Quantitation of hydroxyproline isomers in acid hydrolysates by high-performance liquid chromatography

A method has been developed to rapidly separate and quantitate levels of hydroxy-L-proline isomers in tissue hydrolysates. The procedure incorporates derivatization of the imino acids with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole chloride followed by separation by high-performance liquid chromatography employing two C18 reverse-phase columns connected in series. Conditions for the derivatization procedure have been optimized for the selective reactivity of imino acids. The derivatized imino acid fractions are then quantitated spectrophotometrically at 495 nm. Using this technique, quantities above 40 pmol are readily detected for trans-4-hydroxyl-L-proline, trans-3-hydroxyl-L-proline, proline, and other imino acid analogs. The method is applicable to a wide range of clinical and experimental tissues.