A method for simultaneous determination of six Maillard reaction products in rice and flour products for infant complementary foods

Abstract

The present invention relates to the technical field of detection of infant and young children's complementary food rice and flour products, and in particular to a method for simultaneously determining 6 kinds of Maillard reaction products in infant and young children's complementary food rice and flour products. After the sample of the present invention is extracted with water and oxalic acid solution and protein is precipitated with trichloroacetic acid, it is separated by Eclipse XDB-C18 chromatographic column, acetonitrile-0.1% trifluoroacetic acid solution is used as the mobile phase, gradient elution is performed at a flow rate of 0.6 mL / min, diode array detector is detected, and external standard method is quantitatively determined. The linear range of the 6 components is 0.2~16.0 mg / L, the correlation coefficient is greater than 0.999, and the quantitative limit of the method is 250~1000μg / 100g (S / N=10). The recovery rate of the 6 components ranges from 71.2% to 100%, and the relative standard deviation is 0.9% to 4.9%. The method has high sensitivity and good separation, and is suitable for the simultaneous quantitative detection of Maillard reaction products in infant and young children's complementary food rice and flour products.

Description

Technical Field

[0001] The invention relates to the technical field of detection of rice and flour products for infant complementary food, and in particular to a method for simultaneously determining six Maillard reaction products in rice and flour products for infant complementary food. Background Art

[0002] The Maillard reaction is a natural reaction between carbonyl compounds and amino compounds in food. It is widely present in the thermal processing, storage and transportation of food and is one of the main sources of food flavor substances. Studies have shown that the Maillard reaction will occur during the thermal processing or high-temperature storage of food, and produce furosine, furfural compounds, acrylamide, etc. Among them, furfural compounds are mainly composed of 4 compounds: 5-hydroxymethylfurfural, furfural, furan methyl ketone and methylfurfural. During the Maillard reaction, some amino acids in food will be degraded and some substances that damage nutrition will be produced, some of which may be toxic. For example, 5-hydroxymethylfurfural has been proven to be neurotoxic and can enter the human body through inhalation or skin contact, and accumulate with human proteins to cause poisoning. Therefore, the study of Maillard reaction products has become one of the focuses of food research.

[0003] At present, the methods for detecting Maillard reaction products in food include spectrophotometry, high performance liquid chromatography, liquid chromatography-tandem mass spectrometry, gas chromatography, gas chromatography-mass spectrometry, etc. Among them, the pre-treatment of spectrophotometry is complex and the sensitivity is low, which cannot meet the needs of trace analysis; liquid chromatography-tandem mass spectrometry has high accuracy, but the instrument is expensive and the cost is high; gas chromatography requires pre-column derivatization and the operation is cumbersome; high performance liquid chromatography is the most commonly used instrumental analysis method and is widely used in the analysis and detection of toxic and harmful substances in food. For the detection of Maillard reaction products in food, most of the literature reports on the detection of dairy products, condiments, royal jelly and other foods. At present, the detection and research of Maillard reaction products in rice and flour products is relatively rare. Summary of the invention

[0004] In order to solve the above-mentioned technical problems, the purpose of the present invention is to provide a quantitative, accurate and simple high performance liquid chromatography method for simultaneously determining six Maillard reaction products, namely furanone, 5-hydroxymethylfurfural, furfural, furanone, furanmethyl ketone and methylfurfural, in infant and young children's complementary rice and flour products, and to optimize the pretreatment and liquid chromatography conditions to fill the gap in the detection of Maillard reaction products in infant and young children's complementary rice and flour products, so as to provide technical support for studying the production rules of furanone, furanone and furfural substances during the processing, storage and transportation of infant and young children's complementary rice and flour products, and then evaluating the quality and freshness of rice and flour products.

