Improved high-performance liquid chromatography method for quantitation of proline and hydroxyproline in biological...

Numerous high-performance liquid chromatography systems have been described for the determination of hydroxyproline (Hyp) and proline (Pro) levels in biological materials. These methods are generally complicated and have shortcomings in applicability due to poor separation, low sensitivity or derivatization-associated problems. The large number of chemical components present in biological samples further complicates the analysis of Hyp which usually occurs in extremely low concentrations. The present investigation describes the development of a simple highly sensitive derivatization method which results in good separation of peaks and which is capable of quantitating less than 10 pmol of Hyp and Pro in complex test systems. The method is based on removal of o-phthalaldehyde (OPA) derivatives of primary amino acids using reversed-phase chromatography, pre-column derivatization with OPA and phenylisothiocyanate, and detection of derivatized Hyp and Pro using a UV detection system. The procedure yields good peaks and a 93% recovery of Hyp and Pro provided that the analysis is initiated within 5 min of completion of OPA derivatization. While a 93% recovery of Pro was obtained up to 100 min post-derivatization with OPA, the recovery of Hyp is decreased to approximately 80% within the same time interval.