期刊
ANALYTICAL BIOCHEMISTRY
卷 352, 期 1, 页码 24-32出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2006.02.015
关键词
malonyl-CoA; HPLC; electrospray ionization; mass spectrometry
资金
- NHLBI NIH HHS [P01 HL 074237] Funding Source: Medline
- NIA NIH HHS [P01 AG15885-01] Funding Source: Medline
- NIDDK NIH HHS [R01 DK066107, 1R33DK070291-01] Funding Source: Medline
We present a validated high-performance liquid chromatography/mass spectrometry (HPLC/MS) method for the quantification of malonyl-coenzyme A (CoA) in tissues. The assay consists of extraction of malonyl-CoA from tissue using 10% trichloroacetic acid, isolation using a reversed-phase solid-phase extraction column, HPLC separation, and detection using electrospray MS. Quantification was performed using an internal standard (C-13(3)]malonyl-CoA) and multiple-point standard curves from 50 to 1000 pmol. The procedure was validated by performing recovery, accuracy, and precision studies. Recoveries of malonyl-CoA were determined to be 28.8 +/- 0.9, 48.5 +/- 1.8, and 44.7 +/- 4.4% (averages +/- SD n = 5) for liver, heart, and skeletal muscle, respectively. Accuracy was demonstrated by the addition of known amounts of malonyl-CoA to tissue samples. The malonyl-CoA detected was compared with the malonyl-CoA added, and the resulting relationships were linear with slopes and regression coefficients equal to 1. Precision was demonstrated by repetitive analysis of identical samples. These showed a within-run variation between 5 and 11%, and the interbatch repeatability was essentially the same. This procedure was then applied to rat liver, heart, and skeletal muscle, where the malonyl-CoA contents were found to be 1.9 +/- 0.6, 1.3 +/- 0.4, and 0.7 +/- 0.2 nmol/g wet weight, respectively, for these tissues. This analytical approach can be extended to the quantification of other acyl-CoA species with no significant modification. Published by Elsevier Inc.
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