A rapid, sensitive method for quantitative analysis of underivatized amino acids by liquid chromatography–tandem mass spectrometry (LC–MS/MS)

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2014-01, Vol.944, p.166-174
• Main Authors: Le, Anthony, Ng, Angelina, Kwan, Tony, Cusmano-Ozog, Kristina, Cowan, Tina M.
• Format: Article
• Language: English
• Subjects: Amino acid analysis; Amino acids; Amino Acids - blood; Amino Acids - chemistry; Analyzers; Chromatography; Chromatography, Liquid - instrumentation; Chromatography, Liquid - methods; Correlation analysis; Humans; Limit of Detection; Linear Models; Liquids; Mass spectrometry; Monitoring; Reproducibility of Results; Tandem mass spectrometry; Tandem Mass Spectrometry - instrumentation; Tandem Mass Spectrometry - methods; Underivatized
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2013.11.017

•We developed a robust method for the quantitative analysis of underivatized amino acids by LC–MS/MS.•The analysis is rapid and utilizes inexpensive and easily obtainable reagents.•A unique chromatographic configuration minimizes ion suppression and yields excellent analytic performance.•This method can easily be extended to encompass additional amino acids and sample matrices. The quantitation of free amino acids from physiologic samples is essential for diagnosing and monitoring patients with inherited metabolic disorders. Current methods are hindered by long preparative and/or analysis times, expensive reagents, and often suboptimal performance characteristics. To overcome these challenges, a improved method for amino acid analysis using liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed and validated. Samples were deproteinized with sulfosalicylic acid and supernatants diluted with tridecafluoroheptanoic acid. Chromatographic separation of amino acids occurred using two columns, with conditions favoring resolution of isobaric compounds and minimizing ion suppression. Eluted compounds were detected by selective reaction monitoring, and quantitated by relating peak areas of amino acids to externally run standards. Validation studies evaluated linearity, within- and between-run imprecision, lower limits of detection and quantification for 33 amino acids, and correlation with the Biochrom 30 Amino Acid Analyzer. Total run time including re-equilibration was 15min per sample. Within-run precision averaged 2.8% for all compounds, with an average linear correlation coefficient of 0.995. The majority of compounds were reliably quantitated at ≤0.1μM. Between-run precision averaged 4.0%. Results showed excellent correlation with the Biochrom 30 amino acid analyzer with an average overall correlation of 0.908. We conclude that our method is extremely sensitive, specific and reproducible and represents an improvement over other currently available technologies.