Development and validation of a novel high-performance thin-layer chromatography method for the quantitative estimation of neohesperidin from Citrus aurantium peel extract

A high-performance thin-layer chromatography method was developed for the estimation of neohesperidin from Citrus aurantium peel. CAMAG Linomat ATS 4 sample applicator was used for the application of the sample. Chromatographic separation of the marker was performed over thin-layer chromatography (TLC) plates pre-coated with silica gel 60F254 using ethyl acetate‒methanol‒water‒formic acid (7.1:1.4:1:0.5, V/V) as the mobile phase via a linear ascending technique in twin-trough chamber. Detection and quantification were carried out at a wavelength of 254 nm using a TLC Scanner 4. This method showed good peak symmetry for the marker with a retention factor of 0.54 ± 0.02 for neohesperidin. The calibration curve was linear in the range of 1000‒3000 ng/spot for neohesperidin and the correlation coefficient (r2) was 0.9922. The method was validated according to the International Council for Harmonisation (ICH) Q2 R1 guidelines for linearity, specificity, limit of detection, the limit of quantification, precision, accuracy (recovery), and robustness. In conclusion, the developed method is simple, reliable, and specific for the identification and quantification of neohesperidin.

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