Quantification of Branched-Chain Amino Acids in Plasma by High-Performance Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS)

Hamed Piri-Moghadam, Alan Miller, Debra Pronger, Faye Vicente, Joel Charrow, Shannon Haymond, David C. Lin*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapter


Branched-chain amino acids (BCAA), including valine, alloisoleucine, isoleucine, and leucine, play significant roles in a number of metabolic pathways in the body. Deficiency in branched-chain ketoacid dehydrogenase complex, an enzyme required for metabolism of those amino acids, will lead to elevation and accumulation of BCAA and ketoacids in bodily fluids. This results in maple syrup urine disease (MSUD), a condition estimated to affect 1 in 100,000–300,000 births. If MSUD is not diagnosed in the first few days of life, progression of this disease can lead to intellectual disability, coma, irreversible brain damage, seizures, or even death. If diagnosed early, MSUD can be managed by monitoring the blood concentrations of BCAA and adjusting the patient’s dietary intake accordingly. Therefore, it is critical to have a rapid, accurate, and reliable BCAA assay for confirmation of MSUD in newborns as well as routine monitoring of MSUD patients. Here, we describe a high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method for BCAA measurement which requires only 20 μL of plasma. The sample preparation does not require derivatization and only involves protein precipitation with LC/MS-grade methanol, which contains leucine(13C6;15N), isoleucine(13C6;15N), and valine(13C5;15N) as the internal standards. The final sample extracts do not require dry-down and reconstitution and are readily compatible with the liquid chromatography (LC) method. BCAA are separated using the isocratic gradient method on a mixed-mode Intrada column. Multiple-reaction monitoring (MRM) mode is used for MS/MS detection to monitor the parent-to-daughter transitions m/z 132.2 to 86.4 for leucine, isoleucine, and alloisoleucine; m/z 118.2 to 72.4 for valine; m/z 139.2 to 92.4 for leucine(13C6;15N) and isoleucine(13C6;15N); and m/z 124.2 to 77.4 for valine(13C5;15N).

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Number of pages17
StatePublished - 2022

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029


  • Branched-chain amino acid
  • Liquid chromatography
  • Maple syrup urine disease
  • Mass spectrometry
  • Plasma
  • Quantification

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics


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