HPLC-FLD determination of serum aromatic amino acids: Application in chronic kidney disease patients

Background: The aromatic amino acids (AAA) are very important amino acids in the body and have been suggested to be involved in many diseases. We describe the development and full validation of a high performance liquid chromatography with fluorescence detection (HPLC-FLD) method for simultaneously quantitative determination of serum AAA. Furthermore, we aimed to explore the clinical significances of AAA for chronic kidney disease (CKD). Methods: Serum samples were deproteinized by perchloric acid. Separation was carried out on a Megres C18 column (4.6 mm x 250 mm i.d., 5 mu m). The mobile phase consisted of 10% acetonitrile (v/v) in water and was run at a flow rate of 1.0 ml/min. The eluted AAA was monitored by measuring the fluorescence intensity using the programmed wavelength detection setting. Results: AAA determination in a serum sample was achieved in < 10 min. The linear ranges of the method were 0.55-275 mu mol/l, 3.05-1220.0 mu mol/l and 0.049-49 mu mol/l for Tyr, Phe and Trp, respectively. The limit of detection (LOD) was 0.014 mu mol/l for Tyr, 0.5 mu mol/l for Phe, and 0.0049 mu mol/l for Trp. Recovery and reproducibility were satisfactory. Compared with healthy control subjects, the CKD patients showed lower serum levels of AAA. It should probably available to apply for screening and monitoring of CKD. Conclusions: The method is simple, fast, accurate, and suitable for routine analysis. Crown Copyright (C) 2011 Published by Elsevier BM. All rights eserved.