Quantitative separation of 4-hydroxyproline from skeletal muscle collagen by micellar electrokinetic capillary electrophoresis

The phenylthiohydantoin (PTH) derivatives of 3- and 4-hydroxyproline (Hyp) were separated using micellar electrokinetic capillary electrophoresis (MEKC). The separation protocol was also used to determine Hyp content of bovine skeletal perimysial collagen preparations and whole muscle samples. Amino acids from hydrolyzed tissues were labeled using a two step procedure that involved initial reaction with o-phthalaldehyde (OPA) to modify primary amines followed by their precipitation under acidic conditions. In the second step, imino acids were reacted with phenyl isothiocyanate (PITC). This labeling method was rapid and the Hyp values determined in these biological samples were found to be in close agreement with conventional methods and other published reports.