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Development and validation of a CE‐MS method for the targeted assessment of amino acids in urine
Author(s) -
Rodrigues Karina T.,
Mekahli Djalila,
Tavares Marina F. M.,
Schepdael Ann
Publication year - 2016
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201500534
Subject(s) - formic acid , chromatography , repeatability , chemistry , urine , isoleucine , mass spectrometry , methanol , amino acid , leucine , organic chemistry , biochemistry
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% NH 4 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.