Detection of 4-methylimidazole, a carcinogenic risk substance in beverages

Detection of 4-methylimidazole, a carcinogenic risk substance in beverages
In mid-February 2012, the US Consumer Advocacy Organization's Center for Public Interest Science (CSPI) published a statement claiming that high levels of chemical 4-methylimidazole were found in cola beverages.
4-methylimidazole is a chemical found in caramelides. It is produced when caramel is produced. Caramel can turn the cola drink into a brown color. In the experiment, it was found that 4-methylimidazole can cause long tumors in animals and may cause cancer risk to the human body.
The World Health Organization allows the addition of 4-methylimidazole-containing caramel color to foods, and the dose of 4-methylimidazole is defined as no more than 200 mg per kilogram. The People's Republic of China standard GB 8817-2001 "food additive caramel color (ammonium sulfite method, ammonia method, common law)" stipulates that 4-methylimidazole content in liquid food is less than 0.02%, that is, per kilogram of liquid 4- The methyl imidazole content is less than 200 mg, which is the same as the requirements of the World Health Organization.
In the standard GB 8817-2001 "food additive caramel color (ammonium sulfite method, ammonia method, common method)", the reagents and equipment used in the experiment are required to be analytically pure reagent, distilled water or deionized unless otherwise specified. Water and laboratory equipment. The determination of 4-methylimidazole is mainly carried out by thin layer chromatography.
Thin layer chromatography is fast and simple. However, compared with liquid or gas chromatography, there is a possibility of qualitative error. If the substance to be tested is not the same substance as the standard substance, and their retention time is the same, a qualitative error will occur. Therefore, many places choose to use liquid chromatography to detect 4-methylimidazole, and the corresponding local standards have been introduced. The detection method will be described below according to the Hebei Provincial Local Standard DB13/T 1116-2009 "Determination of 4-Methylimidazole in Caramel Color by High Performance Liquid Chromatography".
Analysis step
1 sample processing
Accurately weigh 3g sample (accurate to 0.001 g), add 15mL water to dissolve, transfer to 250mL separatory funnel, add 100g/L sodium carbonate solution 15mL, add chloroform-anhydrous ethanol extract 60mL Shake vigorously for 5 min. After standing to separate the layers, the organic phase was transferred to another separatory funnel, and the aqueous phase was extracted once again in the same manner, and the organic phases were combined; the extract was washed three times with 30 g/L sodium carbonate solution, 30 mL each time. Then, the organic phase was filtered through a sodium sulfate-free solution into a rotary evaporation flask, concentrated to near dryness at 50 ° C, and taken up, dissolved in water and made to a volume of 10 mL, filtered through a 0.45 μm microporous membrane for HPLC analysis.
2 determination
2.1 HPLC reference conditions
Analytical column: C18, 4. 6 mm × 250 mm particle size 5 μm.
Mobile phase: methanol: 0. 05 mol/L potassium dihydrogen phosphate buffer ((pH 3. 5, containing 0.1% sodium heptanesulfonate) = 5: 95.
Wavelength: 210 nm.
Flow rate: 1.0 mL/min.
2.2 Standard curve: 10 μL of each standard solution was separately analyzed by HPLC, and then the standard curve was drawn by taking the peak area as the ordinate and the content of 4-methylimidazole as the abscissa.
2.3 Determination of sample: Pipette 10 μL of the sample solution after 6.1 treatment for HPLC analysis, determine the peak area, and check the content of 4-methylimidazole in the determination solution from the standard curve.
Result calculation
The content of 4-methylimidazole in the sample is calculated by the following formula:
In the formula:
The content of 4-methylimidazole in X-sample, the unit is milligrams per kilogram (mg/kg);
C1 The amount of 4-methylimidazole in the injection solution was found on the standard curve in micrograms per milliliter (μg/mL);
V-sample dilution total volume in milliliters (mL);
m a sample mass in grams (g);
The calculation results retain two significant digits.
When using liquid chromatography, it is required to use the first grade water in accordance with the People's Republic of China standard GB 6682-2008. The configuration of the Other various standard solutions requires a minimum of secondary water. Ruifeng specializes in the combination of Direct-Pure EDI and PURIST to provide high purity water and ultrapure water for a full range of laboratory water needs. Direct-Pure EDI high-purity water system uses Siemens's EDI, which uses tap water as the influent water to provide stable and high-quality high-purity water, and the products have reached the advanced level of similar products. PURIST series ultra-pure water system is exquisite and compact, easy to use; it uses pure water as water to produce ultra-pure water. The water quality exceeds or meets the GB and ASTM, CAP, CLSI first-grade water standards. The combination package can ensure users have no worries about water use. Ruifeng pure water system has passed various certifications such as FCC and CE. At the same time, it has professional verification engineers, provides the required verification service support, and passes the 3Q certification required by various standards to help users to pass GMP and GLP. System certification, allowing users to enjoy professional and thoughtful service while using high quality products.
In order to better protect the HPLC instrument, the solution should be pre-filtered before loading to remove particles and microorganisms in the solution to prevent blockage of the column and damage to the pump, thus ensuring the operating performance of the instrument and prolonging the service life of the instrument. To this end, Ruifeng offers a variety of needle filters such as PES, PTFE, Nylon, MCE, etc., which are suitable for sample filtration of various organic and aqueous phases. For details, please refer to the official website of Ruifeng: http://
According to experts, although 4-methylimidazole is relatively harmful, as long as it is a substance in the regular food additive caramel, the content is within the allowable range, then it will not cause much damage to the human body. According to people's normal consumption level, if 4-methylimidazole does not exceed the daily allowable intake of the human body, the conclusion that it is harmful to health cannot be obtained. To date, no clinical reports of 4-methylimidazole in human food have been seen.

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