HPLC法同时测定水蛭（蚂蟥）中7个成分的含量

目的：建立HPLC法同时测定水蛭（蚂蟥）药材中尿嘧啶、次黄嘌呤、黄嘌呤、尿苷、水蛭胺C、水蛭胺A、水蛭胺B的含量。方法：采用YMC-Pack ODS-AQ色谱柱（250 mm×4.6 mm，5 μm），以10 mmol·L^-1的磷酸二氢钾溶液为流动相A，乙腈为流动相B，梯度洗脱（0~15 min，0%B；15~40 min，0%B→20%B；40~60 min，20%B），流速为0.8 mL·min^-1，检测波长为254 nm，柱温为30 ℃。结果：尿嘧啶、次黄嘌呤、黄嘌呤、尿苷、水蛭胺C、水蛭胺A、水蛭胺B的线性范围分别为0.01855~0.3710 μg（r=0.9999）、0.04050~0.8100 μg（r=0.9999）、0.01595~0.3190 μg（r=0.9998）、0.01250~0.2500 μg（r=0.9997）、0.01415~0.2830 μg（r=0.9998）、0.01530~0.3060 μg（r=0.9997）、0.01445~0.2890 μg（r=0.9999）；平均加样回收率（n=6）分别为100.2%、99.5%、100.1%、99.0%、100.0%、100.1%、100.6%，RSD分别为0.89%、0.85%、1.2%、1.3%、1.2%、1.2%、1.1%。结论：本文建立的方法经方法学验证可用于评价水蛭（蚂蟥）药材质量。水蛭药材含量测定结果显示，尿嘧啶、次黄嘌呤、黄嘌呤、尿苷、水蛭胺C、水蛭胺A、水蛭胺B为以蚂蟥W.pigra入药的水蛭药材的共有成分，以7个成分的总体含量来控制药材质量更为合理。

Objective: To establish an HPLC method for simultaneous determination of seven components including uracil,hypoxanthine,xanthine,uridine,hirudonucleodisulfide C,hirudonucleodisulfide A and hirudonucleodisulfide B in dried Whitmania pigra. Methods: The chromatographic separation was achieved on a YMC-Pack ODS-AQ column (250 mm×4.6 mm,5 μm) and the column temperature was at 30 ℃.The mobile phase consisted of a water solution containing 10 mmol·L^-1 potassium dihydrogen phosphate (A) and acetonitrile (B) with gradient elution(0-15 min,0%B;15-40 min,0%B→20%B;40-60 min,20%B) at a flow rate of 0.8 mL·min^-1,and the detection wavelength was set at 254 nm.The column temperature was 30 ℃. Results: The calibration curves were linear in the ranges of 0.01855-0.3710 μg for uracil (r=0.9999),0.04050-0.8100 μg for hypoxanthine (r=0.9999),0.01595-0.3190 μg for xanthine (r=0.9998),0.01250-0.2500 μg for uridine (r=0.9997),0.01415-0.2830 μg for hirudonucleodisulfide C (r=0.9998),0.01530-0.3060 μg for hirudonucleodisulfide A (r=0.9997) and 0.01445-0.2890 μg for hirudonucleodisulfide B (r=0.9999).The average recoveries (n=6) of these seven components were 100.2% (RSD=0.89%),99.5% (RSD=0.85%),100.1% (RSD=1.2%),99.0% (RSD=1.3%),100.0% (RSD=1.2%),100.1% (RSD=1.2%) and 100.6% (RSD=1.1%),respectively. Conclusion: The method this paper established can be applied to the quality control of dried Whitmania pigra.according to the methodology validation.The determination results of leech medicinal material content shows that it is more reasonable to control the quality of medicinal materials by detection of the total content of seven components.