Abstract: In this work, trypsin-catalyzed 18O labeling combined with high performance liquid chromatography-linear ion trap/Orbitrap high-resolution mass spectrometry (HPLC-LTQ/Orbitrap MS/MS) was used to study the 18O labeling characteristics of marker peptides in donkey-hide gelatin. Moreover, the method was applied to quantify donkey-hide gelatin and bovine-hide gelatin in their mixtures. Under trypsin catalysis, all the marker peptides of donkey-hide gelatin were labeled by two 18O atoms. The measured ratios for 18O-labeled and 16O-labeled peptide mixtures at ratios of 20:1, 10:1, 5:1, 1:1 and 1:5 were 19.66, 10.24, 5.01, 1.00 and 0.20, respectively. The measured ratios for mixtures of donkey-hide gelatin and bovine-hide gelatin at ratios of 1:10, 1:1 and 10:1 using 18O-labeled characteristic peptides as the internal standard were 0.10, 1.02 and 9.72, respectively. The results showed that trypsin-catalyzed 18O labeling combined with HPLC-LTQ/Orbitrap MS/MS should be a promising method to quantify the target gelatin and control the quality of donkey-hide gelatin product.

Key words: donkey-hide gelatin, bovine-hide gelatin, quantification, trypsin-catalyzed 18O labeling, high-performance liquid chromatography-high-resolution mass spectrometry