Ion-exchange chromatography for physiological fluid amino acid analysis

Abstract

In the past l0 years, reversed-phase high-performance liquid chromatography (RP-HPLC) has been widely accepted as a preferred method for the amino acid analysis of protein hydrolysates. L ~ In contrast, physiological samples are often so varied and complex that application of RP-HPLC to their analysis has always resulted in poor peak resolution. 4-7 Therefore, the reliability of RP-HPLC in the quantitative determination of physiological amino acids remains controversial. 2~-H Analysis of physiological amino acids is traditionally carried out by ion-exchange chromatography (IEC) followed by post-column ninhydrin ~47 or o-phthalaldehyde ~'~ derivatization, instrumentation required for IEC has had the disadvantage of being more expensive and less versatile than RP-HPLC systems. Recently, with the advances in instrumental design a new generation of IE analyzers (System Gold TM analyzer, Beckman Instruments, San Ramon, USA) has emerged. This system offers the advantage of ease of operation and is highly adaptable for analyses of substances other than amino acids. Although the manufacturer of the System Gol& M' analyzer has made strong claims for its wide applicability in amino acid analysis, there have been no reports fully describing the use of the System Gold '~ analyzer for the analysis of free amino acids in physiological fluids. An additional complication to the development of a satisfactory system arises from the manufacturer's unwillingness to disclose the composition of the buffers recommended for use in the system. Thus the user has to purchase commercial buffers at high cost for routine operation. In this study, we describe the preparation of lithium citrate buffers and their application in physiological amino acid analysis on the System Gold :'~ analyzer. Quantitative analysis of results obtained for physiological amino acids was examined in terms of accuracy and precision.