title: A rapid, sensitive method for quantitative analysis of underivatized amino acids by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
abstract: 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.
authors: Department of Pathology, Stanford University, Palo Alto, CA, United States.
authors: Anthony
authors: A
authors: Le
authors: Stanford University Medical Center, Palo Alto, CA, United States.
authors: Angelina
authors: A
authors: Ng
authors: Stanford University Medical Center, Palo Alto, CA, United States.
authors: Tony
authors: T
authors: Kwan
authors: Department of Pathology, Stanford University, Palo Alto, CA, United States; Department of Pediatrics, Stanford University, Palo Alto, CA, United States.
authors: Kristina
authors: K
authors: Cusmano-Ozog
authors: Department of Pathology, Stanford University, Palo Alto, CA, United States. Electronic address: tcowan@stanford.edu.
authors: Tina M
authors: TM
authors: Cowan
mesh_terms: D000596:Amino Acids / Q000097:blood* / Q000737:chemistry; D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D016014:Linear Models; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods*
chemical_list: D000596:Amino Acids
keywords: Amino acid analysis; LC–MS/MS; PGC; SRM; TDFHA; Tandem mass spectrometry; Underivatized; liquid chromatography tandem mass spectrometry; porous graphitic carbon; selective reaction monitoring; tridecafluoroheptanoic acid