[0005] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions:

[0006] A method for simultaneously determining six Maillard reaction products in infant complementary food rice and flour products, characterized in that the six Maillard reaction products are furosine, furanone, 5-hydroxymethylfurfural, furfural, furan methyl ketone and methylfurfural; the method comprises the following steps:

[0007] 1) Sample pretreatment

[0008] Weigh 2g of the sample, accurate to 0.0001g, place it in a 50mL centrifuge tube, add 12mL of water, vortex mix, ultrasonically extract for 30min, and shake for 20min; add 5mL of 0.15mol / L oxalic acid solution, shake for 20min; add 5mL of 40g / 100mL trichloroacetic acid solution, shake for 10min, centrifuge at 4000r / min for 5min, and take the supernatant to a 25mL volumetric flask; add 5mL of 4g / 100mL trichloroacetic acid solution to the remaining residue, vortex mix, shake for 10min, centrifuge at 4000r / min for 5min, combine the supernatant, add 4g / 100mL trichloroacetic acid solution to the scale, shake well, take an appropriate amount of solution and filter through a 0.45μm filter membrane for analysis by high performance liquid chromatography;

[0009] 2) Chromatographic conditions

[0010] Chromatographic column: Agilent Eclipse XDB-C18, 150 mm × 4.6 mm, 5 μm; mobile phase: acetonitrile-0.1% trifluoroacetic acid solution gradient elution; column temperature: 30 ° C; flow rate: 0.6 mL / min; detection wavelength: 280 nm; injection volume: 5 μL;

[0011] The gradient elution procedure is as follows:

[0012] Time min Acetonitrile% 0.1% trifluoroacetic acid solution% 0 1 99 5 1 99 10 10 90 15 15 85 20 50 50 25 1 99 30 1 99

[0013] Preferably, the preparation method of 10 mg / mL standard stock solution is as follows: accurately weigh 0.1000 g, accurate to 0.1 mg, of furosine, furfural, 5-hydroxymethylfurfural, 2-acetylfuran, and 5-methylfurfural standards, fully dissolve them with water and dilute to a 10 mL volumetric flask to prepare a standard stock solution with a concentration of 10 mg / mL; weigh 0.1000 g, accurate to 0.1 mg, of furanone, fully dissolve it with methanol and dilute to a 10 mL volumetric flask to prepare a standard stock solution with a concentration of 10 mg / mL, and store it in a refrigerator at 4°C.

[0014] Preferably, the preparation method of 100 μg / mL standard intermediate solution is as follows: accurately pipette 100 μL of each of the above standard stock solutions into 10 mL volumetric flasks, add water to make up to volume, and prepare a standard intermediate solution with a concentration of 100 μg / mL.

[0015] Preferably, the preparation method of the standard working solution is as follows: accurately pipette a certain amount of the above-mentioned standard intermediate solution, dissolve it with an aqueous solution and dilute it step by step to a series of standard working solutions of 0.2, 0.4, 0.8, 2.0, 4.0 μg / mL furfural, 5-hydroxymethylfurfural, 2-acetylfuran, 5-methylfurfural, 0.4, 0.8, 1.6, 4.0, 8.0 μg / mL furanone, 0.8, 1.6, 3.2, 8.0, 16 μg / mL furosine, and store it in a refrigerator at 1°C to 4°C away from light. The validity period is 3 months.

[0016] Preferably, the method further comprises sequentially entering the prepared series of standard working solutions into liquid chromatography for detection, and performing linear regression with mass concentration as the abscissa (X) and peak area as the ordinate (Y). The obtained linear range, linear equation, correlation coefficient and quantitative limit are as follows:

[0017] Compound Linear range mg / L Linear equations Correlation coefficient Quantitative limit μg / 100g Furosine 0.8-16.0 Y=0.0003X 0.9995 1000 5-Hydroxymethylfurfural 0.2-4.0 Y=0.0008X 0.9992 250 Furfural 0.2-4.0 Y=0.0001X 0.9992 250 Furanone 0.4–8.0 Y=0.0003X 0.9992 500 Furanyl methyl ketone 0.2-4.0 Y=0.0007X 0.9991 250 Methylfurfural 0.2-4.0 Y=0.0009X 0.9995 250

