Dinitrophenyl derivatization of imino acids, spectral characteristics and HPLC analysis: application in urinary peptide-derived hydroxyproline and proline assay

Abstract

Background: Assay of urinary imino acids, in particular peptide derived, is of immense utility in diagnosis of collagen-related disorders. The often-used methods for hydrolysis of urinary peptides need a long time and are cumbersome, hence the need for relatively simpler, but effective methods. Methods: The method described, based on alkaline hydrolysis by autoclaving for 60 min followed by pre-column dinitrophenyl (DNP) derivatization and high-performance liquid chromatography (HPLC) analysis, demonstrates the complete hydrolysis and stability of urinary peptide derived imino acids. Results: DNP derivatives of both imino acids had identical λ max (380 nm) with molar ε of 28.224 x 103 and 17.036 x 103, respectively, for hydroxyproline (Hyp) and proline (Pro). HPLC run, extending up to 18 min, resolved major components of collagen products, namely Hyp, Hyl, Gly, Pro and Lys, with retention times of 6.5, 9.8, 10.5, 11.2 and 12.55 min, respectively. The assay method conformed to linear response for individual amino acid concentrations of 0.5-4.0 nmol per injection, with goodness of fit ( r2 value) 0.99 for both Hyp and Pro, and detection limit of 0.05-4.0 nmol of DNP derivatives. The recovery of Pro and Hyp, when spiked with urine prior to hydrolysis, were found to be 95% and 92%, respectively. Conclusion: Alkaline hydrolysis by autoclaving and DNP derivatization of imino acids followed by HPLC provides a method for the analysis of peptide-derived Hyp and Pro in urine. Hence, it is of utility to study collagen disorders.