A new single-reference quantitative method using liquid chromatography with relative molar sensitivity based on 1H-qNMR for khellactone esters from Peucedanum japonicum root extract

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Khellactone ester (KLE) quantification using the absolute calibration method is difficult owing to the unavailability of standard reagents that can guarantee purity. Herein, a new method was developed to quantify KLEs from Peucedanum japonicum root extracts using liquid chromatography (LC) without utilizing standards. This method used relative molar sensitivity (RMS) and 7-ethoxy-4-methylcoumarin as a single-reference (SR) compound instead of KLE standards. RMS is the sensitivity ratio of SR to analytes, determined using an offline combination of quantitative NMR and LC. LC was performed using a triacontylsilyl silica gel column of superficially porous particles with a ternary mobile phase. The range of the method was 2.60-509 µmol/L. The accuracy and precision were reasonable. This is the first study to apply the RMS method to both conventional LC and ultra-high-performance liquid chromatography using the same mobile phase and column. This method may aid the quality assurance of foods containing KLEs.