[0018] Preferably, the method further comprises the method of adding standard solutions to blank flour samples not containing Maillard reaction products to determine the recovery rate and precision of the method, wherein the addition levels of furosine are 1000, 2000, and 10000 μg / 100g, respectively, the addition levels of 5-hydroxymethylfurfural, furfural, furan methyl ketone, and methylfurfural are 250, 500, and 2500 μg / 100g, respectively, and the addition levels of furanone are 500, 1000, and 5000 μg / 100g, respectively, and the determination is performed in parallel 6 times to calculate the spiked recovery rate and RSD; the recovery rates of the 6 target compounds range from 71.2% to 100%, and the RSD ( n =6) ranges from 0.9% to 4.9%.

[0019] The present invention adopts the above-mentioned technical scheme to establish a method for simultaneously determining furosine, furanone and four furfural compounds (5-hydroxymethylfurfural, furfural, furan methyl ketone, methylfurfural) in infant and young children's complementary rice and flour products by high performance liquid chromatography-diode array detection. After the sample is extracted with water and oxalic acid solution and the protein is precipitated with trichloroacetic acid, it is separated by EclipseXDB-C18 chromatographic column, acetonitrile-0.1% trifluoroacetic acid solution is used as the mobile phase, gradient elution is performed at a flow rate of 0.6mL / min, and the diode array detector is used for detection. The external standard method is quantitative. The linear range of the six components is 0.2~16.0mg / L, the correlation coefficient is greater than 0.999, and the quantitative limit of the method is 250~1000μg / 100g (S / N=10). The recovery rate of the six components ranges from 71.2% to 100%, and the relative standard deviation is 0.9% to 4.9%. This method has high sensitivity and good separation, and is suitable for the simultaneous quantitative detection of Maillard reaction products in rice and flour products for infant and young children. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The effect of four chromatographic columns on the separation of six analytes; A. EclipseXDB-C 18 ; B.ZorbaxSB-C 18 ; C.EclipsePlus-C 18 ;D.ThermoAcclaim TM 120-C 18 ; Chromatographic peaks: 1. furosine; 2. 5-hydroxymethylfurfural; 3. furfural; 4. furanone; 5. furan methyl ketone; 6. methyl furan.

[0021] Figure 2 The effect of four mobile phases on the separation of six analytes: A. acetonitrile-water; B. acetonitrile-0.1% trifluoroacetic acid solution; C. acetonitrile-0.5% trifluoroacetic acid solution; D. acetonitrile-1.0% trifluoroacetic acid solution; Chromatographic peaks: 1. furosine; 2. 5-hydroxymethylfurfural; 3. furfural; 4. furanone; 5. furan methyl ketone; 6. methylfurfural.

[0022] Figure 3 The chromatograms of the standard solution, blank flour sample and added recovery are shown in Figure 2. A. Standard solution, B. Blank sample, C. Added recovery spectrum. Chromatographic peaks: 1. Furosine; 2. 5-hydroxymethylfurfural; 3. Furfural; 4. Furanone; 5. Furan methyl ketone; 6. Methylfurfural. DETAILED DESCRIPTION

[0023] Experimental Section

[0024] 1.1 Instruments, materials and reagents

[0025] High performance liquid chromatograph (Thermo Fisher Scientific, USA, equipped with DAD detector); P300H ultrasonic cleaner (Elmasonic, Germany); MultifugeX1R high-speed refrigerated centrifuge (Thermo, USA); MMS-3020 constant temperature oscillator (Tokyo Rika, Japan); JJ100B electronic balance (Shuangjie Balance, USA); AE260 electronic balance (Mettler Toledo, Switzerland); WH-861 vortex mixer (Taicang Hualida Experimental Equipment Co., Ltd.); Synergy185 ultrapure water instrument (Millipore, USA); microporous filter membrane (0.45µm, aqueous phase); AgilentEclipseXDB-C 18 (150mm×4.6mm, 5μm)

