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

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 micromol/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 micromol/L, correlation coefficients from 0.84-0.99 and Syx from 2-125 micromol/L. Isobaric amino acids were separated by chromatography (e.g. leucine, isoleucine) or by unique fragmentation (e.g., alanine, beta-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.

Rapid determination of amino acids in biological samples using a monolithic silica column

“…With regard to MS detection, amino acids can be determined by MS based on their mass-to-charge ratio (m/z) even when the analytes have the same retention time, implying that complete separation of amino acids is not necessary and a faster analysis is possible. Although amino acids can be analyzed within 10 min by using UPLC and/or MS (Casetta et al 2000;Dietzen et al 2008;Waterval et al 2009), special and expensive instruments are required.…”

Evaluation of Physiological Amino Acids Profiling by Tandem Mass Spectrometry

“…Actually, amino acid analyzers have been used since more than 50 years in biochemical laboratories and still are considered as a reference method (Dietzen et al 2008;Duran 2008). Notwithstanding, the technology suffers some drawbacks; chromatographic separation lasts about 150 min and requires high sample volumes (>100 mL).…”

“…Nowadays, identification and quantification of amino acids by mass spectrometry are rather uncommon in clinical laboratories. Only a few methods have already been developed and validated and are based either on native (Piraud et al 2003(Piraud et al , 2005Waterval et al 2009) or on derivatized (Dettmer et al 2012;Dietzen et al 2008;Harder et al 2011;Held et al 2011) metabolites identification.…”

Determination of Autosomal Dominant or Recessive Methionine Adenosyltransferase I/III Deficiencies Based on Clinical and Molecular Studies

“…was high, it was measured quantitatively in plasma amino acid chromatography using a 3200 QTRAP® mass spectrometer (Applied Biosystems, AB Sciex). For subjects with a Met level >50 μmol/L, plasma Met and plasma total homocysteine levels were determined by mass spectrometer and direct competitive chemiluminescent enzyme immunoassay (ADVIA Centaur, Bayer), respectively (15,16).…”