Quantification of malonyl-coenzyme A in tissue specimens by high-performance liquid chromatography/mass spectrometry

Add time:09/06/2019    Source:sciencedirect.com

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 ([13C3]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.

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