Abstract:

An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method was developed
for the determination of 4-(5-)-methylimidazole (4-Mel) and its isomer, 2-methylimidazole (2-Mel), in beverage. Samples
were diluted with water. An OASIS® MCX solid phase extraction column was used to purify and concentrate the target
analytes. Under the optimal conditions, the analytes were separated by a Waters ACQUITY UPLC® HILIC column (2.1 ×
100 mm, 1.7 mm) using a mobile phase consisting of acetonitrile and 5 mmol/L ammonium acetate aqueous solution. The
analytes were detected in multiple-reaction monitoring (MRM) mode via positive electrospray ionization (ESI+). The
results showed that the calibration curves of 4-Mel and 2-Mel were of good linearity in the range of 9–500 ng/mL. The
limit of quantification of the method was 3.0 mg/kg. The average recovery was 97.3% and 93.7% at four spiked levels (3.0,
25, 50 and 75 mg/kg) for 4-Mel and 2-Mel, respectively, and the relative standard deviation (RSD) was 3.8% and 3.4%,
respectively. These figures of merit indicated that the developed method can be used for the determination of 4-Mel and 2-Mel
in beverage samples. This method was applied to different beverages. 4-Mel or 2-Mel was found in soft drink, caramel
colored beverage and coffee. Contrarily, neither of them was found in energy drinks.

Key words: ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS-MS), 4-(5-)-methylimidazole, 2-methylimidazole, solid phase extraction (SPE), beverage