Mixture Design-driven Statistical Optimization and Method Validation in HPTLC: Targeting Rutin, Quercetin, and Gallic Acid

Rutin (RT), Quercetin (QT), and Gallic Acid (GA) are recognized for their potent antioxidant and anticancer properties, prevalent across numerous plant species. The precise quantification of RT, QT, and GA is pivotal for evaluating the therapeutic potential of plant-based substances. In response, a new, straightforward, cost-effective, and reliable method using High-Performance Thin-Layer Chromatography (HPTLC) has been developed and validated for the quantification of these compounds. The mobile phase optimization employed a mixture design approach, achieving chromatographic separation with a mobile phase mixture of toluene, ethyl acetate, menthol, and formic acid at specific ratios (3.56:3.70:0.94:1.80 v/v/v/v/v/v/v/v). Silica gel 60 F254 HPTLC plates were utilized for the analysis. The retention factors (Rf) observed for RT, GA, and QT were 0.21, 0.58, and 0.74, respectively. This method demonstrated a robust linear relationship for concentrations ranging from 400 to 2000 ng per band, with correlation coefficients (R2) of 0.9921 for RT, 0.9936 for QT, and 0.9912 for GA. The Limits of Detection (LOD) and quantification (LOQ) were established at 100.84, 102.38, and 84.54 ng per band for LOD, and 305.58, 310.25, and 256.18 ng per band for LOQ, respectively, for RT, QT, and GA. This validated HPTLC method developed through a Design of Experiment (DoE) approach was successfully employed for the quantification of GA, QT, and RT from the fruits of Adansonia digitata and leaves of Grewia asiatica ethanolic extracts.