Quantification of the concentration and 13C tracer enrichment of long-chain fatty acyl-coenzyme A in muscle by liquid chromatography/mass spectrometry

Recent diabetes and obesity research has been focused on the role of intracellular lipids in insulin resistance. Fatty acyl-coenzyme A (CoA) esters play a central role in the trafficking of intracellular lipids, but there has not previously been a method with which to quantify their kinetics using tracer methodology. We have therefore developed a high-performance liquid chromatography (HPLC)–mass spectrometry method to simultaneously measure the 13C stable isotopic enrichment of palmitoyl-acyl-CoA ester and the concentrations of five individual long-chain fatty acyl-CoA esters extracted from muscle tissue samples. The long-chain fatty acyl-CoA can be effectively extracted from frozen muscle tissue samples and baseline separated by a reverse-phase HPLC with the presence of a volatile reagent—triethylamine. Negative ion electrospray mass spectrometry with selected ion monitoring was used to analyze the fatty acyl-CoAs to achieve reliable quantification of their concentrations and 13C isotopic enrichment. Applying this protocol to rabbit muscle samples demonstrates that it is a sensitive, accurate, and precise method for the quantification of long-chain fatty acyl-CoA concentrations and enrichment.