[0026] (Agilent Technologies (China) Co., Ltd., USA); AgilentZorbaxSB-C 18(150 mm × 4.6 mm, 5 μm) (Agilent Technologies (China) Co., Ltd., USA); Agilent Eclipse Plus-C 18 (150mm×3.0mm, 3.5μm) (Agilent Technologies (China) Co., Ltd., USA); ThermoAcclaim TM 120-C 18 (150mm×4.6mm, 5μm) (Thermo Corporation, USA).

[0027] Trifluoroacetic acid (analytical grade, Zhejiang Changqing Chemical Co., Ltd.); trichloroacetic acid (analytical grade, Zhejiang Changqing Chemical Co., Ltd.); oxalic acid (analytical grade, Zhejiang Changqing Chemical Co., Ltd.); water was ultrapure water; other reagents used in the experiments were of analytical grade unless otherwise specified.

[0028] Standards: furosine (purity 99.7%), furfural (purity 99.7%), all purchased from Bepure; 5-hydroxymethylfurfural (purity 99.0%), 2-acetylfuran (purity 99.9%), 4-hydroxy-2,5-dimethyl-3-(2H)furanone (purity 99.7%), 5-methylfurfural (purity 99.9%), all purchased from ANPEL;

[0029] 1.2 Standard solution preparation

[0030] Standard stock solution (10 mg / mL): Accurately weigh 0.1000 g (accurate to 0.1 mg) of furosine, furfural, 5-hydroxymethylfurfural, 2-acetylfuran, and 5-methylfurfural standards, fully dissolve them in water and dilute to a 10 mL volumetric flask to prepare a standard stock solution with a concentration of 10 mg / mL; weigh 0.1000 g (accurate to 0.1 mg) of furanone, fully dissolve it in methanol and dilute to a 10 mL volumetric flask to prepare a standard stock solution with a concentration of 10 mg / mL, and store it at 4°C.

[0031] Standard intermediate solution (100 μg / mL): Accurately pipette 100 μL of each of the above standard stock solutions into 10 mL volumetric flasks, add water to make up to volume, and prepare a standard intermediate solution with a concentration of 100 μg / mL.

[0032] Standard working solution: Accurately pipette a certain amount of the above standard intermediate solutions respectively, dissolve them with aqueous solution and dilute them step by step to a series of standard working solutions of 0.2, 0.4, 0.8, 2.0, 4.0 μg / mL (furfural, 5-hydroxymethylfurfural, 2-acetylfuran, 5-methylfurfural), 0.4, 0.8, 1.6, 4.0, 8.0 μg / mL (furanone), 0.8, 1.6, 3.2, 8.0, 16 μg / mL (furonic acid), and store them in a refrigerator at 1℃~4℃ away from light. The validity period is 3 months.

[0033] Sample preparation

[0034] Weigh 2g of the sample (accurate to 0.0001g) in a 50mL centrifuge tube, add 12mL of water, vortex mix, ultrasonically extract for 30min, and shake for 20min; add 5mL of 0.15mol / L oxalic acid solution, shake for 20min; add 5mL of 40g / 100mL trichloroacetic acid solution, shake for 10min, centrifuge at 4000r / min for 5min, and take the supernatant to a 25mL volumetric flask. Add 5mL of 4g / 100mL trichloroacetic acid solution to the remaining residue, vortex mix, shake for 10min, centrifuge at 4000r / min for 5min, combine the supernatant, add 4g / 100mL trichloroacetic acid solution to the scale, shake well, take an appropriate amount of solution and filter through a 0.45μm filter membrane for analysis by high performance liquid chromatography.

