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Anal Biochem. 2013 Aug 15;439(2):116-22. doi: 10.1016/j.ab.2013.05.002. Epub 2013 May 14.

Quantification of branched-chain keto acids in tissue by ultra fast liquid chromatography-mass spectrometry.

Author information

1
Department of Cellular and Molecular Physiology, Penn State College of Medicine, Hershey, PA 17033, USA.

Abstract

Branched-chain keto acids (BCKAs) are associated with increased susceptibility to several degenerative diseases. However, BCKA concentrations in tissues or the amounts of tissue available are frequently at the limit of detection for standard plasma methods. To accurately and quickly determine tissue BCKAs, we have developed a sensitive ultra fast liquid chromatography-mass spectrometry (UFLC-MS) method. BCKAs from deproteinized tissue extractions were o-phenylenediamine (OPD) derivatized, ethyl acetate extracted, lyophilized in a vacuum centrifuge, and reconstituted in 200 mM ammonium acetate. Samples were injected onto a Shimadzu UFLC system coupled to an AB-Sciex 5600 Triple TOF mass spectrometer instrument that detected masses of the OPD BCKA products using a multiple reaction monitoring method. An OPD-derivatized (13)C-labeled keto acid was used as an internal standard. Application of the method for C57BL/6J (wild-type) and PP2Cm knockout mouse tissues, including kidney, adipose tissue, liver, gastrocnemius, and hypothalamus, is shown. The lowest tissue concentration measured by this method was 20 nM, with the standard curve covering a wide range (7.8-32,000 nM). Liquid chromatography-mass spectrometry run times for this assay were less than 5 min, facilitating high throughput, and the OPD derivatives were found to be stable over several days.

KEYWORDS:

2-Keto-3-methylvalerate; 2-Ketoisocaproate; 2-Ketoisovalerate; Branched-chain α-keto acid; Maple syrup urine disease; Ultra fast liquid chromatography–mass spectrometry

PMID:
23684523
PMCID:
PMC3887392
DOI:
10.1016/j.ab.2013.05.002
[Indexed for MEDLINE]
Free PMC Article

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