Determination of Methylimidazole in Tea by High-Resolution Mass Spectrometry and Investigation of its Source

Methylimidazole (MEI), classified as a Group 2B carcinogen by the World Health Organization (WHO), is formed primarily as a Maillard reaction byproduct in foods. Its recent detection in tea has raised concerns regarding potential food safety risks.

This study aims to establish an ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) method for the simultaneous determination of MEIs in tea. The validated method was employed to determine the contents of MEIs across various tea types and to explore their potential sources.

Sample pretreatment was performed using a modified QuEChERS approach with isotope dilution. Chromatographic separation was achieved on a HILIC column using a gradient elution program with mobile phases consisting of 5 mmol/L ammonium acetate containing 0.1% formic acid in water and acetonitrile. Detection was carried out using Q-TOF with SWATH for MS/MS quantification.

The method exhibited excellent linearity (r > 0.999) within the concentration range of 2-50 µg/L. The limits of detection (LODs) ranged from 0.0024 to 0.0045 mg/kg. The recovery ranged from 82 to 100.2%, with RSDs between 1.1 and 4.6%. Statistical analysis indicated that heavily fermented and roasted teas exhibited significantly higher MEI levels than lightly processed teas did (p < 0.001). Laboratory simulations experiments showed that MEI formation increased substantially at the baking temperature of 150°C.

The developed UHPLC-HRMS method is accurate and precise and is suitable for the simultaneous quantification of three MEIs in tea. These results suggest that the MEI content in tea is associated with degree of fermentation and roasting, likely originating from Maillard reaction-derived byproducts.

The proposed HRMS method mitigated the limitations commonly associated with conventional LC-MS approaches, Furthermore, the validated method was successfully employed to quantify MEI contents in tea subjected to various fermentation and roasting processes, thereby contributing to a better understanding of MEI's potential formation pathways.