The Maillard reaction is traditionally subdivided into three stages that start consecutively and run in parallel. Here, we show that N-epsilon-carboxymethyllysine (CML), a compound formed in the late stage of the reaction, can undergo a second glycation event at its secondary amino group leading to a new class of Amadori rearrangement products. When N-alpha-hippuryl-CML was incubated in the presence of reducing sugars such as glucose, galactose, ribose, xylose, maltose, or lactose in solution for 1 h at 75 degrees C, the compound was degraded by 6-21%, and N-epsilon-carboxymethyl-N-epsilon-deoxyketosyl lysine derivatives were formed. Under the same conditions, lysine was 5-10 times more reactive than CML. N-alpha-hippuryl-N-epsilon-carboxymethyl-N-epsilon-(1-deoxyfructosyl)-L-lysine (hippuryl-CMFL) and N-epsilon-carboxymethyl-N-epsilon-(1-deoxyfructosyl)-L-lysine (CMFL) were synthesized, isolated and characterized by MS/MS and NMR experiments. Depending on the reaction conditions, up to 21% of CMFL can be converted to the furosine analogue N-epsilon-carboxymethyl-N-E-furoylmethyl-L-lysine (CM-Fur) during standard acid protein hydrolysis with hydrochloric acid. Incubation of bovine serum albumin (BSA) with glucose for up to 9 weeks at 37 degrees C revealed the formation of CMFL in the protein as assessed by HPLC-MS/MS in the MRM mode. Under these conditions, ca. 13% of lysine residues had been converted to fructosyllysine, and 0.03% had been converted to CMFL. The detection of glycation products of glycated amino acids (heterogeneous multiple glycation) reveals a novel pathway in the Maillard reaction.