Quantitative detection method for allergen in infant food based on isotope labeling mass spectrometry technology

Abstract

The invention relates to a quantitative detection method for allergens in infant food based on an isotope labeling mass spectrometry technology, and belongs to the technical field of chemical analysis and detection. The method comprises the following steps: 1) taking a homogenized sample, fully extracting by using a protein extracting solution, performing enzymolysis by using trypsin to obtain a peptide fragment, and desalting the obtained enzymolysis product; 2) separating and detecting the target peptide fragment by using ultra-high performance liquid chromatography-tandem mass spectrometry; 3, accurate quantitative analysis of the multiple sensitizers is achieved through an isotope labeling internal standard method.The method has the advantages of being easy and convenient to operate, high in detection sensitivity, excellent in quantitative accuracy and the like, efficient and synchronous detection of the multiple sensitizers in infant food can be achieved, and reliable technical support is provided for product safety evaluation and quality control.

Description

Technical Field

[0001] This invention belongs to the field of chemical detection technology, specifically relating to a quantitative detection method for allergens in infant food based on isotope-labeled mass spectrometry. Background Technology

[0002] Infant food is a crucial source of nutrients such as protein, vitamins, and minerals, and its quality and safety directly impact the growth, development, and health of infants and young children. Because infants' immune systems are not yet fully developed, they are extremely sensitive to allergens in food, making food allergies a particularly significant health risk among this population. Although GB7718-2025, the "General Rules for Labeling of Prepackaged Foods," requires the labeling of allergens, the long food processing chain, complex raw material sources, and shared production lines lead to frequent instances of unintentional adulteration or trace cross-contamination of allergens, seriously threatening the lives of infants and young children. Infant food is often produced in highly processed forms such as milk powder, rice cereal, fruit puree, or compound complementary foods, rather than from single ingredients. This makes it extremely difficult to detect potential allergens (such as milk, eggs, peanuts, and fish) by sensory means, as their structure changes or they are masked by the matrix after heat processing, enzymatic hydrolysis, or Maillard reactions. Once sensitive individuals ingest food containing trace amounts of allergens, they are highly susceptible to severe reactions such as rashes, respiratory difficulties, and even anaphylactic shock. As a populous country, my country has a huge market for infant formula. Although national standards are constantly being improved, there are still technical bottlenecks in the accurate quantification of multiple allergens simultaneously in actual production and distribution, which exacerbates the difficulty of food safety supervision. To solve this problem, it is necessary to continuously develop and improve highly sensitive and accurate detection methods to confirm multiple allergens in infant formula and accurately quantify their residues. Therefore, establishing an accurate and sensitive method for simultaneous quantitative detection of multiple allergens is of great practical significance for ensuring the safety of infant diets and avoiding allergy risks.

[0003] Traditional food allergen detection primarily relies on immunological methods (such as enzyme-linked immunosorbent assay, ELISA) and molecular biological methods (such as PCR). However, quantitative detection and evaluation are extremely challenging for highly processed infant foods or complex compound products. With advancements in technology, proteomics identification techniques have become increasingly sophisticated, enabling systematic qualitative and quantitative analysis of allergens in food based on differences in protein levels and peptide specificity. Among current detection methods, ELISA is susceptible to matrix interference leading to false positives and struggles to simultaneously detect multiple targets; PCR, based on DNA detection, cannot directly reflect the actual content of allergenic proteins. In recent years, liquid chromatography-mass spectrometry (LC-MS / MS) based on characteristic peptides has shown broad application prospects in trace analysis and simultaneous multi-component detection of food allergens. High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) combines the excellent separation performance of liquid chromatography with the high sensitivity and accuracy of mass spectrometry. This technique avoids problems such as antigen epitope destruction, cross-reactions, and false negatives caused by protein denaturation during processing in immunoassays, achieving accurate detection of target allergenic proteins. Meanwhile, this invention addresses the complex matrix characteristics of infant food by adding isotopically labeled characteristic peptides of known mass and abundance to the sample as internal standards. This corrects for inaccuracies in detection results caused by sample desalting and purification, chromatographic column performance, ionization degree, and severe matrix effects. Through comprehensive research, a highly efficient and accurate method for simultaneous quantification of five major allergens has been developed. Summary of the Invention

[0004] To address the problems existing in current technologies, the purpose of this invention is to design and provide a technical solution for the quantitative detection of allergens in infant formula based on isotope-labeled mass spectrometry. This method can efficiently, conveniently, and accurately determine the content of milk, shrimp, cod, peanuts, and eggs in target matrices.

