QUANTIFICATION OF PLASMA AMINO ACIDS USING UPLC-MRM MS FOR THE CLINICAL DIAGNOSIS OF METABOLIC DISORDERS

CASES, GABRIEL G; CALINSKI, GUSTAVO J; MÉNDEZ, MARIANA L.; GIMÉNEZ, MARÍA I.; UICICH, RAÚL E.

Rosario

Simposio; 2º LATIN AMERICAN METABOLIC PROFILING SYMPOSIUM; 2016

IBR

The analysis of plasma amino acids profile is a useful tool for detection of metabolic disorders. It is also associated with the study of various pathological conditions and nutritional disorders. Quantification is thus required by a selective and sensitive method that allows the clinical laboratory to obtain reliable results for a better profile to help the metabolomic study.An analytical assay based on Waters MassTrakTM AA Analysis was generated and optimized for using stable isotope labeled internal standards and mass spectrometry as a detection method. Using UPLC-MS/MS in MRM mode associated with stable isotopes dilution (SID) improves the sensitivity and specificity of simultaneous analysis of multiple amino acids. A mixture of twenty 15N enriched amino acids used as internal standard allows to normalize analytical variables like matrix supression and sample storage, and confirms presence of the endogenous analog. By changes in the mobile phase concentration and chromatographic gradient a shorter running time was achieved maintaining the peak resolution of the original method, and allowing the separation of all analytes (42 amino acids).MS acquisition was divided into five MRM functions in order that each analyte had more scanning time thus improving the method sensitivity. For key analytes detection to the diagnosis of metabolic defect such as Maple Syrup Urine Disease (MSUD) where the presence of alloisoleucine is considered pathognomonic of disease, the emphasis was placed in a optimal peak resolution.