[0035] Chromatographic conditions

[0036] Chromatographic column: Agilent Eclipse XDB-C 18 (150mm×4.6mm, 5μm); mobile phase: acetonitrile-0.1% trifluoroacetic acid solution (gradient elution program see Table 1); column temperature: 30℃; flow rate: 0.6mL / min; detection wavelength: 280nm; injection volume: 5μL.

[0037] Table 1 Gradient elution program

[0038] Time min Acetonitrile% 0.1% trifluoroacetic acid solution% 0 1 99 5 1 99 10 10 90 15 15 85 20 50 50 25 1 99 30 1 99

[0039] 2. Results and Discussion

[0040] 2.1 Optimization of extraction conditions

[0041] Since temperature has a great influence on the production of Maillard reaction products, the present invention investigates the influence of water bath temperature and time on the recovery rate during the pretreatment process. The addition recovery experiment was carried out by adding standard solution to 4 blank samples respectively, and the hydrolysis extraction experiments of 6 compounds at different water bath temperatures of 30, 60, 80, 100°C and water bath times of 0, 10, 30, 60min were investigated respectively. The results showed that water bath heating would produce 5-hydroxymethylfurfural in blank samples without target compounds, and with the increase of water bath temperature and time, the content of 5-hydroxymethylfurfural produced would gradually increase, but the other 5 target compounds were not produced in the blank samples, and the recovery rate of 6 target compounds in the addition recovery samples was not significantly different. Therefore, the present invention chose not to perform water bath heating extraction during the extraction process, and adopted normal temperature ultrasonic oscillation extraction.

[0042] Column selection

[0043] The most commonly used chromatographic column for Maillard reaction product detection is C 18 In order to achieve the simultaneous separation of six Maillard reaction products, this experiment compared the EclipseXDB-C 18 、ZorbaxSB-C 18 、EclipsePlus-C 18 and Acclaim TM 120-C 18 The separation effect of the column, the results show that the EclipseXDB-C 18 When the chromatographic column was used, under the optimized gradient elution procedure, each component was well separated, and the chromatographic peaks were symmetrical and sharp ( Figure 1 A); Select ZorbaxSB-C 18 and Acclaim TM 120-C 18 When the chromatographic column was used, all components could flow out of the chromatographic column and all showed chromatographic peaks on the chromatogram. However, the separation of the two chromatographic peaks at 17 minutes was poor, and baseline separation could not be achieved. The baseline at 22 minutes was elevated, which affected the integral quantification of the target compound ( Figure 1 B and Figure 1 D); Choose Plus-C 18 When the chromatographic column was used, the analysis time was short, the components were not completely eluted, and only 5 chromatographic peaks were shown on the chromatogram ( Figure 1 C), when analyzing and measuring actual samples, the first chromatographic peak is easily interfered by sample impurities. Therefore, EclipseXDB-C was selected for the experiment. 18 The column was used as the analytical column.

[0044] Choice of mobile phase

[0045] The experiment investigated the separation of 6 target compounds using different mobile phases: acetonitrile-water, acetonitrile-0.1% trifluoroacetic acid solution, acetonitrile-0.5% trifluoroacetic acid solution, and acetonitrile-1.0% trifluoroacetic acid solution. The results showed that when acetonitrile-water system was used as the mobile phase, the peak time of furosine was 2.25min, which was easily interfered by impurities in the sample, and the separation of the two chromatographic peaks at 7min was poor, and baseline separation could not be achieved ( Figure 2 A). The experiment found that adding a small amount of trifluoroacetic acid to the mobile phase would extend the retention time of the target compound accordingly. When using acetonitrile-0.5% trifluoroacetic acid solution and acetonitrile-1.0% trifluoroacetic acid solution, the baselines of the two chromatographic peaks at 13min and 14min were not completely separated ( Figure 2 C and Figure 2 D), while when acetonitrile-0.1% trifluoroacetic acid solution was selected, the chromatographic peak shape and separation of the 6 target compounds were optimal ( Figure 2B). Considering the separation degree, chromatographic peak shape and analysis time, the acetonitrile-0.1% trifluoroacetic acid solution gradient was selected as the mobile phase.

