Automated and simultaneous two-dimensional micro-high-performance liquid chromatographic determination of proline and hydroxyproline enantiomers in mammals

Establishment of a Two-Dimensional HPLC-MS/MS Method Combined with DCl/D₂O Hydrolysis for the Determination of Trace Amounts of D-Amino Acid Residues in Proteins

“…The five target D-amino acids were selected because these D-amino acids are frequently observed in higher animals (as their free forms) [2][3][4][5]. In the first dimension, the five target NBD-amino acids were separated using a capillary monolithic ODS column, and the elution conditions were optimized.…”

“…All proteinogenic amino acids, except glycine, have optical isomers (enantiomers), namely the D-and L-forms, and L-amino acids are predominant in the higher animals. However, from the 1980s, several D-amino acids have been reported to be present as their free forms in mammalian tissues including human beings, and these D-amino acids are expected to be novel physiologically active substances and biomarkers [1][2][3][4][5]. In the proteins and peptides, D-amino acid residues, especially D-Asp, have also been reported in various disease/aged proteins including Alzheimer's disease and cataract [6][7][8][9][10][11][12].…”

“…In the present study, an analytical method has been established for the determination of D-Ala, D-Asp, D-Glu, D-Pro and D-Ser (these 5 D-amino acids are frequently observed in vertebrates [2][3][4][5]) residues in protein samples. For the analysis, a two-dimensional HPLC-MS/MS system was adopted after the DCl/D 2 O hydrolysis.…”

Analysis of Small Amounts of d-Amino Acids and the Study of Their Physiological Functions in Mammals

“…[29][30][31][32][33][34][35][36] For the sensitive determination, amino acids are precolumn derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole 37 (NBD-F, Fig. 1).…”

“…We have already reported the 2D-HPLC procedures for D-Ala, 31 D-Leu, 29,30 D-Ser, 36 D-Pro analogs 30,33,35 and branchedaliphatic D-amino acids. 34 The values of lower limit of quantitation of D-amino acids using these methods are 500 amol -5 fmol, and the RSD values for within-and between-day precisions are approximately 5% or lower.…”