In-syringe solid-phase extraction coupled with UPLC-MS/MS for simultaneous determination of reducing sugars, animo acids and Amadori compounds in tobacco.

Considering the critical role of Maillard reaction-related compounds in determining the sensory and chemical stability of tobacco, this study develops an in-syringe SPE coupled with UPLC-MS/MS method for simultaneous determination of 43 Maillard reaction-related compounds in tobacco, covering reducing sugars (3), amino acids (17), Amadori compounds (17), and structurally related intermediates (6). The sample pretreatment utilizes HLB-packed self-assembled columns for SPE, followed by methanol elution. The extracted solution is then analyzed using a Titank C18 column (150×2.1 mm, 1.8 μm), with acetonitrile and 0.1% formic acid aqueous solution employed as the mobile phase for gradient elution. Data were acquired in multiple reaction monitoring mode using electrospray ionization. Operation parameters were optimized systematically, including the type and amount of filler, solution pH, loading rate, elution solvent composition, and sample extraction volume. This method demonstrated excellent linearity for all 43 target compounds, with correlation coefficients exceeding 0.9980. The limit of detections was 0.10-0.50 µg/L for animo acids, 1.0×102- 2.5×102 µg/L for reducing sugars, 0.10-5.0 µg/L for related Amadori compounds. This approach was successfully applied to the analysis of tobacco samples and extracts, providing a robust tool for studying the Maillard reaction and contributing to food safety assessment and optimization of food processing techniques.