[0005] This invention is specifically achieved through the following technical solutions: The first aspect of this invention provides a method for quantitative detection of allergens in infant food based on isotope-labeled mass spectrometry, comprising the following steps: 1) Take the homogenized sample, extract it fully with protein extraction solution, and then digest it with trypsin to obtain peptides. Add the characteristic peptides labeled with allergen isotopes as internal standards, and desalt the obtained enzymatic hydrolysis products. 2) The target peptides were separated and detected using ultra-high performance liquid chromatography-tandem mass spectrometry; 3) The isotope labeling internal standard method is used to achieve accurate quantitative analysis of multiple allergens.

[0006] Furthermore, the allergens include any one or more of milk, shrimp, cod, peanuts, and eggs.

[0007] Furthermore, the characteristic peptides of the allergens include AVPYPQR, FLAEEADR, VFEIIDQDK, GTGNLELVAVR, and GGLEPINFQTAADQAR, respectively, and the characteristic peptides of the allergen isotope labeling include AVPYPQR ( 13 C6, 15 N4), FL 13 C6, 15 N)AEEADR、V( 13 C5, 15 N)FEIIDQDK、GTGNLELVAVR( 13 C6, 15 N4), GGLEPINFQTAADQAR ( 13 C6, 15 N4).

[0008] Furthermore, the protein extraction solution described in step 1) comprises 50-150 mM Tris-HCl, 1-3 M urea, and pH=8-10.

[0009] Further, the trypsin hydrolysis in step 1) specifically includes: adding 5-15 mL of 50-150 mM ammonium bicarbonate solution to the protein extract and mixing well; adding 0.5-1.5 mL of 45-55 mM dithiothreitol and incubating at 35-40℃ for 0.5-1.5 h; adding 0.5-1.5 mL of 50-150 mM iodoacetamide and incubating at room temperature in the dark for 20-40 min; adding 50-150 μL of 5-15 mg / mL trypsin solution and incubating at 35-40℃ for 3-5 h; and adding 15-25% formic acid solution to terminate the hydrolysis; the ratio of trypsin to protein is 1:10-30.

[0010] Furthermore, the desalination treatment described in step 1) specifically includes: activating C using acetonitrile and a 0.05-0.1% formic acid aqueous solution. 18 Solid-phase extraction column, the enzymatically hydrolyzed solution is passed through C 18 The target substance was eluted by adding acetonitrile / 0.1% formic acid (80 / 20, v / v) solution to a solid phase extraction column. The eluent was dried with nitrogen, reconstituted with mobile phase A, and then filtered through an aqueous filter membrane.

[0011] Furthermore, the chromatographic conditions for the ultra-high performance liquid chromatography-tandem mass spectrometry method described in step 2) are as follows: column: ACQUITY UPLC BEH C18, 2.1 × 100 mm, 1.7 μm; column temperature: 40℃; injection volume: 3 mL; mobile phase A is 0.1% formic acid aqueous solution, and mobile phase B is acetonitrile, with gradient elution. The mass spectrometry conditions for the ultra-high performance liquid chromatography-tandem mass spectrometry method are as follows: ESI source, positive ion mode; capillary voltage: 3.5 kV; ion source temperature: 120℃; desolvation gas temperature: 450℃; nebulizer gas flow rate: 1000 L / Hr; collision gas flow rate: 150 L / Hr; scanning mode: multiple reaction monitoring.

[0012] Furthermore, the gradient elution program in the chromatographic conditions described in step 2) is as follows: .

[0013] Furthermore, the characteristic peptide of the allergen and its internal standard mass spectrometry parameters mentioned in step 2) are as follows: .

[0014] Furthermore, the internal standard method for quantification described in step 3) specifically includes: (1) Preparation of standard curve: The characteristic peptide standard is prepared into solutions with concentrations of 1.0, 5.0, 10.0, 50.0, 100.0, 500.0, and 1000.0 ng / mL. The internal standard peptide is prepared into a solution with a concentration of 10 μg / mL. Take 10 μL of the internal standard peptide solution and 1 mL of the corresponding characteristic peptide standard solution, mix them well, and then test them on the instrument. Plot the standard curve using the concentration of the characteristic peptide standard solution as the abscissa and the ratio of the peak area of ​​the quantitative ion pair of the characteristic peptide and the internal standard peptide as the ordinate. The software automatically corrects the concentration of the internal standard. (2) Determination of commercially available samples: Take the processed commercially available samples for testing, substitute the peak area obtained from the test into the standard curve of the corresponding substance, and analyze and calculate the content of allergens in the commercially available samples.

