DETERMINATION OF PROLINE, HYDROXYPROLINE, AND N–ETHYLGLYCINE IN URINE BY USING A NEW HPLC LABELING REAGENT, AND ITS APPLICATION IN DETECTION OF TUMOR MARKERS

Abstract

According to previous reports, the concentration of urinary proline (Pro), hydroxyproline (Hyp), and N-ethylglycine (Neg) varies in association with various diseases, especially, Neg as a tumor marker was recently found in association with bone metastasis cancers. We developed a low-cost, highly sensitive precolumn high–performance liquid chromatography (HPLC) method for simultaneous determination of Pro, Hyp, and Neg in urine of patients with cancers, bone metastasis cancers, and bone metastasis cancers that have appeared after radiotherapy and chemotherapy treatment. The analytes in the urine were labeled with 4–methoxybenzenesulfonyl fluoride (MOBS–F) at 40°C. The derivatives were separated on a reversed-phase column by gradient elution and monitored with ultraviolet (UV) detection at 232 nm. The detection limits for Pro, Hyp, and Neg were 6.0 pmol/injection, 4.0 pmol/injection, and 50 pmol/injection (S/N = 3), respectively. To some extent, Neg levels in their urine show there were relevance to their health conditions or therapy progress. We believe that the developed method may be a promising measure for providing a useful reference to diagnose the illness and monitor the results for bone metastasis cancers after radiotherapy and chemotherapy treatment.