Automated Analysis of Primary Amino Acids in Plasma by High-Performance Liquid Chromatography

Abstract

The concentration of primary amino acids (AAs) in plasma can accurately be determined using high-performance liquid chromatography (HPLC). Before the analysis can be performed, several steps have to be regarded. First, the time and method of blood withdrawal, type of blood tube, use of medication, and differences in dietary intake are important factors that should be standardized. Second, the handling of and the way the blood is transported to the laboratory, the time between blood withdrawal and centrifugation, the method of centrifugation, and the temperature and time of plasma storage have to be noticed. Third, the methods used for deproteinization and derivatization may account for varying results between laboratories.

In this chapter, we describe an HPLC method that measures primary amino acids in plasma using automated precolumn derivatization with ortho-phthalaldehyde, and that pays attention to the above-mentioned criteria. This method is relatively fast, simple, sensitive, and reliable. Since with this method we can determine over 40 physiological amino acids with a very good resolution, trace amounts of amino acids can also be determined. In addition, interassay resolution times have very low variation and the use of two internal standards guarantees reliable quantification.