UPLC methodology for identification and quantitation of naturally fluorescent crosslinks in proteins: A study of bone collagen

Methods used to determine collagen crosslinks in different connective tissues require a relatively large amount of material and include a number of experimental steps. We addressed these issues by developing the first ultrahigh-pressure liquid chromatography (UPLC) methodology for detection and quantification of naturally fluorescent enzymatic (pyridinoline, deoxypyridinoline) and senescent (pentosidine) crosslinks using nanogram amounts of acid-hydrolyzed bone and purified bone collagen. Not only the developed set of UPLC methods relies on a single column analysis of all three fluorescent crosslinks in one separation step, but under different separation conditions, the same column is also used to determine hydroxyproline concentration necessary to calculate collagen contents in the samples making this a unique feature of our methodology. The determined detection limit was 10 fmol for the pyridinium crosslinks and 1.5 fmol for pentosidine. The smallest pieces of human cortical bones were 224–240 ng in weight and this is approx. 106-fold less as compared to some high-pressure LC (HPLC) methods that need a minimum of approx. 0.50–1 mg of a bone sample. In general, our UPLC methodology can be applied to analysis of similar crosslinks in various collagenous tissues as well as purified/recombinant proteins of different origin. Thus, in addition to biomedical and bone research, this work is of general importance to other fields including biology, forensic, anthropology and archaeology, where samples could truly be rare, minute and precious.