Systems and Synthetic Biology, Department of Chemical and Biological Engineering, Chalmers University of Technology, Göthenborg, Sweden.
Anal Chem. 2013 May 21;85(10):4912-9. doi: 10.1021/ac3032405. Epub 2013 May 13.
We here present simple and rapid methods for fast screening of yeast lipids in Saccharomyces cerevisiae. First we introduced a microwave-assisted technique for fast lipid extraction that allows the extraction of lipids within 10 min. The new method enhances extraction rate by 27 times, while maintaining product yields comparable to conventional methods (n = 14, P > 0.05). The recovery (n = 3) from spiking of synthetic standards were 92 ± 6% for cholesterol, 95 ± 4% for triacylglycerol, and 92 ± 4% for free fatty acids. Additionally, the new extraction method combines cell disruption and extraction in one step, and the approach, therefore, not only greatly simplifies sample handling but also reduces analysis time and minimizes sample loss during sample preparation. Second, we developed a chromatographic separation that allowed separation of neutral and polar lipids from the extracted samples within a single run. The separation was performed based on a three gradient solvent system combined with hydrophilic interaction liquid chromatography-HPLC followed by detection using a charged aerosol detector. The method was shown to be highly reproducible in terms of retention time of the analytes (intraday; 0.002-0.034% RSD; n = 10, interday; 0.04-1.35% RSD; n = 5) and peak area (intraday; 0.63-6% RSD; n = 10, interday; 4-12% RSD; n = 5).
我们在此介绍了一种用于快速筛选酿酒酵母中酵母脂质的简单快速方法。首先，我们引入了一种微波辅助的快速脂质提取技术，可在 10 分钟内提取脂质。新方法将提取速度提高了 27 倍，同时保持与传统方法相当的产物收率（n = 14，P > 0.05）。从合成标准品中添加的回收率（n = 3）分别为胆固醇 92 ± 6%、三酰基甘油 95 ± 4%和游离脂肪酸 92 ± 4%。此外，新的提取方法将细胞破碎和提取结合在一步中，因此不仅大大简化了样品处理，而且还缩短了分析时间并最大程度地减少了样品制备过程中的损失。其次，我们开发了一种色谱分离方法，可在单次运行中从提取的样品中分离中性和极性脂质。该分离基于三梯度溶剂系统与亲水相互作用液相色谱-HPLC 相结合，然后使用带电气溶胶检测器进行检测。该方法在分析物的保留时间（日内，0.002-0.034%RSD；n = 10；日间，0.04-1.35%RSD；n = 5）和峰面积（日内，0.63-6%RSD；n = 10；日间，4-12%RSD；n = 5）方面均表现出高度重现性。
