title: Development and validation of a CE-MS method for the targeted assessment of amino acids in urine.
abstract: A CE-ESI-MS method was developed and validated for the separation and quantitative analysis of amino acids (AA) in urine. Experimental parameters related to the CE-MS interface, BGE, and mass spectrometer (MS) settings were optimized providing a good separation of 27 AA, including the isomers L-leucine, L-isoleucine, and L-alloisoleucine, in less than 30 min. The sheath liquid was composed by 0.50% formic acid in 60% (v,v) methanol-water delivered at a flow rate of 5 μL/min. The BGE consisted of 0.80 mol/L formic acid at pH 1.96 and 15% methanol. A pH stacking procedure was implemented to enhance sensitivity (a 12.5% NH4 OH solution was injected at 0.5 psi/9 s prior to samples injected at 0.6 psi/20 s). The proposed method was validated according to FDA and ICH protocols exhibiting acceptable parameters. Analytical curves presented coefficients of determination from 0.996 to 0.9997 (with large F statistics and low p-values). LODs and quantification ranged from 0.63 to 29 μmol/L and from 1.9 to 86 μmol/L, respectively. Practical repeatability was obtained for all AA with coefficients of variation better than 0.55% CV (migration time) and 1.7% CV (peak area ratios; methionine sulfone as internal standard). Recoveries of AA in spiked urine ranged from 92.0 to 123% with few exceptions. Moreover, a successful quantification of AA in pooled control and test urine samples, which compose a vesicoureteral reflux cohort, was achieved showing the potential applicability of the proposed method for targeted metabolomics studies using CE-ESI-MS with an Ion Trap as mass analyzer.
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, Pharmaceutical Analysis, KU Leuven-University of Leuven, Leuven, Belgium.|Institute of Chemistry, University of Sao Paulo, Sao Paulo, Brazil.
authors.forename: Karina T
authors.initials: KT
authors.lastname: Rodrigues
authors.affiliation: Department of Development and Regeneration, Molecular and Cellular Signal Transduction, KU Leuven-University of Leuven, Leuven, Belgium.|Department of Pediatric Nephrology, University Hospitals Leuven, Leuven, Belgium.
authors.forename: Djalila
authors.initials: D
authors.lastname: Mekahli
authors.affiliation: Institute of Chemistry, University of Sao Paulo, Sao Paulo, Brazil.
authors.forename: Marina F M
authors.initials: MF
authors.lastname: Tavares
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, Pharmaceutical Analysis, KU Leuven-University of Leuven, Leuven, Belgium.
authors.forename: Ann
authors.initials: A
authors.lastname: Van Schepdael
mesh_terms: D000596:Amino Acids / Q000652:urine*; D002648:Child; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D016014:Linear Models; D055432:Metabolomics / Q000379:methods; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D000596:Amino Acids
keywords: Amino acids; CE-ESI-MS; Targeted metabolomics; Urine