Determination of Unbound Urinary Amino Acids Incorporated with Creatinine Normalization by LC-MS/MS Method with CLAM-2000 Online Sample Pre-treatment

Free or unbound amino acids are important metabolites in human blood and urine [1]. The profile of unbound amino acids in urine are the reference indication of metabolic imbalances and amino acid transport disorders as well as dietary protein adequacy and assimilation. Creatinine produced by muscle metabolism is excreted in the urine, which can be used to normalize the metabolite levels to compensate the large variation due to different intakes of water and fluid food [2-4]. The aim of this study is to develop a reliable LC-MS/MS method for quantitation of 22 free amino acids and creatinine in urine samples. A derivatization-free LC-MS/MS amino acid method [5] with stable isotope labelled IS was employed. An on-line sample pre-treatment module CLAM-2000 coupled with LC-MS/MS makes the analysis fully-automated, which enables from adding internal standards, sample and solvent mixing, shaking for protein-crash and filtration to transferring the final sample solution to LC-MS/MS for analysis.