Newly designed liquid chromatographic method using relative molar sensitivity based on ¹H-qNMR for quantifying...

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 ¹H-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.