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. ; Van Schepdael, Ann

• Document type: Journal article
• Source: ELECTROPHORESIS; v. 37, n. 7-8, p. 9-pg., 2016-04-01.

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 mu L/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% NH4OH 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 mu mol/L and from 1.9 to 86 mu mol/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 ofAAin 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. (AU)

• FAPESP's process: 12/09822-0 - Evaluation of urinary global fingerprint of vesicoureteral reflux for CE-MS
• Grantee: Karina Trevisan Rodrigues
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 14/18789-2 - Evaluation of CE-MS for the urinary targeted metabolomics assessment of vesicoureteral reflux
• Grantee: Karina Trevisan Rodrigues
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate