2-methylimidazole (2-MeI) and 4-methylimidazole (4-MeI) are by-products from the manufacture of caramel colour additives used
in beverages, soy sauces, baked foods, and so on. The International Agency of Research of Cancer classified these two compounds
as "possibly carcinogenic to humans", and proposed the no significant risk level (NSRL) to be 29 µg/day for 4-MeI, while California
listed 4-MeI as a probable carcinogen and proposed a 16 µg/day NSRL. This application note presents a simple, fast, and cost-effective
method to determine 2-MeI and 4-MeI in beverages simultaneously. Beverage samples were degassed, diluted 10 times with acetonitrile
(MeCN), and filtered through a solid-phase extraction (SPE) cartridge with 200 mg FASt sorbent, onto which the undesired matrix
components, such as sugars and organic acids were retained, while the analytes were passed through and collected. A new HILIC
HPLC method was developed to separate the 2-MeI and 4-MeI isomers, which underwent the same MS–MS transitions. Baseline separation
was achieved in an 8-min run.
1. Sample pretreatment
Pour the beverage samples into 500-mL beakers, and degas the samples by stirring at high speed for 2 h.
2. Filter and Shoot (FASt) procedure
a. Transfer 0.1 mL of the degassed samples into test tubes or vials. Add 0.9 mL of MeCN and 100 ng/mL imidazole as internal
standard (IS). Vortex for 10 s.
b. Attach the FASt cartridges (CSFAS203) to a glass block or positive pressure manifold, insert test tubes or 2-mL auto-sampler
vials into the manifold.
c. Transfer the entire diluted samples into the cartridges, apply low vacuum or positive pressure, and collect the filtrates.
d. The samples are ready for LC–MS–MS analysis.
LC–MS–MS: Thermo Scientific Ultimate 3000® LC coupled to Vantage MS/MS system
Injection: 10 µL at 10 °C
LC column: Thermo Scientific Accucore HILIC, 100 × 2.1 mm (2.6 µm), with 10 mm guard column
Column temperature: 40 °C
Mobile phase: 8-min isocratic with 5% of 50 mM ammonium formate in water and 95% of MeCN
Flow: 0.4 mL/min
Polarity: ESI positive
Collision gas: Argon at 0.8 mTorr
Retention times and MS–MS transitions:
a. IS: 1.96 min; 69.07→42.01; 69.07→28.08
b. 4-MeI: 3.18 min; 83.08→56.05; 83.08→42.00
c. 2-MeI: 5.72 min; 83.07→42.04; 83.07→56.05