Analysis of 4-Methylimidazole Using an Agilent InfinityLab Poroshell 120 HILIC-OH5 Column

Importance of the topic

4-Methylimidazole (4-MEI) is a polar by-product found in certain heated foods and caramel colorings used in beverages. Due to its potential health concerns and trace-level occurrence, developing reliable analytical methods for its detection in food matrices is critical to ensure consumer safety and regulatory compliance.

Objectives and Study Overview

This study aimed to establish a rapid, sensitive approach for the separation and quantification of 4-MEI and its isomer 2-MEI in cola-type beverages. By comparing two HILIC stationary phases and coupling with triple quadrupole LC/MS, the work evaluates retention behavior, detection limits, and analytical performance in a real sample matrix.

Methodology and Used Instrumentation

Sample Preparation:

• 3 mL cola adjusted to pH ~7 with ammonium hydroxide and diluted to 5 mL with water.
• 1 mL of this solution mixed with 8 mL acetonitrile, sonicated and filtered.
• Spiked samples prepared by addition of standard prior to processing.

Chromatographic Conditions:

• Column: Agilent InfinityLab Poroshell 120 HILIC-OH5, 2.1×100 mm, 2.7 µm.
• Mobile phase: 10 mM ammonium acetate in water (10 %) and acetonitrile (90 %).
• Flow rate: 0.30 mL/min, temperature: 30 °C, injection volume: 5 µL.

MS Conditions:

• Agilent 6460 triple quadrupole with Jet Stream ESI in positive mode.
• Drying gas: 210 °C, 13 L/min; sheath gas: 400 °C, 12 L/min; capillary voltage: +2 500 V.
• MRM detection with ΔEMV 500 V.

Instrumentation:

• Agilent 1290 Infinity II pump, multisampler, multicolumn thermostat.
• Agilent 6460 triple quadrupole LC/MS and MassHunter software.
• ELGA water system, sonicator, Econofilter PTFE filters.

Main Results and Discussion

The HILIC-OH5 stationary phase delivered superior retention and baseline separation of 4-MEI and 2-MEI compared to HILIC-Z under identical conditions. Limit of detection was determined below 1 ppb at S/N of 3. Spiked sample analysis at 1 and 10 ppb demonstrated clear, interference-free peaks with signal-to-noise ratios exceeding 12 and 69 respectively, confirming method sensitivity and selectivity.

Benefits and Practical Applications

• Effective retention of small polar analytes without derivatization.
• High sensitivity LC/MS detection suitable for regulatory monitoring.
• Fast sample preparation and analysis workflow for routine quality control in food and beverage testing.

Future Trends and Potential Applications

Advances may include integrating this approach with high-resolution MS for broader contaminant screening, automating sample preparation, and extending HILIC-MS workflows to other polar by-products in diverse food matrices. Ongoing improvements in stationary phase design could further enhance selectivity and throughput.

Conclusion

The Agilent InfinityLab Poroshell 120 HILIC-OH5 column coupled with triple quadrupole LC/MS provides a robust, sensitive, and rapid method for simultaneous quantitation of 4-methylimidazole and 2-methylimidazole in cola beverages, achieving detection limits below 1 ppb and reliable separation characteristics.

Reference

Agilent Technologies Application Note 5991-9323EN (2018).