DOI 10.1007/s00726-005-0300-z Amino acid analysis by hydrophilic interaction chromatography coupled on-line to electrospray ionization mass spectrometry

Summary. Collagens form a common family of triple-helical proteins classified in 21 types. This unique structure is further stabilized by specific hydroxylation of distinct lysyl and prolyl residues forming 5-hydroxyly-sine and hydroxyproline (Hyp) isomers, mostly 4-trans and 3-trans-Hyp. The molecular distribution of the Hyp-isomers among the different col-lagen types is still not well investigated, even though disturbances in the hydroxylation of collagens are likely to be involved in several diseases such as osteoporosis and autoimmune diseases. Here, a new approach to analyze underivatized amino acids by hydrophilic interaction chromato-graphy (HILIC) coupled on-line to electrospray ionization mass spectro-metry (ESI-MS) is reported. This method can separate all three studied Hyp-isomers, Ile, and Leu, which are all isobaric, allowing a direct qua-litative and quantitative analysis of collagen hydrolysates. The sensitivity and specificity was increased by a neutral loss scan based on the loss of formic acid (46 u)