| Food allergies can cause acute or chronic disease, and even life-threatening, seriously affecting the health of the public life and the management of food allergy problem is of importantance to ensure food safety. Eggs and milk are rich in protein, some ofwhich are allergenic. So far, there is no effective method in the treatment of allergies and the food potential allergen detection and identification is the internationally accepted means to avoid food allergy. In2012, the national standard of "The GB7718-2011pre packaged food labels" was released in China and the detection and identification of food allergen such as eggs and milk. Therefore, the development of the high accuracy of the egg and milk allergen detection method is the inevitable requirement for the support of the food allergen labeling standards implementation, the avoidance of food allergy and the protection of public health and safety. First of all, a new method of UPLC-Q-TOF had been proposed for the rapid and accurate detection of common amino acids. Without derivatization and using ion-pair regent, amino acids were analyzed fast and accurately. With using high-resolution TOF mass spectrometry, this method avoids the interference of ion overlapping. This study could be applied with both external standard method and ID-MS.14common amino acids could be analyzed in2minutes, which reduced the analysis time greatly, which can improve the accuracy of measurement results, and will be well applied in homogeneity and stability testing of protein certified reference materials.Secondly, based on the amino acid analysis, the HPLC-IDMS method was developed for the quantitation of β-lactoglobulin, β-casein, lactoferrin and lysozyme. Through the optimization of hydrolysis time, the method of isotope dilution mass spectrometry was established for the determination for the above4pure protein content. The calculated results were validated by the purity deduction method. The method has good reproducibility, high accuracy and measured results can be traceable to SI units. This will have a very good application in the pure allergy protein standard development.Furtherly, we developed an IDMS method based on the peptide TK-11to determine the β-lactoglobulin content in the milk powders. Bioinformatics analysis was carried out for the amino acid sequence of the p-lactoglobulin and the specific peptide TK-11was identified and chosen for accurate quantification of β-lactoglobulin in milk powders matrix; the mass fragment of high pure TK-11was also determined with isotope dilution mass spectrometry method; after the optimization and investigation of β-lactoglobulin extraction, pretreatments, restriction effect, enzymic condition, enzyme digestion efficiency, the based on peptide isotope dilution mass spectrometry method was developed for the determination of P-lactoglobulin content in milk powders. By the proposed method, the recovery ranged from86.0%to118.3%with CVs<9.0%. Ten samples from domestic market were analyzed with CVs <5.6%and the relative expanded uncertainties ranged from4.2%to5.9%(k=2). With the CRMs, it is expected that the comparability of β-LG quantitation results will be improved among different laboratories.Finally, the above study on the quantitative method of β-lactoglobulin content with isotope dilution mass spectrometry was applied to the assessment and interoperability research of commercially available β-lactoglobulin ELISA kits on sale. We used their own standards to evaluate the ELISA kits. The results show that they both have good repeatability and high sensitivity which meets the prerequisites of the futher interoperability research. Then, with the β-lactoglobulin certified reference material which was accurately determined by isotope dilution mass spectrometry method, the conversion facters were calculated between the β-lactoglobulin content of two kits, which was2.61and1.93, respectively. As the same sample was detected, the results of β-lactoglobulin content by the two kits were245.03ng·g-1and174.32ng·g-1respectively. After the correction of the conversion factor but the two kits was corrected after their conversion factor, the final results were93.88ng·g-1and90.32ng·g-1, respectively. And the relative deviation was only2.7%. The results can be directly traced to β-lactoglobulin certified reference material, assuring the measuring results of different kits comparable and successfully building interoperability of two ELISA kits. This will provide the technical support for accurate and comparable results of more protein allergen ELISA kits.The methods above are not reported in the literature and have high accuracy, good repeatability; traceability to SI unit etc.They can be applied in the research of certified reference material development and the evaluation and calibration of allergens detections by commercial ELISA kits. It means a lot for the the implementation of national standard allergen labeling, the accurancy and conparablity of food allergen detection results, and the protection for food safety and public health technically. |
