Separation of Natural Collagen Crosslinks Using Buffer and Ion-pairing Agent Free Solvents on Silica Hydride Column for Mass Spectrometry Detection.

In this protocol we describe the separation of collagen crosslinks in biological tissues and samples including skin, tendon, cartilage, bone and urine. The existing methods use either cation exchange chromatography followed by post-column derivatization with ninhydrin or reverse phase chromatography with mass spectrometry detection. The cation exchange chromatography method has limited sensitivity and long run times while reverse phase chromatography requires strong ion-pairing. In this method, the sample containing crosslinks is applied on a diamond hydride column using water and acetonitrile solvents containing 0.1% (w/v) formic acid. Eight crosslinks are eluted separately from the column and detected by mass spectrometry in the sub-pmol range. By using this method, it is possible to separate all crosslinks of collagen in several biological samples without the need for ion-pairing agent or derivatization for detection.