Chromatographic Method Validation for Simultaneous Determination of Bergenin and Alpha‐Lipoic Acid in Skin Penetration Studies

ABSTRACT

This study validates a selective, robust high‐performance liquid chromatography (HPLC) method for the simultaneous determination of bergenin (BER) and alpha‐lipoic acid (ALA) in skin permeation studies. Separation used a Discovery C ₁₈ column (250 × 4.6 mm, 5 μm) and a mobile phase of acetonitrile and aqueous phosphoric acid (pH 3.0). A multistep gradient (10%–30% acetonitrile from 0–2 min; 30%–40% from 2–6 min; and 40%–60% from 6–9 min) at 1.0 mL/min enabled a 20‐min run time with UV detection at 210 nm. Following ICH M10 bioanalytical guidelines, the method showed linearity ( r  > 0.999) from 0.5 to 15 μg/mL for both analytes. Limits of detection were 0.120 and 0.083 μg/mL, and limits of quantification were 0.363 and 0.250 μg/mL for BER and ALA, respectively. Precision and accuracy were satisfactory, with a relative standard deviation (RSD) < 3.5%, while recoveries ranged between 80.8% and 96.2% (RSD < 10.0%) from porcine stratum corneum and viable skin. The method proved robust against variations in temperature, flow rate, and column batch, without endogenous skin interference. In conclusion, this validated method is a reliable tool for supporting the development of topical formulations containing the association of BER and ALA.