Simultaneous Quantification of Amadori and Heyns Compounds Derived from the Same Amino Acid in Tobacco by HPLC-MS/MS with Ion Ratio Strategy

Amadori and Heyns compounds are key Maillard reaction intermediates, resulting from the reaction of a certain amino acid with glucose (Glu) or fructose (Fru), respectively. They play a dual role in augmenting cigarette aroma and attenuating sensory irritation, and their quantification in tobacco is vital for tobacco quality evaluation and Maillard reaction mechanism study. Currently, simultaneous quantification of Amadori and Heyns compounds derived from the same amino acid, which present as isomers, is encumbered by poor chromatographic separation, and reliance on unavailable specific fragment ions. Herein, a method based on high performance liquid chromatography combined with tandem mass spectrometry (HPLC-MS/MS) detection was developed for the simultaneous quantification of Amadori and Heyns compounds derived from the same three amino acid (alanine, proline, phenylalanine, abbreviated as Ala, Pro, and Phe, respectively) with an ion ratio strategy. Under the optimized conditions, the limits of detection ranging from 2.22 to 6.94 ng/mL were obtained for six target analytes (Fru-Phe/Glu-Phe, Fru-Pro/Glu-Pro, Fru-Ala/Glu-Ala). When the method was applied to tobacco leaf powder extracts, target Amadori and Heyns compounds were found in the range of 0.65-56.4 μg/g, with Heyns compounds consistently less abundant than Amadori counterparts. In the recovery test, target analytes showed recoveries of 76.8-111%. Compared with reported methods, the proposed method features universal applicability without requiring characteristic fragment ions or chromatographic separation. It provides a reliable tool for analyzing hard-to-separate sugar-derived Maillard intermediates in tobacco.