反相高效液相色谱法测定蟾酥中的3种蟾毒内酯

建立了一种基于毛细管反相液相色谱-串联质谱联用技术和质谱峰强度数据处理的肽段鉴定和相对定量分析方法。该方法无需对样品中的肽进行化学标记,在对样品进行反相色谱分离和串联质谱分析后,将二级质谱扫描数据进行蛋白质数据库搜索，获得所鉴定肽段的序列、保留时间、质荷比、带电荷数等定性信息；再以此为定位依据，在全扫描质谱数据中提取该肽段对应的离子峰并以该离子峰的峰强度作为定量信息,从而实现对不同样品中的共有肽段进行差异比较分析。以标准蛋白酶解混合肽段为实验对象,以肽段相对强度的相对标准偏差为指标,考察了该方法用于肽段相对定量分析的重现性、检测动态范围以及浓度标准曲线等,为将该方法用于生物样品中内源性肽的差异分析奠定了基础。。

Determination of three bufogenins in toad venom using reversed-phase high performance liquid chromatography

A reversed-phase high performance liquid chromatographic method for the quantitative determination of three bufogenins in toad venom was established. An Alltech Alltima C18 column (250 mm x 4.6 mm, 5 microm) was used. The mobile phase was acetonitrile-0.1 mol/L acetate buffer (pH 3.2) (50:50, v/v), and the flow rate was 0.8 mL/min. The detection wavelength was set at 299 nm. The method has good linearity in the ranges of 0. 25 -0.875 microg for cinobufagin (r = 0.999 0), 0.25 -0.875 microg for resibufogenin (r = 0.999 1), and 0.15 - 0.525 microg for bufalin (r = 0. 999 0). The average recoveries were 100.3%, 100.0% and 98.0% for cinobufagin, resibufogenin, and bufalin, respectively. The results indicate that the method is simple, accurate, reproducible, and can be used for the quality control of bufogenins in toad venom.