Determination of proline-hydroxyproline dipeptide in rat urine by high-performance anion-exchange chromatography coupled with pulsed amperometric detection

A method for determination of proline-hydroxyproline dipeptide (PHP) was developed using high-performance anion-exchange chromatography-pulsed amperometric detection (HPAEC-PAD). This method resulted in good separation of proline (Pro), hydroxyproline (Hyp), and PHP within 20 min using a mobile phase of 1.2 mM Ba(OH)2 + 1.5 mM Ba(OAc)2. The linear dynamic ranges and their detection limits (S/N = 3) were 1-100 (r2 = 0.9990-0.9999) and 0.05-0.3 μM, respectively. Mean recoveries were 91.6-121.3% and 92.2-110.3% for intra- and inter-day assays, respectively. Our HPAEC-PAD method showed clear differences in the corrected PHP levels measured in urine samples from two groups of rats, sham-operated and ovariectomized, without the need for prior acid hydrolysis or sample derivatization.