Simultaneous analysis of 2‐methylimidazole, 4‐methylimidazole,and 5‐hydroxymethylfurfural potentially formed in fermented soy sauce by “quick,easy, cheap,effective, rugged,and safe” purification and UHPLC with tandem mass spectrometry

2‐Methylimidazole, 4‐methylimidazole and 5‐hydroxymethylfurfural are harmful by‐products potentially formed via Maillard reaction in fermented soy sauce. The present study proposed a new method based on “quick, easy, cheap, effective, rugged, and safe” purification and ultra high performance liquid chromatography with tandem mass spectrometry for the simultaneous analysis of 2‐methylimidazole, 4‐methylimidazole and 5‐hydroxymethylfurfural in fermented soy sauce. The sample was dissolved in water after addition of internal standard 4‐methylimidazole‐d₆ and extracted with acetonitrile. After dehydration, it was centrifuged and the supernatant was subsequently purified using two sorbents namely primary‐secondary amine and multi‐walled carbon nanotube. Three target analytes were separated by gradient elution and determined under multiple reactions monitoring mode. The limit of detection, matrix effect, recovery and precision of the developed method were investigated. Results found that three target analytes displayed excellent linearity in concentration range of 1–250 μg/L. Limit of detection was in the range of 0.3–1 μg/kg for three target analytes. The mean recoveries for fermented soy sauce samples at three spiked concentrations were in the range of 91.2–112.5%, and the intra‐ and interday precision were in the ranges of 3.6–9.2 and 7.1–10.8%, respectively. This validated method was successfully applied to determine 2‐methylimidazole, 4‐methylimidazole and 5‐hydroxymethylfurfural concentrations in fermented soy sauce.