A Novel Enzymatic UHPLC-MS/MS Method for Quantifying Free and Trapped 2-Acetyl-1-pyrroline in Fragrant Rice

2-Acetyl-1-pyrroline (2-AP) is the principal aromatic compound responsible for the characteristic popcorn-like scent of fragrant rice. However, accurate quantification of 2-AP is still a considerable challenge due to its low concentration, instability, and tendency to form complexes with starch matrices. In this study, we developed a novel and highly sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method using 4-hydrazinobenzoic acid (HBA) as a derivatization reagent and stable isotope dilution analysis (SIDA). The optimized method demonstrated excellent sensitivity, with an instrumental limit of detection (LOD) of 0.2 μg/kg and a quantification range of 10–2000 μg/kg in rice samples. To improve extraction efficiency, amylase was introduced to hydrolyze starch complexes, facilitating the release of both free and trapped 2-AP. The method showed satisfactory analytical performance, with recoveries of 97–120% for free 2-AP and 97–103% for total 2-AP, together with good reproducibility. Application of this method to ten fragrant rice varieties revealed significant differences in both free and total 2-AP concentrations, with total 2-AP ranging from 65.2 to 1166. 9 μg/kg. Sensory evaluation via quantitative descriptive analysis (QDA) indicated that trapped 2-AP had a stronger correlation with popcorn flavor intensity than free 2-AP, suggesting its greater role in aroma perception. This study provides a robust analytical tool for the accurate quantification of different 2-AP forms in rice and offers new insights into the relationship between aroma compounds and sensory quality in fragrant rice, with implications for breeding, quality control, and flavor enhancement.