Thin-layer chromatography image analysis with leave-1-out method and cross-sectional study for quality assessment of a polyherbal preparation ‘Phikud Navakot’

This paper introduces a new methodology for assessing the quality of a polyherbal preparation using thin-layer chromatography (TLC) image analysis integrated with the leave-1-out method and cross-sectional study. “Phikud Navakot”, a Thai polyherbal preparation for relieving circulatory disorders and dizziness, consisting of nine herbs, was used as a case study. The leave-1-out technique involves systematically excluding one herb at a time from the preparation to generate TLC profiles, which are then analyzed and compared with the original preparation. Another approach, based on a cross-sectional study of the TLC image, was employed by converting all track on the TLC image into red, green, and blue (RGB) profiles simultaneously for direct band-by-band comparison across chromatograms. The leave-1-out approach combined with image analysis enhances band detection clarity, allowing for a more precise assignment of each band to its corresponding herb. Regardless of whether the substance band in polyherbal preparation was distinctly noticeable, faint, closely adjacent, overlapping, or concealed, the approaches based on TLC image analysis integrated with the leave-1-out method and cross-sectional study were demonstrated to be effective analytical techniques for complex herbal preparations and could be applied to other herbal mixtures.