QbD-driven development and validation of an HPTLC method for simultaneous estimation of berberine from <i>Berberis aristata</i> DC ( <i>Berberidaceae</i> ) and conessine from <i>Holarrhena pubescens</i> Wall. ( <i>Apocynaceae</i> ) in herbal capsule

A simple, precise, and robust validated high-performance thin-layer chromatographic (HPTLC) method was developed and validated for the simultaneous estimation of berberine and conessine, the major alkaloids of Berberis aristata DC. (Berberidaceae) and Holarrhena pubescens Wall (Apocynaceae), in a polyherbal capsule formulation using a Quality by Design (QbD) approach. Using ethyl acetate: methanol: diethylamine (6.5:1.0:0.3, v/v) as the mobile phase, conessine and berberine were optimally separated on silica gel GF 254 plates with detection at 350 and 620 nm, respectively. The method was validated as per ICH Q2(R1) guidelines. Berberine and conessine showed well-resolved peaks at Rf 0.22 ± 0.02 and 0.85 ± 0.02, respectively, with linearity (berberine: 100–800 ng/spot, R2 > 0.998; conessine: 2,000–16,000 ng/spot, R2 > 0.998). The proposed QbD-based HPTLC method is accurate, sensitive, and reproducible and can be effectively applied for the routine quality control and standardisation of in-house formulations containing B. aristata and H. pubescens.