QbD-Driven Development and Validation of an Optimized HPTLC Method for Simultaneous Estimation of Berberine and Conessine

Berberine, an isoquinoline-derivative alkaloid from Berberis aristata , and conessine, an alkaloid found in the stem bark of Holarrhena antidysenterica , have long been used in traditional medicine to treat gastrointestinal and reproductive issues. This study focused on developing a high-performance thin-layer chromatography (HPTLC) method for the simultaneous quantification of berberine and conessine, optimized using a Quality by Design (QbD) approach. The method development involved the systematic optimization of critical method parameters (CMPs) such as mobile phase ratio, saturation time, distance travelled, and derivatizing agent concentration, using a Box-Behnken design. The critical analytical attributes (CAAs) evaluated included peak area and retardation factor as indicators of method robustness. Optimal chromatographic separation was achieved with a mobile phase of ethyl acetate, methanol, and diethyl amine in a ratio of 6.5:1.0:0.3 v/v on Silica gel 60GF 254 plates. Berberine and conessine were detected densitometrically at 350 nm and 620 nm, respectively, with Rf values of 0.22 and 0.85. The method was validated as per the ICH recommended conditions, which revealed high degree of linearity, accuracy, precision, sensitivity and robustness. The method was demonstrated to be simple, fast, accurate, resilient, and exact. Also, the method was applied for the estimation of berberine and conessine in inhouse formulations, which indicated no significant change in the retention time. In a nutshell, the studies demonstrated successful development of the HPTLC method for simultaneous estimation of berberine and conessine with improved understanding of the relationship among the influential variables for enhancing the method performance.