Effect of different sample pretreatment methods on the concentrations of excitatory amino acids in cerebrospinal fluid determined by high-performance liquid chromatography

Using high-performance liquid chromatography (HPLC), the effect of different sample pretreatment methods on the concentrations of excitatory amino acids (EAAs, glutamate and aspartate) measured in cerebrospinal fluid (CSF) was investigated. The results showed that the measured values of glutamate and aspartate were constant when the samples were stored at −80 °C and then methanol was used for CSF deproteinization before assay; the values of glutamate (Glu) increased when 0.3 M perchloric acid was used for CSF deproteinization with the CSF subsequently being stored at −20 °C; the values of Glu changed when the samples were stored at −20 °C over 8 weeks with methanol subsequently being used for CSF deproteinization before assay. This reference data suggested that the CSF sample would be better stored at −80 °C. If the sample is stored at −20 °C over 8 weeks, the Glu values change with the storage time. If strong acidic reagents are used for precipitation of protein in the CSF sample and then stored at −20 °C, Glu values are abnormally increased. From this study, an accurate and sensitive reversed-phase high-performance liquid chromatographic method has been developed for anti-excitotoxicity therapy and thorough study of EAAs in a clinical setting.