Edible bird’s nest (EBN) is an expensive health food. There are many adulterants in the market. It remains challenging to discriminate EBN from its adulterants due to a lack of high-specificity markers. Besides, the current markers are confined to soluble fraction of EBN. Here, both soluble and insoluble fractions were analyzed by LC-QTOF-MS/MS. A total of 26 high-specificity peptides that were specific to EBN were selected as qualitative authentication markers. Among them, 10 markers can discriminate EBN from common adulterants, 13 markers discriminate white EBN from grass EBN/common adulterants, and 3 markers discriminate grass EBN from white EBN/common adulterants. Three of them, which showed high signal abundance (Peak area ≥ 106) and satisfactory linearity (R2 ≥ 0.995) with EBN references, were selected as the assay marker; and their peptide sequences were confidently identified by searching database/de novo sequencing. Based on these markers, a qualitative and quantitative analytical method was successfully developed and well-validated in terms of linearity, precision, repeatability, and accuracy. The method was subsequently applied to detect EBN products on the market. The results indicated that more than half of EBN products were not consistent with what the merchants claimed.
