淀山湖水源水微囊藻毒素-RR、-YR和-LR的HPLC测定

目的应用上海水源水淀山湖藻类样品,优化微囊藻毒素(microcystin-RR,-YR,-LR)提取、纯化和HPLC检测方法。方法分别以5%乙酸和75%甲醇为提取溶剂,80%甲醇(加三氟乙酸)为洗脱溶剂,采用不同温度(室温和56℃)和离心条件(10000×g,30min和3500r/min,10min),提取和纯化样品中的微囊藻毒素。应用HPLC,以乙腈、水和TFA为流动相,梯度洗脱检测微囊藻毒素。结果5%乙酸提取冻干藻细胞时,56℃水浴加低速离心是一个简单有效提取MC-RR的方法;80%甲醇洗脱溶剂中加入三氟乙酸能明显增加微囊藻毒素的洗脱;淀山湖藻类样品中微囊藻毒素含量由高到低依次为MC-RR,MC-LR和MC-YR。结论提取冻干藻细胞中的微囊藻毒素,以75%甲醇为提取溶剂,80%甲醇+0·05%TFA为洗脱溶剂MC-YR和MC-LR提取效率高;5%乙酸提取MC-RR效率高。

HPLC analysis of microcystin-RR, YR and LR from Dianshan Lake

OBJECTIVE: To optimize the solid-phase extraction method of isolation, purification and determination of intracellular microcystin-RR, -YR and -LR (MC-RR,-YR,-LR), algal samples were collected from Dianshan Lake, which is a major water source of Shanghai. METHODS: During extraction, Algal samples were treated with different solvent (5% acetic acid and 75% methanol), at different temperature (room temperature and 56 degrees C), centrifuge at 10000 x g, 30 min and 3500 rpm/min, 10 min, eluted with 80% methanol with or without trifluoroacetate acid(TFA). Further, gradient elution were employed to separate microcystins in high performance liquid chromatography (HPLC), the mobile phase consisted of aqueous acetonitrile and 0.05% TFA. RESULTS: When dried algae cells were extracted by 5% acetic acid, 56 degrees C water bath combined with centrifuge at 3500 rpm/min was an effective and simple method for the extraction of microcystin-RR, elutant contains TFA can markedly increase the yield of microcystins extraction; the concentration of microcystin-RR in nature algal samples was higher than MC-LR and MC-YR. CONCLUSION: The extraction efficacy of MC-YR and -LR is higher in 75% methanol aqueous and eluted with 80% methanol containing 0.05% TFA, while the extraction efficacy of MC-RR is higher in 5% acetic acid.