National Food Institute, Technical University of Denmark, Kemitorvet Building 202, 2800 Lyngby, Denmark.
Department of Biotechnology and Biomedicine, Technical University of Denmark, Søtofts Plads, Building 221, 2800 Lyngby, Denmark.
Mar Drugs. 2017 Aug 31;15(9):278. doi: 10.3390/md15090278.
Being able to quantify ichthyotoxic metabolites from microalgae allows for the determination of ecologically-relevant concentrations that can be simulated in laboratory experiments, as well as to investigate bioaccumulation and degradation. Here, the ichthyotoxin karmitoxin, produced by , was quantified in laboratory-grown cultures using high-performance liquid chromatography (HPLC) coupled to electrospray ionisation high-resolution time-of-flight mass spectrometry (HRMS). Prior to the quantification of karmitoxin, a standard of karmitoxin was purified from cultures (80 L). The standard was quantified by fluorescent derivatisation using Waters AccQ-Fluor reagent and derivatised fumonisin B₁ and fumonisin B₂ as standards, as each contain a primary amine. Various sample preparation methods for whole culture samples were assessed, including six different solid phase extraction substrates. During analysis of culture samples, MS source conditions were monitored with chloramphenicol and valinomycin as external standards over prolonged injection sequences (>12 h) and karmitoxin concentrations were determined using the response factor of a closely eluting iturin A2 internal standard. Using this method the limit of quantification was 0.11 μg·mL, and the limit of detection was found to be 0.03 μg·mL. Matrix effects were determined with the use of cultures grown with C-labelled bicarbonate as the primary carbon source.
能够量化微藻中的鱼毒性代谢物，可以确定在实验室实验中可以模拟的生态相关浓度，以及研究生物积累和降解。在这里，使用高效液相色谱（HPLC）与电喷雾电离高分辨率飞行时间质谱（HRMS）联用，对实验室培养的产生的鱼毒素卡米毒素进行了定量。在定量卡米毒素之前，从 培养物（80 L）中纯化了卡米毒素标准品。该标准品通过 Waters AccQ-Fluor 试剂进行荧光衍生化，并以衍生化的伏马菌素 B₁和伏马菌素 B₂作为标准品进行定量，因为它们都含有伯胺。评估了各种全培养物样品的制备方法，包括六种不同的固相萃取基质。在分析培养物样品时，用氯霉素和缬氨霉素作为外部标准监测 MS 源条件，在长时间的注射序列（>12 h）中，使用与紧密洗脱的伊曲康唑 A2 内标物的响应因子来确定卡米毒素浓度。使用该方法，定量下限为 0.11 μg·mL，检测下限为 0.03 μg·mL。使用以 13C 标记的碳酸氢盐作为主要碳源培养的 培养物确定了基质效应。
