Slides used in presentation at the Thompson Rivers University Undergraduate Conference, 2014.

1. Determination of 2- and 4(5)-
Methylimidazole in Food Products by
Capillary Electrophoresis
PRESENTER: ZHI CHAO GUO
SUPERVISOR: DR. KINGSLEY DONKOR
DEPARTMENT OF CHEMISTRY, THOMPSON
RIVERS UNIVERSITY, KAMLOOPS, BC, CANADA

2. OBJECTIVE
Development of a capillary electrophoresis (CE) method
to analyze the quantities of 2-methylimidazole (2-MI) and 4(5)-
methylimidazole (4-MI) in cola and alcoholic beverages and
sauces.
2-MI 4-MI

3. INTRODUCTION

2-MI and 4-MI are by-products of manufacture formation of caramel
color through maillard reaction

Maillard reaction is a chemical reaction between amino acids and
reducing sugars that gives a desirable flavor in browned foods

Caramel color is used as color and flavor additive to several food
products

They have carcinogenic effects on human

4-MI causes convulsant in rabbits, mice and chicks at 360 mg/kg of
body weight

2-MI shows toxity in rats at 1500 mg/kg of body weight

4. 
Β-cyclodextrin is a 7-membered sugar ring molecule

The cavity is hydrophobic in order to interact with hydrophobic molecules

The wall is hydrophilic, so it is soluble in water (solubility = 18.5 mg/ml)

5. INSTRUMENT
TABLE 1. BECKMAN P/ACE MDQ CAPILLARY
ELECTROPHORESIS CONDITIONS
Capillary 60 cm total length
with 49.7 cm effective
length, ~50 μm i.d.
Column temperature 25 °C
Separation voltage 15 kV
Detector UV detection

6. CE OPERATION SCHEMATIC

7. EXPERIMENTAL
TABLE 2. OPTIMIZED CONDITIONS
UV detection wavelength 214 nm
pH of buffer 7.7
Concentration of β-cyclodextrin in buffer 3.0 mM
Concentration of sodium phosphate in
70 mM
buffer
Water (solution/buffer preparation) LC/MS grade water
Solution/sample filter 0.45 μm Nylon
All final optimized conditions selected were based on sensitivities of 2-MI
and 4-MI, and resolutions of both and the internal standard, imidazole.

8. SOLUTION AND SAMPLE PREPARATION

All stock solutions were prepared with LC/MS grade water and then
filtered with 0.45 μm nylon filters, and 2-MI and 4-MI stock solutions
were stored in lamber vials in the fridge

All real samples are purchased from local grocery stores, filtered with
0.45 μm nylon filters and stored in the fridge

9. RESULTS
ELECTROPHEROGRAM OF A STANDARD SOLUTION (1000 ΜG/L OF 2-MI AND 4-MI)
5.8 5.9 6.0 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 7.0 7.1 7.2 7.3 7.4 7.5 7.6 7.7 7.8 7.9 8.0 8.1 8.2 8.3
Minutes
AU
0.00090
0.00085
0.00080
0.00075
0.00070
0.00065
0.00060
0.00055
0.00090
0.00085
0.00080
0.00075
AU
0.00070
0.00065
0.00060
0.00055
UV - 214nm
calibration101913-027
2-MI
4-MI
imidazole

10. CALIBRATION OF 2-MI IN RANGE OF 10 – 3000 ΜG/L
y = 0.002x - 0.2051
R² = 0.9863
6
5
4
3
2
1
0
peak area ratio
-400 100 600 1100 1600 2100 2600 3100
[2-MI] (μg/L)

11. CALIBRATION OF 4-MI IN RANGE OF 10 – 3000 ΜG/L
y = 0.0015x - 0.1016
R² = 0.9819
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
Peak area ratio
-400 100 600 1100 1600 2100 2600 3100
[4-MI] (μg/L)

12. TABLE 3. ANALYTICAL FIGURE OF MERITS FOR 2-MI AND 4-MI
Analyte Regression equationa R2 LOD (μg L-1) LOQ (μg L-1)
2-MI y = 0.002 x – 0.2051 0.9863 113.9 379.8
4-MI y = 0.0015 x – 0.1016 0.9819 128.4 428.1
a y and x stand for peak area ratio of analyte to the internal standard and concentration
of analyte, respectively

13. TABLE 4. QUANTITIES OF 2-MI AND 4-MI IN FOOD PRODUCTS
sample Concentration (μg/L)
2-MI 4-MI
Pepsi - 164.7
Coca Cola – Zero 128.0 138.3
No Name Artificial Vanilla Extract - 170.1
Shaohsing Cooking Wine ND ND
Oigatsuo Tsuyu Soup Base ND ND
Chinkiang Vinegar ND ND
Guinness – Draught beer ND ND

14. CONCLUSION

2-MI and 4-MI in soft drinks could be detected and analyzed, but in
sauces were difficult to detect because the complexity of
ingeradiants

The LOD of 2-MI and 4-MI were 113.9 μg L-1 and 128.4 μg L-1,
respectively

The LOQ were 379.8 μg L-1 for 2-MI, and 428.1 μg L-1 for 4-MI

15. FUTURE WORK

Use preconcentration or solid-phase extraction to
prepare the real samples and then analyze them
with this method

Build a CE/MS method to quantify and identify 2-
MI and 4-MI in food products

16. ACKNOWLEDGMENT
I would like to thank dr. Kingsley Donkor for all of his
guidance and support in this project; Laiel C. Soliman for her
assistance; as well as the support from the TRU Department
of Chemistry.

17. REFERENCES
1. Petruci, J. F. S., Pereira, E. A., Cardoso, A. A. Determination of
2-methylimidazole and 4-methylimidazole in Caramel Colors by Capillary
Electrophoresis. J. Agric. Food Chem. 2013, 61, 2263−2267.
Thank YOU!!!