HPLC in determination of hypoxanthine,xanthine,uridine and uracil in Shanghai Pheretima and Pheretima aspergillum(E.Perrier)

Objective To establish an effective method for the determining hypoxanthine,xanthine,uridine and uracil in earthworm in Shanghai Pheretima and Pheretima aspergillum(E.Perrier),contributing to quality control of the medicinal material.Methods Hypoxanthine,xanthine,uridine and uracil were extracted from the earthworms with 0.9% NaCl by ultrasonic and determined by HPLC.The chromatographic conditions were:SORBAX SB-Aq column(250 mm×4.6 mm,5μm,Aglient Co.,Ltd),5 mmol/L KH2PO4(pH 2.9)as the mobile phase with a flow rate of 1 mL/min,the detection wavelength was 254 nm,the column temperature was set at 30℃,and the injection volume was 10μL.Hypoxanthine,xanthine,uridine and uracil were determined by HPLC at the same time.Results The linear range was 0.5-100μg(r=0.999 9)for hypoxanthine,with the average recovery being 99.37%,RSD=1.36%(n=6).The linear range was 0.5-100μg(r=0.993 1)for xanthine,with the average recovery being 91.57%,RSD=1.40%(n=6).The linear range was 0.5-100μg(r=0.999 9)for uridine,with the average recovery being 95.31%,RSD=1.64%(n=6).The linear range was 0.5-100μg(r=0.999 9)for uracil,with the average recovery being 100.21%,RSD=1.98%(n=6).Conclusion The current method is reproducible and has satisfactory recovery,and it can be used to determine hypoxanthine,xanthine,uridine and uracil in earthworm medicinal material.