Abstract
Japan’s Specifications and Standards for Food Additives (JSFA) lists the monograph of the specifications for Sorbic Acid, which is primarily composed of sorbic acid (SoA, (2E,4E)-hexa-2,4-dienoic acid), as a preservative. The current assay in JSFA for Sorbic Acid is a comprehensive quantification method for SoA and the three stereoisomers of SoA. Although high-performance liquid chromatography (HPLC) is an effective analytical technique for separately detecting SoA and the stereoisomers, accurate quantification by HPLC requires corresponding reference materials because the sensitivity of each analyte to the detector is dependent upon the chemical structure. However, reagents for stereoisomers, which are poorly certified, in short supply, and generally expensive, are not available as reference materials. In the present study, we developed single-reference quantification methods using HPLC with ultraviolet/visible absorption spectrophotometry and relative molar sensitivity (RMS) of the stereoisomers to the reference substance. By optimizing the HPLC conditions for the absolute separation of SoA and the stereoisomers, we determined the RMSs by referring to SoA or caffeine, which were selected as internal references, based on the results of quantitative 1H-NMR spectrometry (1H-qNMR). HPLC quantification conducted by utilizing the determined RMSs and calibration curves of SoA or caffeine provided 99.6 – 103.4% of the accurate contents of the stereoisomers obtained from 1H-qNMR. These results demonstrate that our single-reference HPLC method is suitable for accurate simultaneous quantification of the stereoisomers of SoA.