[0015] The present invention has the following beneficial effects: (1) This invention develops and establishes a method for analyzing allergens in infant food using ultra-high performance liquid chromatography-tandem mass spectrometry. This method has the advantages of simple and efficient pretreatment, good accuracy, small matrix interference effect, high recovery rate, and good stability. It overcomes the barriers and difficulties of traditional analytical techniques and provides a basic experimental approach for the determination of other types of allergens in infant food and even allergens in other food matrices.

[0016] (2) The present invention exhibits good linearity within the detection range of 1-1000 ng / mL, with a linear correlation coefficient R0. 2 Greater than 0.999. The limit of detection and limit of quantitation were 0.3 ng / mL and 1.0 ng / mL, respectively, and the recovery rate was 80.00%-110.00%.

[0017] (3) The characteristic peptides showed good reproducibility. In rice flour, carrot and green vegetable matrices, the intra-day and inter-day relative standard deviations (RSD) were all less than 15%.

[0018] (4) Because LC-MS / MS is easily affected by matrix effects, and from the perspective of ionization technology, ESI is more susceptible to ion suppression than APCI. This method is improved to address the interference of matrix effects by introducing isotopic internal standards to eliminate the influence.

[0019] (5) C 18 Solid-phase extraction column purification reduces matrix interference and improves detection accuracy. Attached Figure Description

[0020] Figure 1 This is a standard curve of the characteristic peptides of milk allergenic proteins in the examples; Figure 2 This is a standard curve of the characteristic peptides of crustacean allergenic proteins in the examples; Figure 3 The standard curve for the characteristic peptides of cod allergenic protein in the examples; Figure 4 This is a standard curve of the characteristic peptides of peanut allergenic protein in the examples; Figure 5 This is a standard curve of the characteristic peptides of the egg allergenic protein in the examples; Figure 6 The ion chromatograms show the limits of quantitation (1 ng / mL) for the characteristic peptides of the five sensitizing proteins in the examples. Figure 7 The ion chromatograms for high concentrations (1000 ng / mL) of the characteristic peptides of the five sensitizing proteins in the examples are shown. Figure 8 The following is a chromatogram of ion flow in rice matrix spiked with the five allergenic proteins in the example. Detailed Implementation

[0021] The present invention will be further illustrated below with reference to the embodiments.

[0022] Example 1

[0023] 1. Main instruments and reagents Waters UPLC I-Class / Xevo TQ-s ultra-high performance liquid chromatography-tandem mass spectrometry system (Waters Corporation, USA), Milli-Q ultrapure water system, ST16R high-speed refrigerated centrifuge (Thermos Corporation), VG3 S025 vortex mixer (IKA Corporation), T25 high-speed homogenizer (IKA Corporation), and GM200 blade grinder (Retsch Corporation).

[0024] Acquity BEH C18 columns (1.7 μm, 2.1 × 100 mm) and Sep-Pak solid-phase extraction columns were purchased from Waters (USA); 0.22 μm polyethersulfone (PES) filter membranes were purchased from Hangzhou Shuangtian Biotechnology Co., Ltd. (China). Synthetic peptides AVPYPQR, FLAEEADR, VFEIIDQDK, GTGNLELVAVR, GGLEPINFQTAADQAR, and AVPYPQR ( 13 C6, 15 N4), FL 13 C6, 15 N)AEEADR、V( 13 C5, 15 N)FEIIDQDK、GTGNLELVAVR( 13 C6, 15 N4), GGLEPINFQTAADQAR ( 13 C6, 15 N4). Purchased from Qiangyao Biotechnology Co., Ltd. (China).

[0025] 2. Sample Preparation The samples were purchased from Tmall Supermarket or Tmall Flagship Store online.

