Reversed-phase high performance liquid chromatography assay for determining 4 kinds of markers of diabetic nephro- pathy in urine

AIM: To establish a new reversed-phase high performance liquid chromatography (RP-HPLC) method for assay 4 kinds of non-protein-nitrogen markers of human diabetic nephro- pathy (DN), creatine (Cr), creatinine (Cn), urea (U) and uric acid (Ua) in DN patients' urines. METHODS: The urine samples were assayed by RP-HPLC on C18 column (200 mm× 4.6 mm, id 5 μm) at the ambient temperature and a wave length of 200 nm, using a 10 mmol/L pH 6.86 phosphate buffer [consisted 30%(V/V) methanol] as mobile phase with the flow rate of 0.9 mL/min. RESULTS: Baseline separation of the urine could be easily achieved in 5 min after a simple sample preparation. The method also displayed a high reproducibility (RSD values of retention time and peak area were no more than 3%) and the calibration curves showed good linearities in the range of 5-300 μmol/L, 10-200 μmol/L, 1-30 μmol/L, 10-1500 μmol/L for Cr, Cn, U and Ua respectively. CONCLUSION: This method has great potential for routine assay of clinical urine samples and monitoring DN early.