Abstract:Objective To establish an ultra-high performance liquid chromatography tandem isotope dilution mass spectrometry method for the determination of three major allergens, such as lysozyme, egg transferrin and ovalbumin, from eggs in baked food. Methods The specific peptides of lysozyme, ovotransferrin and ovalbumin were first screened, and then the specific peptides and isotopic markers were synthesized. Samples were homogenized in a buffer solution of 200 mmol/L Tris-HCl (pH=8.0) for 2-3 times. The sample solution was mixed with trypsin in a proportion of 1∶50 (trypsin/protein ratio) and then placed on a shaking bed and hydrolyzed for 12 h at 37 ℃. The enzymatic hydrolysate was detected by the electrospray positive ion (ESI+) with multiple reaction monitoring (MRM) mode and quantified by an isotope dilution method. Results Good linear relationships within the ranges of 1-100 nmol/L with linear correlation coefficients (r2) above 0.995 was achieved for the calibration curves of nine specific peptides standards. The limits of quantitation(LOQ)of the three proteins in baked food were 0.4-10.0 μg/g. The recoveries of target proteins in baked food ranged from 65.0% to 108.5% with relative standard deviations (RSDs) less than 15.0%. Conclusion The method is sensitive, specific and accurate. It is suitable for the detection of egg allergen proteins in baked food.