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National food safety standard —
Determination of 1-methylimidazole, 2-methylimidazole and 4-methylimidazole in foods
1 Scope
This standard specifies the liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the determination method of 1-methylimidazole, 2-methylimidazole and 4-methylimidazole in foods.
This standard is applicable to the determination of 1-methylimidazole, 2-methylimidazole and 4-methylimidazole in condensed milk, frozen drinks, jams, bean products, cocoa and chocolate products, candies, wheat flour products, biscuits, fillings and surface paste, meat products, seasoning syrup, condiments (i.e., soy sauce, vinegar, sauce and sauce products, cooking wine, compound seasonings), beverages, alcoholic beverages, jellies and puffed foods.
2 Principle
After added with isotope-labeled internal standards (ILISs), the specimen are first extracted with water or acid solution, purified and concentrated with a solid phase extraction column, and then determined by liquid chromatography-tandem mass spectrometer, and quantified by isotope-labeled external standard method.
3 Reagents and materials
Unless otherwise specified, analytical reagents and Class-I water specified in GB/T 6682 are adopted for the purpose of this method.
3.1 Reagent
3.1.1 Acetonitrile (CH3CN): chromatographically pure.
3.1.2 Methanol (CH3O): chromatographically pure.
3.1.3 Formic acid (HCOOH): chromatographically pure.
3.1.4 Ammonia (NH4OH): 25% ~ 28%.
3.1.5 n-Hexane (CH3(CH2)4CH3): chromatographically pure.
3.1.6 Ammonium acetate (CH3COONH4): chromatographically pure.
3.1.7 Sodium chloride (NaCl).
3.2 Preparation of reagents
3.2.1 2% Formic acid aqueous solution: Measure 20 mL of formic acid, add it into 980 mL of water, and mix them well.
3.2.2 Ammonia-methanol solution: Measure 50 mL of ammonia, add it into 950 mL of methanol, and mix them well.
3.2.3 Ammonium acetate solution (5 mmol/L): Weigh 0.39 g of ammonium acetate, dissolve it in 1,000 mL of water and mix them well.
3.2.4 Acetonitrile-ammonium acetate solution (9+1): Mix acetonitrile and ammonium acetate solution (5 mmol/L) well in a volume ratio of 9:1.
3.3 Standards
3.3.1 1-Methylimidazole standard (C4H6N2, CAS No.: 616-47-7): purity ≥ 98%; or a reference material certified by the nation and granted with a reference material certificate.
3.3.2 2-Methylimidazole standard (C4H6N2, CAS No.: 693-98-1): purity ≥ 98%; or a reference material certified by the nation and granted with a reference material certificate.
3.3.3 4-Methylimidazole standard (C4H6N2, CAS No.: 822-36-6): purity ≥ 98%; or a reference material certified by the nation and granted with a reference material certificate.
3.3.4 ILIS 1-methylimidazole-D6 (C4H6N2, CAS No.: 285978-27-0): purity ≥ 98%.
3.3.5 ILIS 2-methylimidazole-D6 (C4H6N2, CAS No.: 1173022-19-9): purity ≥ 98%.
3.3.6 ILIS 4-methylimidazole-D6(C4H6N2, CAS No.: 1219804-79-1): purity ≥ 98%.
3.4 Preparation of standard solutions
3.4.1 Stock standard solution (1 mg/ml): Weigh 25 mg of 1-methylimidazole standard (accurate to 0.01 mg) accurately, add 1 mL of water to dissolve it, transfer it to a 25mL brown volumetric flask with acetonitrile, dilute it to the scale, and shake well. Weigh 25 mg (accurate to 0.01 mg) of 2-methylimidazole and 4-methylimidazole standards accurately, dissolve it with acetonitrile, transfer it to a 25 mL brown volumetric flask, dilute it to the scale, and shake well. The solution shall be stored in the dark place below -20°C, with validity of 6 months.
3.4.2 Mixed standard working solution (2.0 μg/mL): Pipet 100 μL of the stock standard solutions mentioned above into the same 50 mL brown volumetric flask, dilute it with acetonitrile to the scale, shaking well to obtain 2.0 μg/mL of mixed standard solution. The solution shall be stored in the dark place below 4°C, with validity of 3 months.
3.4.3 ILIS stock solution: Weigh 10 mg of 1-methylimidazole-D6, 2-methylimidazole-D6 and 4-methylimidazole-D6 standards accurately (accurate to 1 mg), dissolve them with acetonitrile, transfer to a 10 mL brown volumetric flask, dilute to the scale, and shake well. The solution shall be stored in the dark place below -20°C, with validity of 6 months.
3.4.4 ILIS mixed standard working solution (1.0 μg/mL): Pipet 100 μL of the ILIS stock solutions mentioned above into the same 100 mL brown volumetric flask, dilute it with acetonitrile to the scale, shaking well to obtain 1.0 μg/mL of ILIS mixed standard working solution. The solution shall be stored in the dark place below 4°C, with validity of 3 months.
3.4.5 Standard series solutions: Pipet 25 μL, 75 μL, 150 μL, 300 μL, 600 μL and 1,000 μL of mixed standard working solutions to 5 mL brown volumetric flasks, add 250 μL of ILIS mixed working solution into them respectively, dilute them with acetonitrile-ammonium acetate solution (9+1) to the scale, and prepare 1-methylimidazole, 2-methylimidazole and 4-methylimidazole mixed standard series solutions with mass concentrations of 10 ng/mL, 30 ng/ml, 60 ng/mL, 120 ng/mL, 240 ng/mL and 400 ng/mL. In actual sample determination, the additional amount and dilution multiple of ILIS mixed working solution may be adjusted by sample concentration.
