Rapid and reliable analysis of underivatized amino acids in urine using tandem mass spectrometry

Abstract

Nowadays there is a great interest in the development of fast and reliable methods for the determination of amino acids (AAs) in biological samples due to their biological importance. In the present work, a method based on the use of a guard column (gC) prior to tandem mass spectrometry (gC-MS/MS) is proposed for the determination of proteinogenic AAs in urine. Heptafluorobutyric acid (HFBA) is used as ion pairing reagent. Comparison of the gC-MS/MS method versus tandem mass spectrometry (MS/MS) in stand-alone mode showed improved sensitivity and peak shape, and solved some problems related to interfering compounds, with a total analysis time of 2.8 min. All the proteinogenic AAs were adequately determined using the gC-MS/MS method, except glutamic acid (Glu).To confirm quantitative results obtained with gC-MS/MS for individual AAs, an ion-pair liquid chromatography tandem mass spectrometry method (LC-MS/MS) has also been developed. Both methods (gC-MS/MS and LC-MS/MS) were validated using synthetic urine. For the gC-MS/MS method, LODs and LOQs values were found to be between 0.004 and 0.425 mg/L and 0.01 and 1.40 mg/L, respectively. Aspartic acid (Asp) showed the highest LOD and LOQ values (3 mg/L and 9 mg/L, respectively). A one-point standard addition method and internal standard normalization were used for the quantification because matrix effects were observed. L-alanine-1-13C (Ala-13C) and L-leucine-1-13C (Leu-13C) were used as isotopically labeled internal standards.To demonstrate the applicability of the gC-MS/MS method in the reliable determination of AAs in real samples, urine from eighteen healthy volunteers were analyzed using both gC-MS/MS and LC-MS/MS methods. Similar quantitative results were obtained for individual AAs with both of them. In addition, possible differences in AAs concentrations related to sex were checked, but the results did not show significant differences for the evaluated compounds.