Abstract:

A high performance liquid chromatographic (HPLC) method was proposed to simultaneously determine four common nonprotein nitrogen substances, including creatine (Cr), creatinine (Cn), uric acid (Ua) and pseudouridine (Pu) in urine. After proteins being removed by acetone precipitation method, freeze drying and redissolving, the urine samples were analyzed by HPLC. Chromatographic separation was performed on a Waters RP18 Column (150 mm×4.60 mm, 3.5 μm) in gradient elution mode using 10.0 mmol/L KH₂PO₄ solution (pH 4.78) and acetonitrile as mobile phases at a flow rate of 0.8 mL/min. The samples were detected at 220 nm. Rapid separation was achieved within 7 min. Under the optimized conditions, good linearities of four common nonprotein nitrogen substances were obtained in the range of 0.1-250 mg/L. The detection limits were 9.31 (Cr), 26.19 (Cn), 4.70 (Ua), and 6.30 (Pu) μg/L and the recoveries were in the range of 81%-111% with the relative standard deviations of 0.23%-2.78% (n=3). The results demonstrate that this method is simple, rapid and accurate with good reproducibility, and can provide early diagnosis and preliminary judgment for type 2 diabetes mellitus (T2DM) patients with renal damage.

Key words: creatine, creatinine, high performance liquid chromatography (HPLC), pseudouridine, uric acid, urine