Development and validation of an HPTLC method for quantitative estimation of quercetin and its application in Bauhinia variegata Bark Extract Keywords: Quercetin; HPTLC; Bauhinia variegata; method validation; ICH Q2(R1); phytochemical standardization

Bauhinia variegata Linn. (Fabaceae) is a widely used medicinal plant whose bark exhibits antioxidant, anti-inflammatory, antimicrobial, hepatoprotective, and anticancer activities, largely attributed to its flavonoid content, of which quercetin is a key bioactive marker. Since no validated method previously existed for quantifying quercetin specifically in B. variegata bark, the present study aimed to develop and validate a simple, sensitive, and reproducible HPTLC method for this purpose, in accordance with ICH Q2(R1) guidelines. Chromatographic separation was achieved on silica gel 60 F₂₅₄ plates using a mobile phase of ethyl acetate: toluene: methanol: formic acid (3.5: 6: 0.5: 0.5, v/v/v/v), with densitometric detection at 383 nm, giving a well-resolved band at Rf 0.54. The method showed good linearity over 100–700 ng/band (y = 23.063x − 430.75, R² = 0.991), with an LOD of 20 ng/band and LOQ of 80 ng/band. Precision (%RSD < 2% for repeatability, intraday, and interday studies), accuracy (mean recovery 98.57–100.65%), robustness, and specificity all met acceptance criteria, confirming the reliability of the method. Application to the methanolic bark extract of B. variegata revealed a quercetin content of 0.012% w/w, with consistent results across replicate analyses. This validated HPTLC method offers a rapid, cost-effective, and reliable tool for the routine quality control, standardization, and phytochemical evaluation of B. variegata bark and its extracts. Keywords: Quercetin; HPTLC; Bauhinia variegata; method validation; ICH Q2(R1); phytochemical standardization