Development and validation of a high-performance thin-layer chromatographic method for the quantitative analysis of...

Citrus peel and pomace, major byproducts of citrus processing, are rich in polymethoxyflavones (PMFs), such as nobiletin, tangeretin, and sinensetin. In this study, a cost-effective and reliable high-performance thin-layer chromatography (HPTLC) method was developed and validated for the simultaneous quantification of these PMFs in eleven citrus waste varieties. Separation was achieved on silica gel 60 F₂₅₄ plates using an optimized mobile phase of hexane:1-butanol: formic acid (80:20:1, v/v/v), with densitometric detection at 254 nm. The method exhibited excellent linearity over 0.078-2.50 µg band⁻¹ (R² = 0.9912-0.9986), high precision (intra- and interday %RSD < 2%), satisfactory recoveries (98.14%-100.97%), and robustness against deliberate chromatographic variations, in accordance with International Council for Harmonization (ICH) guidelines. The application of the method to eleven citrus waste varieties revealed pronounced interspecies variability in PMF content. Overall, the validated method provides a rapid, reproducible, and low-solvent analytical platform suitable for routine analysis and high-throughput analytical screening of citrus processing byproducts.