Amino acid analysis of physiological fluids by high-performance liquid chromatography with phenylisothiocyanate derivatization and comparison with ion-exchange chromatography.

The suitability of pre-column derivatization with phenylisothiocyanate followed by high-performance liquid chromatography was investigated as a means of analyzing free amino acids in plasma and other physiological fluids. A comparison was made between this method and a conventional ion-exchange method. The correlation coefficient for all the amino acids tested was greater than 0.9, except for proline and tryptophan. Various forms of sample preparation were tried for plasma and amniotic fluid; it was finally decided that protein precipitation with acetonitrile was most suitable. Ultrafiltration was used for cerebrospinal fluid preparation while urine was treated the same as a standard mixture. The retention times relative to the internal standard (nor-leucine) are given for over 90 compounds. Some of these were chromatographed underivatized because they are known to be present in some physiological fluids and absorb at 254 nm because of their aromaticity. The imprecision for this method compared favourably with the standard ion-exchange method although each had specific amino acids for which the imprecision was poor. The technique is suitable for the same routine clinical analysis purposes as high-resolution ion-exchange chromatography. It also offers the advantages of speed of analysis, sensitivity and equipment versatility over the conventional ion-exchange methods.