高效液相色谱分析鱼毒性藻类海洋卡盾藻溶血毒素

Analyses of Hemolytic Toxin from Ichthyotoxic Phytoplankton Chattonella marina(Hong Kong Strain) by High Performance Liquid Chromatography

采用高效液相色谱技术(HPLC)分离和分析了海洋卡盾藻(香港株)溶血毒素的甲醇粗提物,通过对比不同流动相、检测波长、等度和梯度洗脱方式,初步建立了分析其溶血毒素的HPLC方法。结果表明,采用梯度洗脱的方法,海洋卡盾藻溶血毒素的粗提物在50 min内取得了较好地分离。色谱条件为C8硅胶柱(4.6 mm×150 mm,5μm),流动相为乙腈和水,梯度洗脱(0～10 min,60%→80%乙腈;10～40 min,80%→90%乙腈;40～50 min,90%乙腈);紫外检测器检测波长为205和448 nm;流速为0.8 mL/min;柱温为25℃。用兔血红细胞法对各个主要色谱峰进行溶血活性检测显示,海洋卡盾藻(香港株)的溶血毒素至少含有5种成分。光谱分析显示一种成分的紫外吸收高峰在448 nm,一种未完全分离的混合组分的紫外吸收高峰为440和446 nm,另外3种成分的紫外吸收高峰为205 nm。

Chattonella marina is a typical fish-killing HAB（Harmful Algae Bloom） species,which could produce hemolytic toxin and result in mass mortality of fishes.In order to understand the transmission and transformation of hemolytic toxin in food chain,high performance liquid chromatography（HPLC） with gradient elution has been used to separate hemolytic toxin extracted from C.marina.Different solvent systems,detection wavelength,isocratic elution and gradient elution for HPLC were compared.The separation is carried out on a C8 column（4.6 mm×150 mm,5 μm） with acetonitrile/water as mobile phase,gradient elution（0-10 min 60%-80% acetonitrile,10-40 min 80%-90% acetonitrile,40-50 min 90% acetonitrile） with a flow rate of 0.8 mL/min.The detection wavelength is 205 nm and 448 nm,and the column temperature is 25 ℃.Hemolysis bioassay shows that there are at least five components in C.marina（Hong Kong stain）.One component had the maximum ultraviolet（UV） absorption at 448 nm,another that was not separated completely had the maxima at 440 nm and 446 nm,and the others all had the maximal peak at 205 nm.