An HPLC method using FMOC-ADAM for determination of hydroxyproline in muscle

An HPLC method using 9-fluorenylmethyl chloroformate-1-amino-adamantane (FMOC-ADAM) for the determination of hydroxyproline (HYP) in muscle hydrolyzates is described. The HYP was converted to a stable fluorescent derivative with FMOC after removal of all primary amino acids with ortho-phthaldialdehyde and 2-mercaptoethanol. Excess FMOC was reacted with a hydrophobic amine, ADAM, thus eliminating the need for pentane or diethyl ether extraction. The method was performed without the use of controlled temperature derivatizations or extra equipment (e.g. column switching valves). Analysis of HYP was complete after 3 min, with a total analysis time of 7·5 min. The HYP content of various muscle samples obtained with the HPLC procedure compared very well with values obtained using a colorimetric assay.