Comprehensive identification and comparative analysis of protein-unbound Amadori products in powdered infant formulas: Integrating untargeted and targeted metabolomics with LC-Q-Orbitrap-MS/MS

Infant formula is prone to the Maillard reaction, forming early-stage Amadori products and advanced glycation end products (AGEs). Despite extensive research on protein-bound Amadori products and AGEs, studies on protein-unbound Amadori products in infant formula are limited. This study, for the first time, reveals that Amadori products derived from mono and oligohexose exhibit distinct MS/MS neutral loss patterns. Using these characteristics, a combined targeted and untargeted strategy for rapid and comprehensive analysis of mono- and oligohexose derived Amadori products was developed. A total of 40 protein-unbound Amadori products were identified, resulting from reactions between glucose, lactose, galactose, and 17 amino acids along with 3 other amino compounds. Novel Amadori products like fructosyl-O-phosphorylethanolamine/glycerophosphorylethanolamine and lactulosyl-O-phosphorylethanolamine/glycerophosphorylethanolamine were identified. Further comparative analysis of free Amadori product contents in 5 standard infant formula samples revealed significant variations. However, the levels of Amadori products and their corresponding precursor compounds showed no significant correlation. These findings offer valuable insights into the complexity and diversity of Amadori products in infant formulas. Moreover, the identified Amadori products show promise as potential markers for differentiating infant formulas from different manufacturers. • Mono and oligohexose derived free APs show distinct neutral loss patterns • Methods for screening mono and oligohexose derived APs were established • A panel of novel free APs were identified in infant formulas • Levels of free APs and their corresponding precursors lack correlation • Free APs are potential quality markers for differentiating infant formula brands