A Novel Determination Method of Thirty-Seven<i> o</i>-Phthalaldehyde-Derivatized D/L-Amino Acids with Complementary Use of Two Chiral Thiols by High Performance Liquid Chromatography

Abstract

Simultaneous separation of 37 proteinogenic D/L-amino acids excluding proline with HPLC is generally difficult. In this study, two sets of o-phthalaldehyde (OPA)-based fluorescent diastereomers of D/L-amino acids were formed using two chiral thiols of N-acetyl-L-cysteine (NAC) and N-isobutyryl-L-cysteine (NIBC) independently. It is expected that obtained two sets of OPA-derivatized D/L diastereomers show different retention selectivity in reversed phase chromatography because NAC and NIBC have different hydrophobicities. The two independent HPLC methods were designed to obtain complementary separations for the two sets of fluorescent diastereomers of the proteinogenic D/L-amino acids. The obtained two complementary chromatograms provided complete determination for diastereomers of proteinogenic D/L-amino acids. After the optimization of HPLC separation conditions, two types of automated analytical procedures were investigated. One was for OPA/NAC and OPA/NIBC derivatization of amino acids using originally equipped pretreatment functions of auto-sampler. The other was for switching the two HPLC separation methods during the consecutive batch run. Through these re-consideration for automated procedures, finally obtained method afforded satisfactory peak area repeatability of 1.5%RSD or less and calibration curve linearity of 0.999 or greater for each diastereomers. The established methods were applied to the real liquor samples and successful results were obtained.

Development of Sensitive and Simultaneous Determination Method for Thirty-Seven D/L-Amino Acids by Automatic Pre-column <i>o</i>-Phthalaldehyde- Derivatization with Chiral Thiol Using High Performance Liquid Chromatography

“…In the previously established method, the 37 D/L-amino acids were separated complementarily from the obtained both results using two different types of derivatizing reagents and taking advantage of the difference in hydrophobicity [29]. In this study, the 37 D/L-amino acids were separated within 66 minutes under a set of analytical conditions of OPA/NIBC-derivatized diastereomers by increasing the methanol ratio of mobile phase and keeping the column temperature to 20 o C. Using C8 as the stationary phase in reversed phase chromatography, good separation of D/L-Asp diastereomers having the most hydrophilicity was difficult without the nearly 100% aqueous mobile phase, and simultaneous separation took a long time.…”

“…Obtained two sets of derivatized proteinogenic D/L-amino acids showed different retention selectivities in reversed phase chromatography. The complete individual determinations of proteinogenic D/L-amino acids were achieved by executing two independent HPLC analyses on the same sample [29]. Developing a simple operational method that provides good D/L-amino acid separation in a short time, it is necessary to separate and determine the D/L-amino acid in a shorter time by analyzing once per sample.…”

Design of Novel Mixed-Mode Columns for Analysis of Amino Acids Derivatized with 4-Fluoro-7-nitro-2,1,3-benzoxadiazole and the Evaluation of Retention Properties

“…For the chiral amino acid analysis, various methods, such as gas chromatography (GC) [19], capillary electrophoresis (CE) [20,21] and high-performance liquid chromatography (HPLC) [22][23][24][25], have been reported. Concerning the methods utilized for the real biological samples, the combination of LC separation with mass spectrometric detection (LC-MS and LC-MS/MS) is one of the useful tools and various methods have been developed and applied to food and clinical samples including humans [26][27][28][29][30].…”