Development of validated HPTLC and UHPLC methods for quantification of Withanolide in extract and formulation

Abstract

High performance thin layer chromatography (HPTLC) and Ultra-High-Performance Liquid Chromatography (UHPLC) techniques were developed and validated to quantify Withanolide in extract and formulation. On Al-backed silica gel 60 F254 TLC plates (10 cm × 10 cm, layer thickness 0.2 mm), which had been prewashed with methanol, HPTLC separation was carried out. Dichloromethane: Methanol: Toluene: Acetone in various ratios produced good separation in mobile phase (5:1:1:0.5 v/v). Camag TLC scanner densitometric scanning at 365 nm determined and quantified. This approach produced compact Withanolide spots at Rf 0.48. ICH guidelines verified HPTLC's precision, reproducibility, and accuracy. Withanolide linearity was 500-3000 ng/spot with R2= 0.9994. LOD & LOQ were found to be 9.48 & 28.73 ng respectively. For UHPLC, Cosmosil C18 was used with acetonitrile: water (0.2 % OPA) (70:30, v/v) mobile phase. Flow rate was 1.5 mL/min. Under optimal chromatographic conditions, Withanolide was retained for 5.9 min and detected at 254 nm. ICH guidelines verified UHPLC's precision, repeatability, and accuracy. Withanolide linearity was 10-60 μg/mL with R2= 0.9994. LOD along with LOQ were 0.411 and 1.245 μg. HPTLC and UHPLC procedures utilized for regular quality control and quick screening of active components from plant extracts.