Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan.
Anal Bioanal Chem. 2013 Oct;405(25):7993-9. doi: 10.1007/s00216-013-7034-7. Epub 2013 May 25.
In this study, a fast and quantitative determination method for branched-chain amino acids (BCAAs), namely leucine, isoleucine, and valine, was developed using a pillar array column. A pillar array column with low-dispersion turns was fabricated on a 20 × 20-mm(2) microchip using multistep ultraviolet photolithography and deep reactive ion etching. The BCAAs were fluorescently labeled with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), followed by reversed-phase separation on the pillar array column. The NBD derivatives of the three BCAAs and an internal standard (6-aminocaproic acid) were separated in 100 s. The calibration curves for the NBD-BCAAs had good linearity in the range of 0.4-20 μM, using an internal standard. The intra- and interday precisions were found to be in the ranges of 1.42-3.80 and 2.74-6.97%, respectively. The accuracies for the NBD-BCAA were from 90.2 to 99.1%. The method was used for the analysis of sports drink and human plasma samples. The concentrations of BCAAs determined by the developed method showed good agreements with those determined using a conventional high-performance liquid chromatography system. As BCAAs are important biomarkers of some diseases, these results showed that the developed method could be a potential diagnostic tool in clinical research.
本研究采用柱阵列柱开发了一种快速定量测定支链氨基酸（BCAAs），即亮氨酸、异亮氨酸和缬氨酸的方法。使用多步紫外光刻和深反应离子刻蚀，在 20×20mm(2)微芯片上制造了具有低分散转弯的柱阵列柱。BCAAs 用 4-氟-7-硝基-2,1,3-苯并恶二唑（NBD-F）荧光标记，然后在柱阵列柱上进行反相分离。三种 BCAAs 和内标（6-氨基己酸）的 NBD 衍生物在 100 秒内得到分离。使用内标，NBD-BCAAs 的校准曲线在 0.4-20 μM 范围内具有良好的线性。日内和日间精密度分别在 1.42-3.80%和 2.74-6.97%范围内。NBD-BCAA 的准确度为 90.2-99.1%。该方法用于运动饮料和人血浆样品的分析。用开发的方法测定的 BCAAs 浓度与用常规高效液相色谱系统测定的浓度具有良好的一致性。由于 BCAAs 是某些疾病的重要生物标志物，这些结果表明，该方法可能成为临床研究中的一种潜在诊断工具。
