Newly designed liquid chromatographic method using relative molar sensitivity based on 1H-qNMR for quantifying polymethoxyflavones from Kaempferia parviflora

Quality control of six polymethoxyflavones in Kaempferia parviflora (KP) extract, a functional food widely used in Japan, is limited by the availability of high-purity reference standards. To address this, we developed a new analytical method with relative molar sensitivity based on 1 H-quantitative nuclear magnetic resonance spectroscopy. This approach enables simultaneous quantification of six polymethoxyflavones using 5,7-dimethoxyflavone as a single-reference standard, combined with ultrahigh-performance liquid chromatography employing a superficially porous particle column and a ternary mobile phase (detection at 265 nm). The method achieved a limit of quantification of 2.00 μmol/L, and specificity was confirmed by comparison with liquid chromatography–tandem mass spectrometry. Accuracy and precision met the acceptance criteria established by AOAC guidelines. By reducing reliance on multiple standards, this method provides a practical solution for the quality assurance of KP-derived products. Its validation, aligned with internationally recognized frameworks, further supports its potential for application in other regions.