[Simultaneous determination of neopterin and biopterin in urine by high performance liquid chromatography]

A method was established for simultaneous determination of neopterin (NP) and biopterin (BP) in urine by high performance liquid chromatography-fluorescence detection.

column, Hypersil BDS C18 (4.6 mm i. d. x 200 mm, 5 microm); mobile phase, methanol-water (10:90, v/v); flow rate, 0.5 mL/min; column temperature, 20 degrees C; excitation wavelength, 360 nm; emission wavelength, 440 nm. Urine samples were first precipitated with trichloroacetic acid, then centrifuged at 12 000 r/min for 15 min at 4 degrees C, and finally added with NaOH to neutralize the supernatant. Injection volume was 30 microL. The linear ranges of determination of the standards NP and BP were 0.12 - 1000 microg/L and 0.48 -1000 microg/L, with the detection limits of 0.01 microg/L and 0.1 mictog/L, the relative standard deviations (RSDs) in urine of 1.09% and 3.39%, and average recoveries of 100.7% and 100.3%, respectively. The concentrations of NP and BP, and concentration ratio of NP and BP in urines were detected and compared between controls and uremia patients. The concentration of creatinine (Cr) in urines was detected with picric acid method. The concentration ratios of NP and Cr, NP and BP in uremia group increased more significantly than that in normal group (P < 0.05). The method is simple, fast, accurate and convenient. It can be used to detect NP and BP for clinical urine samples and provide a more convenient and accurate tool for the diagnosis of patients.