[0046] Standard curve, linear range and limit of quantitation

[0047] The 10 mg / mL standard solutions of various furfurals were diluted in sequence to prepare standard working solutions with mass concentrations of 0.2, 0.4, 0.8, 2.0, 4.0 mg / L (furfural, 5-hydroxymethylfurfural, 2-acetylfuran, 5-methylfurfural), 0.4, 0.8, 1.6, 4.0, 8.0 mg / L (furanone), and 0.8, 1.6, 3.2, 8.0, 16 mg / L (furonic acid). Under the optimized best chromatographic conditions, the prepared series of standard solutions were sequentially subjected to liquid chromatography detection, and linear regression was performed with mass concentration as the abscissa (X) and peak area as the ordinate (Y). The obtained linear range, linear equation, correlation coefficient and quantification limit are shown in Table 2.

[0048] Table 2 Linear range, linear equation, correlation coefficient and quantification limit of each compound

[0049] Compound Linear range / (mg / L) Linear equations Correlation coefficient Limit of quantification / (μg / 100g) Furosine 0.8-16.0 Y=0.0003X 0.9995 1000 5-Hydroxymethylfurfural 0.2-4.0 Y=0.0008X 0.9992 250 Furfural 0.2-4.0 Y=0.0001X 0.9992 250 Furanone 0.4–8.0 Y=0.0003X 0.9992 500 Furanyl methyl ketone 0.2-4.0 Y=0.0007X 0.9991 250 Methylfurfural 0.2-4.0 Y=0.0009X 0.9995 250

[0050] 2.5 Recovery rate

[0051] The recovery rate and precision of the method were determined by adding standard solutions to blank flour samples without Maillard reaction products. The addition levels of furosine were 1000, 2000, and 10000 μg / 100 g, respectively. The addition levels of 5-hydroxymethylfurfural, furfural, furan methyl ketone, and methylfurfural were 250, 500, and 2500 μg / 100 g, respectively. The addition levels of furanone were 500, 1000, and 5000 μg / 100 g, respectively. The determination was repeated 6 times, and the spiked recovery rate and RSD were calculated. The relevant chromatograms are shown in Figure 2. Figure 3 The results are shown in Table 3. The recoveries of the six target compounds ranged from 71.2% to 100%, with RSD ( n =6) ranged from 0.9% to 4.9%. The recovery and precision meet the requirements of SANTE / 12682 / 2019

[30] , can meet the analytical requirements of infant and young children's complementary rice and flour products, and can be used for daily analysis.

[0052] Table 3 Spiked recoveries and relative standard deviations of 6 analytes in blank flour samples ( n =6)

[0053]

[0054] 2.6 Determination of actual samples

[0055] In order to investigate the effectiveness and practicality of this method, the established method was applied to detect six Maillard compounds in 67 randomly selected samples of rice and flour products for infants and young children (including 12 samples of rice flour, 22 samples of noodles, and 33 samples of biscuits). The results showed that 5-hydroxymethylfurfural was detected in two biscuit samples, with contents of 335μg / 100g and 436μg / 100g, respectively, and no other samples were detected.