[0026] Take the edible portion of the sample and homogenize it using a grinder. Add 10 mL of protein extraction buffer (100 mM Tris-HCl, 2 M urea, pH=9.0) to 1 g of sample, homogenize the sample and extract using a homogenizer, and stir in an ice bath for 30 min. Centrifuge at 10000 r / min for 20 min and collect the supernatant. Take 10 mL of protein extraction buffer (100 mM Tris-HCl, 2 M urea, pH=9.0), add 10 mL of 100 mM ammonium bicarbonate solution and mix well, then add 1 mL of 50 mM DTT solution, incubate at 37°C for 1 h, then add 1 mL of 100 mM IAA solution and incubate at room temperature in the dark for 30 min. Subsequently, add trypsin solution (10 mg / mL, dissolved in 50 mM acetic acid solution) to the system at an enzyme:substrate ratio of 1:20 and incubate at 37°C for 8 h. Add 300 μL of 20% formic acid solution to terminate the enzymatic hydrolysis. Add 10 μL of a stable isotope-labeled peptide solution containing 100 μg / mL AVPYPQR to the peptides obtained from the enzymatic hydrolysis. 13 C6, 15 N4), FL 13 C6, 15 N)AEEADR、V( 13 C5, 15N)FEIIDQDK、GTGNLELVAVR( 13 C6, 15 N4), GGLEPINFQTAADQAR ( 13 C6, 15 (N4). Further desalting and purification of the enzymatic hydrolysate was performed. 18 mL of acetonitrile, 18 mL of 0.1% formic acid, and 20 mL of sample were added sequentially to the SPE column. After eluting impurities from the sample with 18 mL of 0.1% formic acid, the peptides were eluted with a 6 mL acetonitrile / 0.1% formic acid (80 / 20, v / v) mixture, and the eluent was collected. The eluent was dried using a nitrogen evaporator, and 1 mL of 0.1% formic acid was added to redissolve the peptides. The peptide solution was filtered through a 0.22 μm PES filter membrane, and the filtrate was transferred to a sample vial for analysis.

[0027] 3. Ultra-high performance liquid chromatography-tandem mass spectrometry detection Chromatographic conditions: The chromatographic column was an ACQUITY UPLC BEH C18, 2.1 × 100 mm, 1.7 μm. Column temperature: 40℃. Injection volume: 3 μL. Mobile phase A was 0.1% formic acid solution, and mobile phase B was acetonitrile; gradient elution details are shown in Table 1.

[0028] Table 1. Gradient elution program for mobile phase

[0029] Mass spectrometry conditions: ESI source, positive ion mode; capillary voltage: 3 kV; ion source temperature: 120 ℃; desolvation gas temperature: 450 ℃; nebulizer gas flow rate: 800 L / Hr; collision gas flow rate: 150 L / Hr; scan mode: multiple reaction monitoring (MRM). The internal standard mass spectrometry parameters for the characteristic peptides of the five allergens are detailed in Table 2.

[0030] Table 2. Main mass spectrometry parameters of the compounds

[0031] 4. Quantitative analysis of allergens in the sample to be tested. (1) Preparation of standard curve: The characteristic peptide standards were prepared into solutions with concentrations of 1.0, 5.0, 10.0, 50.0, 100.0, 500.0, and 1000.0 ng / mL. The internal standard peptide was prepared into a solution with a concentration of 10 μg / mL. 10 μL of the internal standard peptide solution and 1 mL of the corresponding characteristic peptide standard solution were mixed and analyzed. A standard curve was plotted using the instrument's software, with the concentration of the characteristic peptide standard solution on the x-axis and the ratio of the peak areas of the quantitative ion pairs of the characteristic peptide and the internal standard peptide on the y-axis. The software automatically corrected for the internal standard concentration. Figures 1-5As shown, within the concentration range of 1-1000 ng / mL, the five characteristic peptides exhibited good linear correlation. The regression equations are shown in Table 3, with LOD and LOQ of 0.3 ng / mL and 1.0 ng / mL, respectively. Figure 6 and Figure 7 As shown, the five characteristic peptides exhibited good separation and peak shape at the limit of quantitation and the highest point of the standard curve. To evaluate the accuracy of this method, allergen spike tests were conducted using rice flour, carrots, and green vegetables as blank matrices at concentrations of 5.0 ng / g, 50.0 ng / g, and 500.0 ng / g. The recoveries were found to be 80.00%–110.00%, demonstrating the good accuracy of this method. Figure 8 As shown, the candidate peptides exhibited good separation in the spiked recovery experiment.

[0032] Table 3 Linear Regression Equations

[0033] (2) Test reagent samples: The content of five allergenic protein characteristic peptides was detected in the processed commercially available infant food samples. The peak areas obtained from the tests were substituted into the standard curves of the corresponding substances, and the content of the five allergenic protein characteristic peptides in the eight commercially available samples was calculated. The results are shown in Table 4 below.

[0034] Table 4. Content of characteristic peptides of sensitizing proteins in actual samples

[0035] Note: ND indicates not detected.

[0036] As shown in Table 4 above, allergens were detected in all samples, indicating that this method has good application prospects in the field of detecting allergens in infant formula.

