Quantitation of free amino acids in biological samples by high-performance liquid chromatography : Application of the method in evaluating amino acid levels in cerebrospinal fluid and plasma of patients with multiple sclerosis

Abstract

An automatic on-line high-performance liquid chromatographic method based on a precolumn derivatization with o-phthalaldehyde has been developed to quantitate levels of free amino acids in cerebrospinal fluid (CSF) and plasma samples from 12 patients with multiple sclerosis (MS) and 12 controls. The analytical method gave reproducible results with relative standard deviations of 0.5–3% for all amino acids. The separation of 24 amino acids was performed on a reversed-phase C 18 column, using two solvents and a multiple-step gradient. Each chromatographic experiment was completed within 40 min. The results showed higher levels of Glu, Gln, Gly and Ala and lower levels of Met, Val, Phe and Lys in plasma of MS patients. In CSF, increased levels of Gln, Arg, Ser and Tyr and decreased levels of Asp, Glu, Met, γ-aminobutyric acid and Phe were found in MS patients, whereas the levels of other amino acids remained more or the less same in both groups.