Claims

1. A method for simultaneously determining six Maillard reaction products in infant and young children's complementary rice and flour products, characterized in that: The six Maillard reaction products are furosine, furanone, 5-hydroxymethylfurfural, furfural, furan methyl ketone and methylfurfural; the method comprises the following steps: 1) Sample pretreatment Weigh 2 g of the sample to an accuracy of 0.0001 g, place it in a 50 mL centrifuge tube, add 12 mL of water, vortex mix, ultrasonically extract for 30 min, and shake for 20 min; add 5 mL of 0.15 mol / L oxalic acid solution and shake for 20 min; add 5 mL of 40 g / 100mL trichloroacetic acid solution, shake for 10 min, centrifuge at 4000 r / min for 5 min, and take the supernatant to a 25 mL volumetric flask; add 5 mL of 4 g / 100mL trichloroacetic acid solution to the remaining residue, vortex mix, shake for 10 min, centrifuge at 4000 r / min for 5 min, combine the supernatant, add 4 g / 100mL trichloroacetic acid solution to the scale, shake well, take an appropriate amount of solution and filter it through a 0.45 μm filter membrane for analysis by high performance liquid chromatography; 2) Chromatographic conditions Column: Agilent Eclipse XDB-C 18 , 150 mm×4.6 mm, 5 μm; mobile phase: acetonitrile-0.1% trifluoroacetic acid solution gradient elution; column temperature: 30 ℃; flow rate: 0.6 mL / min; detection wavelength: 280 nm; injection volume: 5 μL; The gradient elution procedure is as follows: 。 2. The method for simultaneously determining six Maillard reaction products in infant and young children's complementary rice and flour products according to claim 1, characterized in that: The preparation method of 10 mg / mL standard stock solution is as follows: accurately weigh 0.1000 g, accurate to 0.1 mg, of furosine, furfural, 5-hydroxymethylfurfural, 2-acetylfuran, and 5-methylfurfural standards, fully dissolve them in water and dilute to a 10 mL volumetric flask to prepare a standard stock solution with a concentration of 10 mg / mL; weigh 0.1000 g, accurate to 0.1 mg, of furanone, fully dissolve it in methanol and dilute to a 10 mL volumetric flask to prepare a standard stock solution with a concentration of 10 mg / mL, and store it in a refrigerator at 4 °C.

3. The method for simultaneously determining six Maillard reaction products in infant and young children's complementary rice and flour products according to claim 2, characterized in that: The preparation method of 100 μg / mL standard intermediate solution is as follows: accurately pipette 100 μL of each of the above standard stock solutions into 10 mL volumetric flasks, add water to make up to the volume, and prepare a standard intermediate solution with a concentration of 100 μg / mL.

4. The method for simultaneously determining six Maillard reaction products in infant and young children's complementary rice and flour products according to claim 3, characterized in that: The preparation method of the standard working solution is as follows: accurately pipette a certain amount of the above standard intermediate solution respectively, dissolve it with aqueous solution and dilute it step by step to a series of standard working solutions of 0.2, 0.4, 0.8, 2.0, 4.0 μg / mL furfural, 5-hydroxymethylfurfural, 2-acetylfuran, 5-methylfurfural, 0.4, 0.8, 1.6, 4.0, 8.0 μg / mL furanone, and 0.8, 1.6, 3.2, 8.0, 16 μg / mL furosine, and store it in a refrigerator at 1°C to 4°C away from light. The validity period is 3 months.

5. The method for simultaneously determining six Maillard reaction products in infant and young children's complementary rice and flour products according to claim 4, characterized in that: The method also includes sequentially placing the prepared series of standard working solutions into liquid chromatography for detection, and performing linear regression with mass concentration as the abscissa X and peak area as the ordinate Y. The obtained linear range, linear equation, correlation coefficient and quantitative limit are as follows: 。 6. The method for simultaneously determining six Maillard reaction products in infant and young children's complementary rice and flour products according to claim 1, characterized in that: The method also includes the method of adding standard solutions to blank flour samples that do not contain Maillard reaction products to determine the recovery rate and precision of the method. The addition levels of furosine are 1000, 2000, and 10000 μg / 100g, respectively. The addition levels of 5-hydroxymethylfurfural, furfural, furan methyl ketone, and methylfurfural are 250, 500, and 2500 μg / 100g, respectively. The addition levels of furanone are 500, 1000, and 5000 μg / 100g, respectively. The determination is carried out in parallel 6 times, and the spiked recovery rate and RSD are calculated. The recovery rates of the 6 target compounds range from 71.2% to 100%, and the RSD ranges from 0.9% to 4.9%. n =6.

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