HPLC-EC DETERMINATION OF FREE PRIMARY AMINO-ACID-CONCENTRATIONS IN CAT CISTERNAL CEREBROSPINAL-FLUID

This paper describes an HPLC-EC method for measuring the concentrations of 9 free primary amino acids in cerebrospinal fluid (CSF) withdrawn from the cisterna magna of Nembutal-anesthetized adult cats. Amino acid derivatives were formed with o-phthalaldehyde and beta-mercaptoethanol; subsequently, excess thiol reagent was removed with iodoacetamide. During elution through a C18 5-mu-m column, the electrochemical detector's sensitivity was switched to accommodate the wide ranges of CSF amino acid concentrations. The analysis was acceptably precise and linear at and above the CSF levels and did not require CSF deproteinization. During the 23 min elution, the concentrations of 8 CSF amino acids were determined: alanine, asparagine, glutamate, glutamine, glycine, serine, taurine, and tyrosine; measurable concentrations were between 1 and 800-mu-M. The concentration of GABA was below its detection limit (0.5-mu-M). To assess the ability to detect small concentration increases which might occur due to experimental manipulations, the minimum detectable increments in CSF amino acid concentrations above endogenous levels were determined.