Claims

1. A method for quantitative detection of allergens in infant food based on isotope-labeled mass spectrometry, characterized in that, Includes the following steps: 1) Take the homogenized sample, extract it fully with protein extraction solution, and then digest it with trypsin to obtain peptides. Add the characteristic peptides labeled with allergen isotopes as internal standards, and desalt the obtained enzymatic hydrolysis products. 2) The target peptides were separated and detected using ultra-high performance liquid chromatography-tandem mass spectrometry; 3) The isotope labeling internal standard method is used to achieve accurate quantitative analysis of multiple allergens.

2. The method as described in claim 1, characterized in that, The allergens mentioned include any one or more of milk, shrimp, cod, peanuts, and eggs.

3. The method as described in claim 2, characterized in that, The characteristic peptides of the allergens include AVPYPQR, FLAEEADR, VFEIIDQDK, GTGNLELVAVR, and GGLEPINFQTAADQAR, respectively. The characteristic peptides of the allergen isotope-labeled peptides include AVPYPQR ( 13 C6, 15 N4), FL 13 C6, 15 N)AEEADR、V( 13 C5, 15 N)FEIIDQDK、GTGNLELVAVR( 13 C6, 15 N4), GGLEPINFQTAADQAR ( 13 C6, 15 N4).

4. The method as described in claim 1, characterized in that, The protein extraction solution described in step 1) includes 50-150 mM Tris-HCl, 1-3 M urea, and pH 8-10.

5. The method as described in claim 1, characterized in that, The trypsin hydrolysis described in step 1) specifically includes: adding 5-15 mL of 50-150 mM ammonium bicarbonate solution to the protein extract and mixing well; adding 0.5-1.5 mL of 45-55 mM dithiothreitol and incubating at 35-40℃ for 0.5-1.5 h; adding 0.5-1.5 mL of 50-150 mM iodoacetamide and incubating at room temperature in the dark for 20-40 min; adding 50-150 μL of 5-15 mg / mL trypsin solution and incubating at 35-40℃ for 3-5 h; and adding 15-25% formic acid solution to terminate the hydrolysis; the ratio of trypsin to protein is 1:10-30.

6. The method as described in claim 1, characterized in that, The desalination process described in step 1) specifically includes: activating C with acetonitrile and a 0.05-0.1% formic acid aqueous solution. 18 Solid-phase extraction column, the enzymatically hydrolyzed solution is passed through C 18 The target substance was eluted by adding acetonitrile / 0.1% formic acid (80 / 20, v / v) solution to a solid phase extraction column. The eluent was dried with nitrogen, reconstituted with mobile phase A, and filtered through an aqueous filter membrane.

7. The method as described in claim 1, characterized in that, The chromatographic conditions for the ultra-high performance liquid chromatography-tandem mass spectrometry method described in step 2) are: ACQUITY UPLC BEH C18 column, 2.1 × 100 mm, 1.7 μm; Column temperature: 40℃; injection volume: 3 mL; mobile phase A is 0.1% formic acid aqueous solution, and mobile phase B is acetonitrile, with gradient elution; The mass spectrometry conditions for the ultra-high performance liquid chromatography-tandem mass spectrometry method are as follows: ESI source, positive ion mode; capillary voltage: 3.5 kV; ion source temperature: 120 °C; desolvation gas temperature: 450 °C; nebulizer gas flow rate: 1000 L / Hr; collision gas flow rate: 150 L / Hr; scanning mode: multiple reaction monitoring.

8. The method as described in claim 1, characterized in that, Step 3) of the internal standard method for quantification specifically includes: (1) Preparation of standard curve: The characteristic peptide standard is prepared into solutions with concentrations of 1.0, 5.0, 10.0, 50.0, 100.0, 500.0, and 1000.0 ng / mL. The internal standard peptide is prepared into a solution with a concentration of 10 μg / mL. Take 10 μL of the internal standard peptide solution and 1 mL of the corresponding characteristic peptide standard solution, mix them well, and then test them on the instrument. Plot the standard curve using the concentration of the characteristic peptide standard solution as the abscissa and the ratio of the peak area of ​​the quantitative ion pair of the characteristic peptide and the internal standard peptide as the ordinate. The software automatically corrects the concentration of the internal standard. (2) Determination of commercially available samples: Take the processed commercially available samples for testing, substitute the peak area obtained from the test into the standard curve of the corresponding substance, and analyze and calculate the content of allergens in the commercially available samples.

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