3.5 Materials
3.5.1 Mixed cation exchange solid phase extraction column: 150 mg, 6 mL of N-vinylpyrrolidone-divinylbenzene copolymer matrix-sulfonic acid group, or the equivalent. Activate it with 5 mL of methanol and rinse it with 5 mL of water for balance before use.
3.5.2 Homogenizer: suitable for 50 mL centrifuge tubes.
3.5.3 Organic filter membrane: with an aperture of 0.22 μm.
Note: Materials in 3.5.1, 3.5.2 and 3.5.3 shall be tested before use, and shall not be used until there is no interference.
4 Apparatus
4.1 Liquid chromatography-tandem mass spectrometer: equipped with electrospray ionization (ESI).
4.2 Analytical balance: with sensitivity of 0.001 g and 0.00001 g.
4.3 Pipettor: with the maximum range of 200 μL and 1,000 μL.
4.4 Homogenizing device.
4.5 Vortex mixer.
4.6 Constant temperature water bath.
4.7 Ultrasonic cleaner: with power ≥ 250 W.
4.8 High-speed centrifuge: with a maximum rotation speed ≥10,000 r/min.
4.9 Solid phase extraction unit.
4.10 Nitrogen blowing thickener.
5 Analytical procedures
5.1 Sample pretreatment
5.1.1 Preparation of specimen
Take a proper amount of representative samples, beverage and other liquid uniform samples and shake them directly; uneven samples shall be homogenized or crushed evenly, and frozen beverage shall be stirred well after melting at room temperature, if necessary, heat and stir in 30°C ~ 40°C water bath, grind or homogenize sauces, or melt chocolate at 50°C ~ 60°C, stirring well while it is hot.
5.1 Extraction of specimen
5.1.2.1 Liquid specimen (e.g., soy sauce, vinegar, beverages, alcoholic beverages, etc.)
Weigh 2 g of specimen (accurate to 0.001 g), place them in a 20 mL calibrated colorimetric tube, add 100 μL of 1.0 μg/mL ILIS mixed working solution, dilute with 2% formic acid aqueous solution to 20 mL, shaking well, place them in a centrifuge tube, centrifuge at 1,0000 r/min for 5 min, and the supernatant will be purified.
5.1.2.2 Semi-fluid specimen (such as sauce, oyster sauce, seasoning syrup, frozen beverage) and gum-based candy and powder balls
Weigh 2 g of specimen that has been mixed well (accurate to 0.001 g), place it in a 50 mL centrifuge tube, add 100 μL of 1.0 μg/mL ILIS mixed working solution, mixing well, add 20 mL of water accurately, swirling for 1 min. After heating in 70°C water bath for 10 min, cool to room temperature, perform ultrasonic extraction for 20 min, centrifuge at 10,000 r/min and 4°C for 5 min, and the supernatant will be purified.
5.1.2.3 Jelly
Weigh 2 g of specimen that has been mixed well (accurate to 0.001 g), place it in a 50 mL centrifuge tube, add 100 μL of 1.0 μg/mL ILIS mixed working solution, mixing well, add 2 g of sodium chloride and 20 mL of water accurately, swirling for 1 min. After heating in 70°C water bath for 10 min, cool to room temperature, perform ultrasonic extraction for 20 min, centrifuge at 10,000 r/min and 4°C for 5 min, and the supernatant will be purified.
5.1.2.4 Condensed milk, cocoa and chocolate products
Weigh 2 g of specimen that has been mixed well (accurate to 0.001 g), place it in a 50 mL centrifuge tube, add 100 μL of 1.0 μg/mL ILIS mixed working solution, mixing well, add 20 mL of 2% formic acid aqueous solution accurately. After heating in 60°C water bath for 5 min, add two homogenizers, swirling for 5 min, centrifuge at 10,000 r/min for 5 min, and the supernatant will be purified.
5.1.2.5 Other specimen like soy products, sauce and sauce products
Weigh 2 g of specimen that has been mixed well (accurate to 0.001 g), place it in a 50 mL centrifuge tube, add 100 μL of 1.0 μg/mL ILIS mixed working solution, mixing well, add 20 mL of water accurately. After homogenizing for 1 min, centrifuge at 10,000 r/min and 4°C for 5 min, and the supernatant will be purified.
5.1.3 Purification of specimen
5.1.3.1 Soy sauce, vinegar, beverages, alcohol, condensed milk, cocoa and chocolate products
Pipet 10 mL of supernatant, pass through solid phase extraction column, rinse with 5 mL of 2% formic acid aqueous solution and 5 mL of methanol in turn, and then elute with 10 mL of ammonia-methanol solution. Collect all the eluents, blow with nitrogen below 45°C to nearly dry, and add 1 mL of acetonitrile-ammonium acetate solution (9+1) accurately, swirling for 30 s, pass through 0.22 μm organic filter membrane, and the filtrate will be determined by liquid chromatography-tandem mass spectrometer.
1 Scope
2 Principle
3 Reagents and materials
4 Apparatus
5 Analytical procedures
6 Expression of analysis results
7 Precision
8 Others
Annex A Reference mass spectrometry conditions
Annex B Standards chromatogram
