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Method for the Analysis of 4-methylimidazole by LC/MS and LC/MS/MS

Kahler, Ty W.; Kowalski, Julie; Lupo, Sharon; Lake, Rick; Kozel, Steve; Denicola, Chris Restek Corporation, Bellefonte, PA, USA

4-methylimidazole (4MI or 4-MEI) is a by-product formed during the production of caramel colorings. These colorings are added to many foods including beverages (cola), coffee, beer, whisky, baked goods, soy and Worcestershire sauces and meats. To date, studies have not provided a clear indication as to the toxicity of 4MI but the National Cancer Institute has nominated 4MI as a candidate for toxicity and carcinogenicity studies. In addition, the EFSA is currently considering changing the allowable 4MI levels in food products. The chromatographic analysis of 4MI has traditionally been accomplished by GC/MS with derivatization, RP-HPLC with ion pairing, and now HILIC. None of which are simple or easily reproducible. In this presentation we utilize our Ultra PFPP column to analyze 4MI using typical LC/MS mobile phases, water and methanol with formic acid, and isocratic conditions in ESI+. We also explore the retention mechanism of the PFPP phase that allows the retention of small polar compounds such as 4MI.

Retention Mechanism of PFP Propyl stationary phase

U-Shaped retention profile as organic is varied demonstrates anhydrous normal phase retention mechanism

0

Tunable Retention using Ultra PFP Propyl column

4-methylimidazole with 75% mobile phase B

XIC of +MRM (7 pairs): 82.900/56.100 Da ID: 4-MI 1 from Sample 23 (4MI 10ppb 75_25) of 052611.wiff (Turbo Spray), Smoothed 2.28 7.0e4 6.5e4 6.0e4 5.5e4 5.0e4 4.5e4 4.0e4 3.5e4 3.0e4 2.5e4 2.0e4 Max. 7.1e4 cps.

H3C

2.28 min

NH N

S/N = 60.6

Ymax=4.9e+3 cps Ymin=3.8e+3 cps

Isocratic: 75% B

Peak Int.(Subt.)=6.7e+4

Mobile Phase A: 0.1% Formic Acid in H2O Mobile Phase B: 0.1% Formic Acid in MeOH Flow: 0.4mL/min Injection: 5µL of 10 ppb in mobile phase Temperature: 30ºC Instrument: ABI 4000 (ESI+) MRM transitions 83 -56, 83-42, 83-68

30

At low pH, imidazole ring has a positive (+) charge, making retention by traditional RPLC difficult or impossible

%B MeOH

60

Intensity, cps

1.5e4 1.0e4 5000.0 0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0 Time, min

4.5

5.0

5.5

6.0

6.5

7.0

7.5

Figure 3. 4-methylimidazole on an Ultra PFP Propyl 50mm x 2.1mm, 3µm column

90 0 Time (min) 15

4-methylimidazole with 85% mobile phase B

XIC of +MRM (7 pairs): 82.900/56.100 Da ID: 4-MI 1 from Sample 28 (4MI 100ppb 85_15) of 052611.wiff (Turbo Spray), Smoothed 2.6e5 2.5e5 2.4e5 2.3e5 2.2e5 Max. 2.6e5 cps.

3.96 min

By increasing the % organic in the mobile phase, we utilize the aqueous normal phase retention mechanism to increase retention

3.98

Ymax=6.7e+3 cps Ymin=4.8e+3 cps Isocratic: 85% B

S/N = 134.2 Peak Int.(Subt.)=2.5e+5

Figure 1. Retention profile of 4-MI on PFP Propyl phase ­ UV at 214nm 4.6 x 150, 5µm by varying organic

Intensity, cps

2.1e5 2.0e5 1.9e5 1.8e5 1.7e5 1.6e5 1.5e5 1.4e5 1.3e5 1.2e5 1.1e5 1.0e5 9.0e4 8.0e4 7.0e4 6.0e4 5.0e4 4.0e4 3.0e4 2.0e4 1.0e4 0.0

Mobile Phase A: 0.1% Formic Acid in H2O Mobile Phase B: 0.1% Formic Acid in MeOH Flow: 0.4mL/min Injection: 5µL of 100 ppb in mobile phase Temperature: 30ºC Instrument: ABI 4000 (ESI+)

Decrease in acid concentration causes increase in retention of charged-species, demonstrates cation-exchange mechanism

The same increase in retention can be done by decreasing the acid concentration in the mobile phase, for this we utilize the cation-exchange mechanism of the phase

MRM transitions 83 -56, 83-42, 83-68

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0 Time, min

4.5

5.0

5.5

6.0

6.5

7.0

7.5

Figure 4. 4-methylimidazole on an Ultra PFP Propyl 50mm x 2.1mm, 3µm column with increased organic mobile phase

RESULTS and CONCLUSIONS: ·PFP Propyl allows tunable retention based on % of organic in mobile phase as well as acid concentration for 4MI ·Method is simple as it does not require derivatization, ion-pairing or HILIC. ·Charged state of imidazole is utilized to aid retention.

Figure 2. Retention profile of 4-MI on PFP Propyl phase and the effects of acid concentration on retention

XIC of +MRM (7 pairs): 231.300/153.000 Da ID: THI 2 from Sample 29 (4MI THI 100ppb 75_25) of 052611.wiff (Turbo Spray), Smoothe... 3.0e5 2.8e5 2.6e5 2.4e5 2.2e5 2.0e5 1.8e5 Intens cp ity, s 1.6e5 1.4e5 1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 2.0e4 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 Time, min 4.5 5.0 5.5 6.0 6.5 7.0 7.5 Max. 2.9e5 cps. 0.44

THI 0.44 min

4-MI 2.27 min

·A 2ppb standard of 4MI was tested and resulting S/N of approximately 10 ·Additional studies need to be conducted on potential carcinogen 2acetyl-4(5)-tetrahydroxybutylimidazole (THI)

Figure 5. 4-methylimidazole (4MI) and 2-acetyl-4(5)tetrahydroxybutylimidazole (THI) on an Ultra PFP Propyl 50mm x 2.1mm, 3µm column. Same conditions used in Figure 3.

The goal of this work was to utilize the unique retention properties of the PFP Propyl phase to analyze the potential carcinogen 4-methylimidazole

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Method for the Analysis of 4-methylimidazole by LC/MS and LC/MS/MS