RESUMEN
An automated method for high-throughput amino acid analysis, using precolumn derivatization high-performance liquid chromatography/electrospray mass spectrometry (HPLC/ESI-MS), was developed and evaluated. The precolumn derivatization step was performed in the reaction port of a home-built auto-sampler system. Amino acids were derivatized with 3-aminopyridyl-N-hydroxysuccinimidyl carbamate, and a 3 microm Wakosil-II 3C8-100HG column (100 x 2.1 mm i.d.) was used for separation. To achieve a 13 min cycle for each sample, the derivatization and separation steps were performed in parallel. The results of the method evaluation, including the linearity, and the intra- and inter-precision, were sufficient to measure physiological amino acids in human plasma samples. The relative standard deviations of typical amino acids in actual human plasma samples were below 10%.
Asunto(s)
Aminoácidos/análisis , Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Aminoácidos/sangre , Aminoácidos/metabolismo , Cromatografía Líquida de Alta Presión/instrumentación , Humanos , Espectrometría de Masa por Ionización de Electrospray/instrumentaciónRESUMEN
Sugar analysis by postcolumn labeling using phenylhydrazine with high-performance liquid chromatography is herein introduced. Phenylhydrazine reacts not only with the reducing end of oligosaccharides, but also with mono-sugar components with degraded glycosyl bonds. N-acethylhexosamine is highly sensitive at a sub-pico molecular range, and this method is useful for detecting glycans on glycoproteins. The detection limit of fetuin, a protein possessing 3 N-glycans and 3 O-glycans, was 0.17 pmol by infusion analysis, and chromatograms of glycopeptides separated on an ODS column were obtained. These results suggest that this method is useful for the preparation of glycopeptides for glyco-proteomics research.