Measuring long‐chain acyl‐coenzyme A concentrations and enrichment using liquid chromatography/tandem mass spectrometry with selected reaction monitoring

Abstract

Long‐chain acyl‐coenzymes A (acyl‐CoAs) (LCACoA) are the activated forms of long‐chain fatty acids and serve as key lipid metabolites. Excess accumulation of intracellular LCACoA, diacylglycerols (DAGs) and ceramides may create insulin resistance with respect to glucose metabolism. We present a new method to measure LCACoA concentrations and isotopic enrichment of palmitoyl‐CoA ([U‐13 C]16‐CoA) and oleoyl‐CoA ([U‐13 C]18:1‐CoA) using ultra‐performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) to quantitate seven different LCACoA (C14‐CoA, C16‐CoA, C16:1‐CoA, C18‐CoA, C18:1‐CoA, C18:2‐CoA, C20‐CoA). The molecules are separated on a reversed‐phase UPLC column using a binary gradient with ammonium hydroxide (NH4OH) in water and NH4OH in acetonitrile (ACN). The LCACoA are quantified using selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer in positive electrospray ionization (ESI) mode. All LCACoA ions except enriched palmitate enrichment of palmitoyl‐CoA ([U−13C]16‐CoA) and oleoyl‐CoA ([U−13C]18:1‐CoA) using ultra‐performance liquid chromatography/mass spectrometry (UPLC/MS/MS) to quantitate seven different LCACoA (C14‐CoA, C16‐CoA, C16:1‐CoA, C18‐CoA, C18:1‐CoA, C18:2‐CoA, C20‐CoA). The molecules are separated on a reversed‐phase UPLC column using a binary gradient with ammonium hydroxide (NH4OH) in water and NH4OH in acetonitrile. The LCACoA are quantified using selected reaction monitoring (SRM) on a triple quadrupolemass spectrometer in positive electrospray ionization (ESI) mode. All LCACoA ions except enriched palmitate and oleate were monitored as [M + 2 + H]+ and [U13C]16‐CoA and [U13C]18:1‐CoA were monitored as [M + 16 + H]+ and [M + 18 + H]+, respectively. The method is simple, sensitive and efficient (run time as short as 5 min) and allowed us to measure the concentration and detect enrichment in intramyocellular [U13C]16‐CoA and [U13C]18:1‐CoA during a low dose intravenous infusion of [U13C]palmitate and [U13C]oleate in adults undergoing either a saline control experiment or an insulin/glucose infusion experiment. This technique should allow investigators to measure the trafficking of extracellular fatty acids to the intracellular LCACoA pool. Copyright © 2011 John Wiley & Sons, Ltd.