Authentication of Agave Products through Isotopic Intramolecular 13C Content of Ethanol: Optimization and Validation of 13C Quantitative NMR Methodology

Agave syrup has become a popular sweetener among consumers because of its low glycemic index and pleasant taste, but substitution by cheaper sugar syrups makes it vulnerable to fraud. A ¹³C site-specific natural isotopic fractionation (SNIF)-NMR approach had been developed to allow the detection of C₄ plants (cane, maize) in agave products, with the drawback of a long NMR acquisition time. In this work, an INEPT (insensitive nuclei enhanced by polarization transfer) pulse sequence was implemented to significantly reduce acquisition time while accurate and reproducible results are obtained. Apparent site-specific isotopic deviations were correlated to values acquired with single pulse method. The analytical method was validated following the normative procedure NF V03-110, from sample preparation to data correction. With a single pulse method as a reference, performances were at least equally good and measurement time was halved, opening new methodological ways for isotopic ratio monitoring of carbon in the ethanol molecule by NMR, also applicable to fermented and fermentable beverages. In parallel, the method was extended to agave inulin characterization after a preliminary controlled hydrolysis.