Automated Analysis of O-Phthalal-Dehyde Derivatives of Amino Acids in Physiological Fluids by Reverse Phase High Performance Liquid Chromatography

An automated method is described for the determination of free amino acids in biological fluids using precolumn deriva-tization with o-phthalaldehyde and reverse phase high performance liquid chromatography. Chromatographic separation of amino acids is accomplished in 24 minutes (cycle time 44 minutes). As little as 1.5 pmol of most commonly occurring amino acids can be accurately quantified. Accuracy and reproducibility are optimized by automating the derivatization-injection sequence and by correcting for variations in the fluorescence response of each amino acid in each run. A total of 31 analyses can be completed in 24 hours on a single column (7 standards and 24 unknowns). The method can be used in the general determination of free amino acids in biological fluids, or can be further accelerated and used for the quantitation of specific amino acids simply by altering the elution conditions.