Integrated Microplate and HPTLC Strategy for Determining and Profiling Total Phenolics and Flavonols in Citrus Peel...

Phenolic compounds and flavonoids significantly contribute to the antioxidant activity and functional value of citrus by-products. However, conventional colorimetric assays, while widely used, lack chromatographic separation and may be affected by chemical interferences. This protocol presents an integrated analytical workflow that combines microplate spectrophotometric assays with high-performance thin-layer chromatography (HPTLC) for the determination and profiling of total phenolic content (TPC) and total flavonols plus 5-hydroxyflavones content (TFHC) in citrus peel extracts. Ethanolic extracts of lime (Citrus aurantifolia) and mandarin (Citrus reticulata) peels were evaluated using Folin-Ciocalteu (FC) and aluminum chloride (AlCl₃) assays in a 96-well microplate format, followed by HPTLC analysis employing two sequential mobile phases and dual post-chromatographic derivatization. Microplate measurements yielded TPC values of 74.2 mmol/g for lime and 72.8 mmol/g for mandarin, while TFHC values were 15.5 mmol/g and 6.0 mmol/g, respectively. HPTLC separation resolved four phenolic bands in lime and six in mandarin. Two lime bands were selectively visualized after AlCl₃ derivatization, consistent with flavonols or 5-hydroxyflavones. The sum of phenolic bads estimated by HPTLC was 22.0 and 20.8 mmol/g dry matter for lime and mandarin, respectively, while TFHC reached 13.4 mmol/g in lime and was not detected in mandarin. These findings demonstrate that microplate assays provide rapid and sensitive estimation of total content, whereas HPTLC enhances selectivity through compound separation and functional-group-specific derivatization. The combined strategy enables high-throughput screening followed by chromatographic confirmation, offering a practical and reproducible platform for phytochemical characterization of complex plant matrices.