Rapid and sensitive high-performance liquid chromatographic method for the analysis of tryptophan, tyrosine and phenylalanine in biological samples

In order to overcome problems related to the determination of free tryptophan in biological fluids using conventional methods, we have developed an accurate and reliable procedure based on a specific pretreatment of samples followed by a very rapid and sensitive reversed-phase high-performance liquid chromatographic analysis. The pretreatment consists of adding to the sample of a very low amount of 3 M phosphate buffer to maintain pH in the physiological range followed by ultrafiltration. The precision, reproducibility and sensitivity of our method were also evaluated. The recovery of each amino acid was greater than 92%. The use of a microbore column allows the detection of up to 0.2 pmol/μl of amino acid. The method has been applied to the analysis of samples obtained from 25 normal and 10 phenylketonuric subjects.