Determination of D- and L-aspartate in cell culturing medium, within cells of MPT1 cell line and in rat blood by a column-switching high-performance liquid chromatographic method
Determination of D- and L-aspartate in cell culturing medium, within cells of MPT1 cell line and in rat blood by a column-switching high-performance liquid chromatographic method
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DOI：
10.1016/s0378-4347(01)00311-5
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发表时间：
2001-09-15
影响因子：
3
通讯作者：
Homma, H
中科院分区：
文献类型：
--
作者：
Long, ZQ;Nimura, N;Homma, H
建立了生物样品中痕量D-氨基酸的高效液相色谱荧光分析方法。在这项研究中，我们建立了一个方便的柱切换荧光高效液相色谱系统，以改善D-氨基酸，特别是D-天冬氨酸(Asp)的分析。我们的系统包括用NBD-F进行氨基酸的荧光衍生化和两个色层步骤，一个是以反相模式分离单个氨基酸，另一个是以正相模式分离每个氨基酸的手性形式。两个分离步骤通过自动柱切换系统通过捕集柱连接。此外，还简化和改进了样品的制备，用三氯乙酸法进行脱蛋白，用pH 9.5的硼酸盐缓冲液进行荧光衍生化。培养基中D-天冬氨酸的检出限为5 nM。由此产生的峰高与D-天冬氨酸和L-天冬氨酸的浓度范围从12.5nM到Z50nM有很好的相关性。应用本方法测定了细胞培养上清液中D-天冬氨酸和L-天冬氨酸的含量。检测到的D-天冬氨酸和L-天冬氨酸的细胞水平与我们以前的方法检测到的一致。此外，用该方法测定了大鼠血中D-天冬氨酸和L-天冬氨酸的含量，结果与文献报道值一致。(C)2001 Elsevier Science B.V.保留所有权利。
HPLC fluorometric methods have been used to analyze trace amounts Of D-amino acids in biological samples. In this study, we established an expedient column-switching fluorometric HPLC system that would improve the analysis Of D-amino, acids, in particular D-aspartate (Asp). Our system consists of the fluorogenic derivatization of amino acids with NBD-F and two chromatographic steps, one that separates individual amino acids in reverse phase mode and another that separates the chiral forms of each amino acid in normal-phase mode. The two separation steps are linked through a trapping column by an automated column-switching system. In addition, sample preparation is simplified and improved, where trichloroacetic acid is used for deproteinization, and borate buffer, pH 9.5 is employed for the fluorescent derivatization. The detection limit for D-Asp in culturing medium is 5 nM. The resulting peak heights correlated well with concentrations that ranged from 12.5 to Z50 nM for both D- and L-Asp. The present method was applied to determine D- and L-Asp levels in cell culturing medium, and within cells of MPT1 cell line. The detected cellular levels Of D- and L-Asp agree with those detected by our previous method. In addition, this method was used to measure D- and L-Asp levels in rat blood samples, and the results are consistent with the reported values. (C) 2001 Elsevier Science B.V. All rights reserved.