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

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.