Development and validation of quality by design guided HPTLC method for estimation of β-sitosterol in Celastrus paniculatus seed oil and its nano-formulation with greenness evaluation

• Eco-friendly HPTLC method for β-sitosterol in CPSO and nanoformulation. • Greenness confirmed via AGREE, Complex GAPI, and BAGI eco-safety metrics. • QbD-based method optimized using Design Expert software. • Method validated as per ICH guidelines parameters. • Promotes green chemistry for sustainable qualitative and quantitative analysis. Celastrus paniculatus is an ethnomedicinally important shrub, belonging to the Celastraceae family. Its seed oil demonstrates diverse pharmacological activities. However, no Quality by Design (QbD)-based analytical method has been reported to date to estimate of β-sitosterol in Celastrus paniculatus seed oil (CPSO). In this study, a HPTLC method was developed using QbD-based approach to estimate β-sitosterol in CPSO and its nano-formulation, followed by its validation parameters, such as calibration, specificity, sensitivity, precision, reproducibility, recovery, robustness, according to the ICH Q2 (R1) guidelines. CPSO was further characterized for its phytochemical profile through HPTLC fingerprinting and assessing the developed method’s greenness though AGREE scale, BAGI and ComplexGAPI tools. HPTLC method was developed by utilizing factorial design and T:EA:GAA:E (6:2:1:0.8)v/v/v/v was selected as the optimized method. The std, CPSO and formulation were then subjected to various validation parameters such as LOD (1.4 µg/ml) and LOQ (4.33 µg/ml), showing optimal results with a Rf value of 0.81. HPTLC fingerprinting revealed the presence of flavonoids, alkaloids, phenols, sterols and terpenoids. Greenness assessment based on AGREE score (0.79), ComplexGAPI and BAGI (77.5) of the developed HPTLC method highlighted its environmental safety. A QbD-driven HPTLC method for CPSO was developed and validated, offering rapid, robust, and eco-friendly analysis, enabling phytochemical profiling, and reliable routine quality control.