Rapid measurement of free amino acids in serum and CSF using high-performance liquid chromatography

Abstract

A simple, rapid method for measuring individual amino acids in physiological fluids using high-performance liquid chromatography [HPLC]/fluorescence detection has been characterized. Solutions containing free amino acids are first derivatized with orthophthalaldehyde, and then chromatographed on C 18 reversed-phase columns. Sixteen amino acids are readily separated and quantitated, with a run time of about 60 min, and a sensitivity of at least 10 pmoles. Applying this technique to solutions of amino acid standards, and methanol supernatants of a large pool of blood, we find within-assay and between-assay coefficients of variation generally to be as good as those obtained using a standard amino acid analyzer. Serum amino acid measurements have also been obtained under a number of experimental conditions in rats, and yielded values comparable to those obtained using the amino acid analyzer. Samples of cerebrospinal fluid have also been analyzed via this new procedure. This technique offers numerous advantages over the oftentimes laborious, expensive, and time-consuming procedures required by conventional amino acid analyzers, and thus should find widespread application.