Rapid comprehensive amino acid analysis by liquid chromatography/tandem mass spectrometry: Comparison to cation exchange with post-column ninhydrin detection

Dennis J. Dietzen, Annette L. Weindel, Mary O. Carayannopoulos, Michael Landt, Ellen T. Normansell, Tyler E. Reimschisel, Carl H. Smith

Research output: Contribution to journalArticlepeer-review

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Abstract

Ion-exchange chromatography with ninhydrin detection remains the gold standard for detecting inborn errors of amino acid catabolism and transport. Disadvantages of such analysis include long chromatography times and interference from other ninhydrin-positive compounds. The aim of this project was to develop a more rapid and specific technique using liquid chromatography/tandem mass spectrometry (LC/MS/MS). Optimal fragmentation patterns for 32 amino acids were determined on a triple quadrupole mass spectrometer following butylation. Chromatographic characteristics of each of the amino acids were determined using C8 reversed-phase chromatography with 20% acetonitrile/0.1% formic acid as isocratic mobile phase. Quantitation using eleven deuterated internal standards was compared to cation exchange and ninhydrin detection on a Beckman 7300 system. Following methanol extraction and butylation, determination of 32 amino acids required 20 min. The dynamic range of each amino acid was generally 1-1000 μmol/L. Imprecision ranged from 7 to 23% (CV) over 6 months and recovery ranged from 88-125%. Deming regression with the Beckman 7300 yielded slopes from 0.4-1.2, intercepts from -21 to 65 μmol/L, correlation coefficients from 0.84-0.99 and Syx from 2-125 μmol/L. Isobaric amino acids were separated by chromatography (e.g. leucine, isoleucine) or by unique fragmentation (e.g., alanine, β-alanine). LC/MS/MS is comparable to traditional LC-ninhydrin detection. Mass spectral detection shortens analysis times and reduces potential for interference in detecting inborn metabolic errors.

Original languageEnglish
Pages (from-to)3481-3488
Number of pages8
JournalRapid Communications in Mass Spectrometry
Volume22
Issue number22
DOIs
StatePublished - Nov 30 2008

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