Methods for Absolute Quantification of Human Plasma Free Amino Acids by High-Performance Liquid Chromatography/Electrospray Ionization Mass Spectrometry Using Precolumn Derivatization

Abstract

Plasma free amino acid (PFAA) concentrations in humans are affected by various diseases. However, the variations caused are not dramatic, so a high accurate and precise method for analyzing PFAAs is required. The PFAA analysis protocol described in this chapter covers blood sampling, sample pretreatment, amino acid derivatization, and LC-MS analysis. Each procedure is important for accurate and precise quantification. In the protocol, a human blood sample is collected using an EDTA-2Na or 2K vacuum collection tube and then immediately cooled in water mixed with crushed ice. The sample is then centrifuged on cooling to allow a plasma sample to be removed. A stable-isotope-labeled internal standard solution is added to the plasma, and then the plasma is deproteinized with acetonitrile. The amino acids in the plasma are then derivatized using 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APDS) reagent which is designed for LC-MS analysis. The derivatized amino acids are separated by reverse-phase HPLC and detected by electrospray ionization mass spectrometry. Using this method, 21 amino acids in human plasma can be analyzed with a 12 min cycle. The accuracy and precision are both better than the required criteria given by the US Food and Drug Administration in guidance of Bioanalytical Method Validation.