Amino acid anslysis by high-performance liquid chromatography with methanesulfonic acid hydrolysis and 9-fluorenylmethylchloroformate derivatization

Abstract

Experiments were undertaken to verify a method for complete amino acid analysis of plant and animal tissues and waste products from a single hydrolysis and high-performance liquid chromatographic run. Using methanesulfonic acid, hydrolysis of cytochrome c at 115°C for 22 h yielded recoveries equal to or higher than hydrolysis at 115°C for 70 h or at 150°C for 22 h. Triple evacuation of the hydrolysis tube alternated with nitrogen flush gave recovery improvements over single evacuation. Refrigerated storage of samples under vacuum for up to 4 days between hydrolysis and further analysis was not different from immediate analysis. However, recoveries of several amino acids were reduced by refrigerated storage in air. Recoveries of individual amino acids were determined by hydrolysis of biological samples with and without added cytochrome c. Although recoveries from biological samples were lower for several amino acids, precision was sufficient to allow quantitation after correction for incomplete recoveries. Derivatization with 9-fluorenylmethylchloroformate (FMOC) was chosen because derivatives are formed with both primary and secondary amino acids, derivatives are quite stable, and detection may be either UV absorbance or fluorescence. Derivative yield is sensitive to the pH of the reaction mixture. A pH of 8.0 gave reproducible derivative yield for all physiological amino acids. Solvent extraction of excess FMOC, when compared to addition of amantadine to react with excess FMOC, gave both higher recoveries and greater precision. Following derivatization, samples could be kept at 4°C for at least 24h before high-performance liquid chromatographic analysis without loss of response. Derivative yield and detector response were constant across a wide range of molar ratio of FMOC to total amino acids. Gradient elution was required to separate FMOC derivatives on a reversed-phase column. The capability of the pumping system to produce exponential gradients permitted rapid and easy fine-tuning of the gradient.