大田软海绵酸和鳍藻毒素-1纯化样品的分析

Analysis of Purified Okadaic Acid and Dinophysis Toxin 1 from Prorocentrum LimaDodge

采用高效液相色谱-荧光检测、高效液相色谱-紫外检测、流动注射进样-质谱检测和高效液相色谱-质谱联用分析等方法对从有毒利玛原甲藻(Prorocentrum lima Dodge)中分离纯化的大田软海绵酸(OA)和鳍藻毒素-1(DTX1)毒素样品进行定性鉴定和纯度分析,为毒素标准品的制备提供材料和方法依据。通过柱前衍生、HPLC-FLD分析,可在样品中检测到OA和DTX1与4-溴甲基7-甲氧基香豆素(BrMmc)的衍生物,表明样品中存在OA和DTX1毒素。HPLC-UV方法在200nm下的检测结果表明,OA和DTX1是样品色谱图中主要的色谱峰,OA样品和DTX1样品纯度可以达到95%和98%。根据正、负离子模式下流动注射-质谱分析结果,毒素样品和标准品质谱图中信号最强离子的m/z相同,应为OA和DTX1毒素的[M+Na]+和[M#H]#离子,其裂解方式也基本一致。通过HPLC-MS方法,对样品进行了m/z200～1000的全扫描分析,发现OA样品中可检测到少量杂质,而DTX1样品中仅检测到DTX1毒素成分。分析结果表明,前期分离纯化的OA和DTX1毒素样品纯度较高,可以在腹泻性贝毒(DSP)毒素液相色谱和液相色谱-质谱联用分析中用作标准毒素。

Okadaic acid （OA） and dinophysis toxins （DTXs） are representative toxins corresponding for the diarrhetic shellfish poisordng （DSP） incidents. In the current study, the samples of OA and DTX1, which were purified previously from the cultured algal cells of dinoflagellate Prorocentrum lima Dodge, were analyzed for the identity and purity with high performance liquid chromatography coupled with fluorescence detector （HPLC-FLD） and ultraviolet detector （HPLC-UV）, mass spectrometry （MS） and HPLC-MS. Commercially available toxin standards of OA and DTX1 were also analyzed to compare with the purified samples. After derivatized with 4-bromomethyl-7-methoxycoumarin （BrMme）, OA and DTX1 were detected in the purified samples with the HPLC-FLD method. In the HPLC-UV chromatogrphy detected at 200 nm, OA and DTX1 were found to be the most predominant peaks, and the purity calculated based on the peak area could reach 95% and 98% for the samples of OA and DTX1, respectively. In the MS spectrums of the purified samples and toxin stand- ards, the highest signals in the positive mode or the negative mode had the same value of m/z, which could be identified as [M＋Na]＋ and [M-H]- of OA and DTX1. The [-M＋Na]＋ and [M-HI ions of the toxin samples showed similar fragmentation pattern as those of the toxin standards, further confirming the presence of OA and DTX1 in the purified samples. In the LC-MS analysis of the puri- fied samples with the full-scan m/z range at 100-1300, a small amount of impurities in the OA sample was found, and no impurity presented in the DTX1 sample. The results suggested that the OA and DTX1 samples prepared previously had high purity and could be used as standards for the analysis of OA and DTX1 with HPLC-FLD and HPLC-MS methods in management and protection of seafood.