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

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

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

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)

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

CE OPERATION SCHEMATIC

TABLE 2. OPTIMIZED CONDITIONS

UV detection wavelength 214 nmpH of buffer 7.7Concentration of β-cyclodextrin in buffer 3.0 mMConcentration of sodium phosphate in buffer

70 mM

Water (solution/buffer preparation) LC/MS grade waterSolution/sample filter 0.45 μm Nylon

EXPERIMENTAL

All final optimized conditions selected were based on sensitivities of 2-MI and 4-MI, and resolutions of both and the internal standard, imidazole.

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

RESULTSELECTROPHEROGRAM OF A STANDARD SOLUTION (1000 ΜG/L OF 2-MI AND 4-MI)

Minutes

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

AU

0.00055

0.00060

0.00065

0.00070

0.00075

0.00080

0.00085

0.00090

AU

0.00055

0.00060

0.00065

0.00070

0.00075

0.00080

0.00085

0.00090

UV - 214nmcalibration101913-027

2-MI

4-MI

imidazole

CALIBRATION OF 2-MI IN RANGE OF 10 – 3000 ΜG/L

-400 100 600 1100 1600 2100 2600 31000

1

2

3

4

5

6

f(x) = 0.00202011811388056 x − 0.205109496969742R² = 0.986309188294667

[2-MI] (μg/L)

peak

are

a ra

tio

CALIBRATION OF 4-MI IN RANGE OF 10 – 3000 ΜG/L

-400 100 600 1100 1600 2100 2600 31000

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

f(x) = 0.00152052891297152 x − 0.10156953794097R² = 0.98194323452532

[4-MI] (μg/L)

Peak

are

a ra

tio

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

TABLE 4. QUANTITIES OF 2-MI AND 4-MI IN FOOD PRODUCTS

sample Concentration (μg/L)2-MI 4-MI

Pepsi - 164.7Coca Cola – Zero 128.0 138.3

No Name Artificial Vanilla Extract - 170.1Shaohsing Cooking Wine ND ND

Oigatsuo Tsuyu Soup Base ND NDChinkiang Vinegar ND ND

Guinness – Draught beer ND ND

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

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

ACKNOWLEDGMENTI 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.

REFERENCES1. 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!!!