Rapid quantification of underivatized amino acids in plasma by hydrophilic interaction liquid chromatography (HILIC) coupled with tandem mass-spectrometry.
Amino acidopathies are a class of inborn errors of metabolism (IEM) that can be diagnosed by analysis of amino acids (AA) in plasma. Current strategies for AA analysis include cation exchange HPLC with post-column ninhydrin derivatization, GC-MS, and LC-MS/MS-related methods. Major drawbacks of the current methods are time-consuming procedures, derivative problems, problems with retention, and MS-sensitivity. The use of hydrophilic interaction liquid chromatography (HILIC) columns is an ideal separation mode for hydrophilic compounds like AA. Here we report a HILIC-method for analysis of 36 underivatized AA in plasma to detect defects in AA metabolism that overcomes the major drawbacks of other methods.



A rapid, sensitive, and specific method was developed for the analysis of AA in plasma without derivatization using HILIC coupled with tandem mass-spectrometry (Xevo TQ, Waters).



Excellent separation of 36 AA (24 quantitative/12 qualitative) in plasma was achieved on an Acquity BEH Amide column (2.1×100 mm, 1.7 μm) in a single MS run of 18 min. Plasma of patients with a known IEM in AA metabolism was analyzed and all patients were correctly identified.



The reported method analyzes 36 AA in plasma within 18 min and provides baseline separation of isomeric AA such as leucine and isoleucine. No separation was obtained for isoleucine and allo-isoleucine. The method is applicable to study defects in AA metabolism in plasma.
Section Metabolic Diagnostics, Department of Genetics, University Medical Center (UMC) Utrecht, KC.02.069.1, Lundlaan 6, 3584 EA, Utrecht, The Netherlands. b.prinsen@umcutrecht.nl.
Hubertus C M T
HCMT
Prinsen
Section Metabolic Diagnostics, Department of Genetics, University Medical Center (UMC) Utrecht, KC.02.069.1, Lundlaan 6, 3584 EA, Utrecht, The Netherlands.
B G M
BGM
Schiebergen-Bronkhorst
Section Metabolic Diagnostics, Department of Genetics, University Medical Center (UMC) Utrecht, KC.02.069.1, Lundlaan 6, 3584 EA, Utrecht, The Netherlands.
M W
MW
Roeleveld
Section Metabolic Diagnostics, Department of Genetics, University Medical Center (UMC) Utrecht, KC.02.069.1, Lundlaan 6, 3584 EA, Utrecht, The Netherlands.
J J M
JJM
Jans
Section Metabolic Diagnostics, Department of Genetics, University Medical Center (UMC) Utrecht, KC.02.069.1, Lundlaan 6, 3584 EA, Utrecht, The Netherlands.
M G M
MGM
de Sain-van der Velden
Department of Metabolic Diseases, University Medical Center (UMC) Utrecht, Utrecht, The Netherlands.
G
G
Visser
Department of Metabolic Diseases, University Medical Center (UMC) Utrecht, Utrecht, The Netherlands.
P M
PM
van Hasselt
Section Metabolic Diagnostics, Department of Genetics, University Medical Center (UMC) Utrecht, KC.02.069.1, Lundlaan 6, 3584 EA, Utrecht, The Netherlands.
N M
NM
Verhoeven-Duif
D000592:Amino Acid Metabolism, Inborn Errors / Q000097:blood / Q000175:diagnosis; D000596:Amino Acids / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008661:Metabolism, Inborn Errors / Q000097:blood / Q000175:diagnosis; D010949:Plasma / Q000737:chemistry*; D013057:Spectrum Analysis / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods
D000596:Amino Acids