Natural crosslinks are important for stabilizing the collagen structure. However, because of their low concentration in collagen compared to amino acids, their isolation, characterization and quantitation are very challenging. To the best of our knowledge, no comparative study of the isolation and characterization of natural crosslinks in different animal skins has been reported. Herein, we describe an efficient and rapid method for the isolation of crosslinks from animal skins by liquid partition chromatography and preparative TLC followed by Reverse Phase High Performance Liquid Chromatography (RP-HPLC) and characterization using Electrospray Ionization-Mass Spectrometry (ESI-MS).

Skin is first treated with sodium borohydride to stabilize the crosslinks to prevent their destruction by acid hydrolysis. Reduced skin is then hydrolyzed using 6 M HCl at 105^oC for 24 hours. Crosslinks are separated from the bulk of the amino acid residues by liquid partition chromatography on fibrous cellulose using butanol:water:acetic acid and water as solvents. Water fractions were monitored by thin layer chromatography (TLC) and fractions containing crosslinks were pooled, concentrated, and purified using preparative TLC with ethylacetate:water:acetic acid as solvent. The purity of the crosslinks was analysed by RP-HPLC and the exact mass of each crosslink measured using ESI-MS. Three main crosslinks were identified in sheep, goat, deer and cow skins; hydroxylysinenorleucine (HLNL, 292.1736 m/z), histidinolysinonorleucine (HHL, 445.2207 m/z) and histidinohydroxymerdesmosine (HHMD, 574.2969 m/z). Non-reduced skin lacked HHMD crosslinks and cartilage showed a different crosslinking profile to skin. It contained dihydroxylysinonorleucine (DHLNL, 309.1040 m/z) and pyridinoline (Pyr, 429.1887 m/z) and lacked the ones in skin. This method allows direct analysis of crosslinks HLNL, HHL and HHMD without interfering compounds such as basic amino acids and salts which has been a drawback of previously reported methods. The crosslinks found in sheep, goat, deer and cow skins will be presented and discussed in terms of their contribution to skin strength.