Fast and quantitative analysis of branched-chain amino acids in biological samples using a pillar array column

Abstract

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.

Advances in and prospects of microchip liquid chromatography

“…Alternatively, COP chips with a pillar array column containing 10 μm diameter diamond pillars (4 μm inter-pillar distance) embossed from a silicon master with the hydrophobic nature of the substrate acting as a stationary phase for LC separation [61]. Bioanalytical applications utilizing pillar array columns with reversed phase functionality have included separations of DNA strands [62] and branched chain amino acids [63]. …”

Amino Acid Analysis Using a Cartridge-Type Monolithic Silica Column

“…Sample derivatization was performed according to previous studies [10][11][12]. A volume of 20 μL of the sample was added to 180 μL of a 0.2 M borate buffer (pH 8.5).…”

Retention and Bandwidth Predictions by Fast Gradient Elution Chromatography Using a Pillar Array Column

“…The microchip fabrication has been described in previous studies [7][8][9][10]. The 20 mm × 20 mm microchip was composed of a cross-Tesla mixer, a separation channel, and a sample channel.…”

“…In addition, pillar array columns have been utilized to separate biological compounds. Six fluorescently labeled amino acids were analyzed within 100 s by isocratic elution using the pillar array column [7,8]. Furthermore, to separate the components of complex biological samples, a gradient elution system was developed to accelerate the elution of strongly retained solutes using pressure-driven liquid chromatography (LC) on a microchip [9,10].…”