.5% v/v). Robustness was evaluated using RSM-CCD, and environmental sustainability of the method was assessed using AGREE, ComplexGAPI, and Analytical Eco-Scale tools. The method provided well-resolved and compact bands with Rf values of 0.61 (gallic acid) and 0.76 (quercetin). The method showed excellent linearity (R2 = 0.9943 for gallic acid and 0.9984 for quercetin), high precision, and good accuracy (98.90-105.15% for gallic acid and 97.33-104.70% for quercetin). The LOD values were 26.17 ng/band for gallic acid and 11.70 ng/band for quercetin, while the LOQ was estimated to be 79.29 ng/band for gallic acid and 35.44 ng/band for quercetin. Quantitative analysis revealed that PRCC leaves contained 0.4507% w/w gallic acid and 0.1548% w/w quercetin at λmax of 277 nm for gallic acid and 290 nm for quercetin. The developed HPTLC technique is reliable, precise, and environmentally considerate, offering an effective tool for the standardisation and routine quality control of polyherbal preparations containing gallic acid and quercetin.