Herrero Miguel, Ibáñez Elena, Fanali Salvatore, Cifuentes Alejandro
Institute of Industrial Fermentations (CSIC), Madrid, Spain.
Electrophoresis. 2007 Aug;28(15):2701-9. doi: 10.1002/elps.200600599.
In this work, chiral and nonchiral MEKC methods have been combined with LIF detection (MEKC-LIF) to identify and quantify a group of D- and L-amino acids (D/L-aa) in different microalgae samples. The combination of the nonchiral and chiral-MEKC-LIF methods made the identification of the microalgae amino acids easier, previously derivatized with FITC, providing a double proof on the correct detection of these analytes. Three microalgae species, Spirulina platensis, Dunaliella salina, and Tetraselmis suecica, were compared in terms of their content in D-Arg, L-Arg, D-Lys, L-Lys, D-Ala, L-Ala, D-Glu, L-Glu, D-Asp, and L-Asp. Also, a comparison between two Spirulina platensis samples dried under different conditions (i.e., hot air or lyophilized) was carried out in order to investigate the effect of the thermal processing on the amino acid content. Moreover, two procedures for the extraction of amino acids from microalgae (i.e., a classical procedure and pressurized liquid extraction (PLE)) together with different conditions for amino acid derivatization were studied in order to increase the sensitivity of the whole analytical method. By using the selected chiral-MEKC-LIF conditions (100 mM sodium tetraborate, 30 mM SDS, and 20 mM beta-CD at pH 9.7) the main microalgae D/L-aa are separated in less than 25 min with efficiencies up to 840 000 plates/m and good sensitivity (i.e., 330 ng of D-Arg per gram of microalga could be detected by this procedure for an S/N of 3). Several D-aa were detected in all the microalgae, observing interesting differences in their D/L-aa profiles, what corroborates the usefulness of the chiral-MEKC-LIF approach to characterize different microalgae species as well as different microalgae drying processes. Moreover, the use of PLE can selectively extract different free amino acids from microalgae.
在本研究中，手性和非手性毛细管电泳-激光诱导荧光检测法（MEKC-LIF）相结合，用于鉴定和定量不同微藻样品中的一组D-和L-氨基酸（D/L-aa）。非手性和手性MEKC-LIF方法的结合使得微藻氨基酸的鉴定更加容易，这些氨基酸之前用异硫氰酸荧光素（FITC）进行了衍生化，为这些分析物的正确检测提供了双重验证。比较了三种微藻，即钝顶螺旋藻、盐生杜氏藻和瑞典四爿藻中D-精氨酸、L-精氨酸、D-赖氨酸、L-赖氨酸、D-丙氨酸、L-丙氨酸、D-谷氨酸、L-谷氨酸、D-天冬氨酸和L-天冬氨酸的含量。此外，还对在不同条件下（即热风干燥或冻干）干燥的两种钝顶螺旋藻样品进行了比较，以研究热处理对氨基酸含量的影响。此外，研究了两种从微藻中提取氨基酸的方法（即经典方法和加压液体萃取（PLE））以及不同的氨基酸衍生化条件，以提高整个分析方法的灵敏度。通过使用选定的手性MEKC-LIF条件（100 mM硼酸钠、30 mM十二烷基硫酸钠和20 mMβ-环糊精，pH 9.7），主要的微藻D/L-aa在不到25分钟内分离，柱效高达840000理论塔板数/米，灵敏度良好（即每克微藻中330 ng的D-精氨酸可通过该方法检测到，信噪比为3）。在所有微藻中均检测到了几种D-氨基酸，观察到它们的D/L-aa谱存在有趣的差异，这证实了手性MEKC-LIF方法在表征不同微藻物种以及不同微藻干燥过程方面的有用性。此外，使用PLE可以从微藻中选择性地提取不同的游离氨基酸。
