Development of a Standard-Free Analytical Quantitative Method Based on Liquid Chromatography Using Relative Molar...

Recently, a chromatographic quantitative method using relative molar sensitivity (RMS), the so-called RMS method, was developed for the determination of target analytes in food additives, drugs, and supplements. By determining the RMS value of the analyte to a different reference compound, this method avoids the need for an analytical standard of the analyte itself to plot calibration curves for quantification. In this collaborative study performed by 10 laboratories, we demonstrated the robustness and reliability of the RMS method in the quantitative analysis of chlorogenic acid (5-O-caffeoylquinic acid, 5CQA) in apple juice. Reagent-grade 5CQA derived from natural sources is commercially available, but its purity, stability, and hygroscopic properties still need to be clarified. Therefore, there is always a risk of bias in the quantitative results, even when the calibration curve is prepared using reagent-grade 5CQA as the analytical standard. The RMS method overcomes this problem by using caffeic acid (CA) with high purity and stability as a reference compound. Prior to the collaborative study, a laboratory documented the standard operating procedure for method validation, which was then implemented in all laboratories to determine the RMS value of 5CQA to CA and quantify 5CQA in five apple juice samples. A comparison between the results obtained using a calibration curve and the RMS method validates the RMS method using CA as a reference compound for the quantitative analysis of 5CQA without an analytical standard.