Quantification of malonyl-coenzyme A in tissue specimens by high-performance liquid chromatography/mass spectrometry.
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 ([(13)C(3)]malonyl-CoA) and multiple-point standard curves from 50 to 1000pmol. 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+/-0.2nmol/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.
Louis Stokes Department of Veterans Affairs Medical Center, Medical Research Service, Cleveland, OH 44106, USA.
Paul E
PE
Minkler
Janos
J
Kerner
Takhar
T
Kasumov
William
W
Parland
Charles L
CL
Hoppel
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D008099:Liver / Q000166:cytology / Q000378:metabolism; D008297:Male; D008316:Malonyl Coenzyme A / Q000032:analysis* / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism; D009206:Myocardium / Q000166:cytology / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results
D008316:Malonyl Coenzyme A