Abstract
An amino acid analysis method for protein hydrolysates, using precolumn phenylisothiocyanate (PITC) derivatization and liquid chromatography, was modified for its application in rapid analysis of commonly occurring free amino acids in serum and other physiological samples. The modifications included changes in column temperature (47.5 degrees C compared to 25-35 degrees C used in analyzing protein hydrolysates), method of preparing standard and test samples, and gradient conditions. By using a Waters Pico-Tag amino acid analysis 15 cm long column (which is also used for analyzing protein hydrolysates), separation of 27 PTC-amino acids in human serum and rat liver, brain, or heart was completed in 20 min by the modified method. The total time for analysis and equilibration was 30 min. The modified method was much faster than the traditional ion-exchange methods (2-3 h) or the existing liquid chromatographic methods using PITC derivatization (66-80 min) for determining nutritionally important free amino acids in physiological fluids and tissues. Variability of the method (expressed as coefficients of variation) for the determination (including deproteinization, derivatization, and liquid chromatography) of all amino acids was less than 5%, which compared favorably with the reproducibility of ion-exchange methods.
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