Systematic Development and Validation of a Thin-Layer Densitometric Bioanalytical Method for Estimation of Mangiferin Employing Analytical Quality by Design (AQbD) Approach

The present work aims at the systematic development of a simple, rapid and highly sensitive densitometry-based thin-layer chromatographic method for the quantification of mangiferin in bioanalytical samples. Initially, the quality target method profile was defined and critical analytical attributes (CAAs) earmarked, namely, retardation factor (R_f), peak height, capacity factor, theoretical plates and separation number. Face-centered cubic design was selected for optimization of volume loaded and plate dimensions as the critical method parameters selected from screening studies employing D-optimal and Plackett-Burman design studies, followed by evaluating their effect on the CAAs. The mobile phase containing a mixture of ethyl acetate: acetic acid: formic acid: water in a 7: 1: 1: 1 (v/v/v/v) ratio was finally selected as the optimized solvent for apt chromatographic separation of mangiferin at 262 nm with R_f 0.68 ± 0.02 and all other parameters within the acceptance limits. Method validation studies revealed high linearity in the concentration range of 50-800 ng/band for mangiferin. The developed method showed high accuracy, precision, ruggedness, robustness, specificity, sensitivity, selectivity and recovery. In a nutshell, the bioanalytical method for analysis of mangiferin in plasma revealed the presence of well-resolved peaks and high recovery of mangiferin.