Abstract:

Cow’s milk protein allergy （CMPA） has become a common public health concern and hypoallergenic formula （partially or extensively hydrolyzed formula） is considered as an alternative during infancy. Alcalase and papain are widely used in industrial production of hydrolysates， as they can modify or destroy the linear and conformational epitopes of cow’s milk allergens （CMAs）， thus leading to decreased allergenicity. However， despite considerable emphasis has been laid on the manufacturing technique and nutritional value of hypoallergenic formulas， whether there are traces of CMAs in hypoallergenic formulas， remains unknown. Consequently， the need for a quantitative method of detecting multiple CMAs at a time is rising. Traditionally， enzyme-linked immunosorbent assay （ELISA）-based methods have proved to be advantageous with a wide availability of commercial kits for CMAs detection. Moreover， polymerase chain reaction （PCR）， as one of DNA-based methods， has shown exceptional specificity and high sensitivity. However， they both may encounter false-positive results and high-throughput detection is challenging. In the past two decades， mass spectrometry （MS）-based proteomic technology has advanced， including improvements in computational capability， algorithm model， as well as mass spectrometric platforms. MS is gaining more and more attention in the field of food allergen detection， allowing multiplex allergen detection with high sensitivity， accuracy and selectivity in a single run. In this study， an analytical method was developed for the simultaneous quantitation of six CMAss （namely， α-lactalbumin， β-lactoglobulin， αS1-casein， αS2-casein， β-casein， and κ-casein） in hypoallergenic formulas using ultra-high performance liquid chromatography-tandem mass spectrometry （UHPLC-MS/MS）. Proteins of spiked hypoallergenic formula were extracted with ammonium bicarbonate and digested with trypsin. After digestion， the samples were purified with a C₁₈ spin column， and analyzed by UHPLC-MS/MS （AB SCIEX Triple Quad 6500⁺）. Separation was achieved on a Waters ACQUITY UPLC Peptide CSH C₁₈ column （150 mm×2.1 mm， 1.7 μm）. The mobile phase consisted of 0.1% formic acid aqueous solution and 0.1% formic acid in acetonitrile， and the flow rate was 0.3 mL/min. The sample injection volume was 10 μL， and the column temperature was set to 40 ℃. Mass spectrometer was operated in positive electrospray ionization （ESI⁺） mode， and multiple reaction monitoring （MRM） mode was adopted. Precursor ions and product ions of marker peptides were predicted by the Skyline software. Mass spectrometric parameters such as declustering potential （DP） and collision energy （CE） were also optimized. Afterwards， by taking allergen epitopes and mass spectrometric responses into consideration， six peptides were retained for quantitative analysis. Calibration curves were constructed with good linearity in the range of 0.05-500 mg/kg， and correlation coefficients （r²） greater than 0.99. The limits of detection （LODs） were 0.05-5.0 mg/kg， and limits of quantification （LOQs） were 0.1-10.0 mg/kg， respectively. The recoveries of the developed method in incurred hypoallergenic formulas ranged from 74.8% to 93.4%. The intraday and interday accuracies ranged from -25.09% to -6.09% and from -24.0% to -5.41%， with precisions between 1.08%-5.05% and 1.35%-4.85%， respectively. Screening of commercial hypoallergenic formula samples confirmed existence of CMAs in three partially hydrolyzed formulas （pHF） and two extensively hydrolyzed formulas （eHF）， with contents ranging from 0.48 to 27.36 mg/kg. The analytical method proposed in this study exhibited high sensitivity， high-throughput capability， and can be used to monitor traces of CMAs in hypoallergenic formulas.

Key words: cow’s milk allergens （CMAs）, ultra-high performance liquid chromatography-tandem mass spectrometry （UHPLC-MS/MS）, peptides, label-free quantification （LFQ）, hypoallergenic infant formula