Improving reproducibility of HPTLC analysis for cranberry supplements through digitization and chemometric preprocessing

Authors: ZHANG, MENGLIANG - The Ohio State University; Sun, Jianghao; CORWIN, ELIZABETH - National Sanitation Foundation (NSF); Harnly, James

Submitted to: Journal of AOAC International
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/2/2025
Publication Date: 6/30/2025
Citation: Zhang, M., Sun, J., Corwin, E., Harnly, J.M. 2025. Improving reproducibility of HPTLC analysis for cranberry supplements through digitization and chemometric preprocessing. Journal of AOAC International. Article qsaf063. https://doi.org/10.1093/jaoacint/qsaf063.
DOI: https://doi.org/10.1093/jaoacint/qsaf063

Interpretive Summary: High performance thin layer chromatography (HPTLC) is method commonly used in industry for authentication of botanical raw materials and supplements. Samples are extracted, chromatographically separated on thin layer gel plates, chemically derivatized, and then illuminated with visible or ultraviolet light. As many as 15 samples can be run in parallel which allows the samples to be directly compared to reference standards on the same plate. Common usage is visual interpretation of the similarity of samples and standards on the same plate, but comparison between plates is difficult. This research used electronic digitization of the chromatograms with sample normalization and retention time alignment to produce digital profiles of the chromatograms which can be accuartely compared between plates and analyzed by statistical and pattern recognition programs. The visually subjective interpretation is replaced by objective mathematical analysis with improved analytical accuracy and facility of data interpretation.

Technical Abstract: Background High-performance thin-layer chromatography (HPTLC) is widely used for the identification and quality assessment of botanical supplements. However, traditional interpretation methods are subjective, and variability between plates hinders reproducibility and inter-plate comparisons. Objective This study aimed to enhance the reproducibility and analytical utility of HPTLC by digitizing chromatograms and applying chemometric preprocessing to cranberry dietary supplement analysis. Method Cranberry supplements of diverse dosage forms were extracted and analyzed using a standardized HPTLC protocol. Plates were derivatized with natural products and anisaldehyde reagents and imaged under multiple lighting conditions. Digital chromatograms were processed using normalization and retention factor (RF) alignment. Chemometric methods, including principal component analysis (PCA) and analysis of variance principal component analysis (ANOVA-PCA), were applied to assess variability and improve classification. Results The digitization and preprocessing workflow significantly reduced plate-related variability while enhancing classification accuracy. RF alignment lowered between-plate variance from 23 to 11%, while increasing sample-type variance from 59 to 79%. Combining data from multiple derivatization and imaging conditions improved chemical fingerprinting and enabled tighter clustering in PCA models. Conclusions The integration of digitized HPTLC data with chemometric preprocessing modernizes the analytical workflow, improves reproducibility, and enables more robust and interpretable botanical fingerprinting. This approach supports improved quality control of botanical products and aligns with emerging standards for data transparency and reusability. Highlights Digitization and alignment reduce HPTLC variability and enhance reproducibility. Combined profiles from multiple derivatization conditions improve sample classification. Chemometric analysis enables better interpretation and data-driven quality control and assessment for botanicals.