Department of Chemistry and Biochemistry, Mendel University in Brno, Zemědělská 1, 613 00 Brno, Czech Republic.
J Chromatogr A. 2010 Dec 17;1217(51):7956-65. doi: 10.1016/j.chroma.2010.07.020. Epub 2010 Jul 16.
New hyphenated technique for the extraction and determination of isoflavones in sea and freshwater algae and cyanobacteria was developed. The method consists of sonication sample pretreatment, extraction by supercritical CO(2) modified by 3% (v/v) of MeOH/H(2)O mixture (9:1, v/v) at 35 MPa and 40°C for 60 min, fast chromatography analysis by the means of Agilent 1200 Series Rapid Resolution and MS/MS determination. Agilent 1200 Series RRLC was used with Zorbax SB-CN chromatographic column (100 mm × 2.1mm, particle size 3.5 μm), 3μl injection volume, mobile phase consisting of 0.2% (v/v) acetic acid in water (solvent A) and acetonitrile (solvent B) and used with linear gradient (30% B at 0 min, from 0 min to 3 min up to 50% B, from 3 to 6 min up to 80% B and from 6 to 10 min down to 30% B). The flow-rate was 0.4 mL/min, column oven temperature 35°C. MS detector Agilent Technologies 6460 Triple quadrupole LC/MS with Agilent Jet Stream was used in a negative ESI mode under following conditions: gas temperature 350°C, gas flow 13 L/min, nebulizer gas pressure 50 psi, sheath gas temperature 400°C, sheath gas flow 12L/min, capillary voltage was 4 kV. Samples were analysed in the multiple reaction monitoring (MRM) mode. Eight isoflavone compounds were found for the first time in seven real samples of sea algae and in three control samples of freshwater algae and cyanobacteria. Usual optimisation study of extraction parameters was performed. Pressure and temperature optima for algae matrix are different from those obtained sooner for other matrices for most of the analytes, but the results of modifier optimisation study are in good accordance with those obtained sooner for spiked samples and red clover matrix. It seems that matrix has very small or no effect on the modifier selection. Two different approaches of sonication pretreatment were tested: sonication bath and the thorn instrument. In longer extraction time experiments, thorn sonication was more efficient and recovery of following supercritical fluid extraction was higher.
开发了一种用于提取和测定海淡水藻类和蓝藻中异黄酮的新型双功能技术。该方法包括超声样品预处理、超临界 CO2 提取，提取溶剂为 3%（v/v）甲醇/水（9:1，v/v）混合物，在 35 MPa 和 40°C 下提取 60 分钟，采用安捷伦 1200 系列快速分辨率和 MS/MS 进行快速色谱分析。Agilent 1200 系列 RRLC 采用 Zorbax SB-CN 色谱柱（100mm×2.1mm，粒径 3.5μm），3μl 进样量，流动相由 0.2%（v/v）水中的乙酸（溶剂 A）和乙腈（溶剂 B）组成，采用线性梯度（0min 时 30%B，0min 至 3min 时上升至 50%B，3min 至 6min 时上升至 80%B，6min 至 10min 时下降至 30%B）。流速为 0.4mL/min，柱温 35°C。采用安捷伦技术 6460 三重四极杆 LC/MS 与安捷伦喷气流在负电喷雾模式下的 MS 检测器，条件如下：气体温度 350°C，气体流量 13L/min，雾化器气体压力 50psi，鞘气温度 400°C，鞘气流量 12L/min，毛细管电压为 4kV。样品在多重反应监测（MRM）模式下进行分析。在七种实际海藻类样品和三种淡水藻类和蓝藻的对照样品中首次发现了八种异黄酮化合物。对萃取参数进行了常规优化研究。对于藻类基质，压力和温度的最佳条件与之前获得的其他基质的最佳条件不同，但修饰剂优化研究的结果与之前对加标样品和红三叶草基质的研究结果非常一致。似乎基质对修饰剂的选择几乎没有影响或没有影响。测试了两种不同的超声预处理方法：超声浴和刺针仪器。在较长的提取时间实验中，刺针超声更有效，后续超临界流体萃取的回收率更高。
