Liquid-liquid extraction coupled with LC/MS/MS for monitoring of malonyl-CoA in rat brain tissue

The formation of malonyl-CoAis catalyzed by acetyl-CoA carboxylase(ACC), the rate-limiting enzyme of de novo fatty acid synthesis. Monitoring the changes of malonyl-CoAconcentration in the brain in response to treatments such as pharmaceutical intervention (via ACC inhibitors) or different dietary conditions (such as varied feeding regimes) is of great interest and could help increase the understanding of how this molecule contributes to feeding behavior and overall energy balance. We have developed a sensitive analytical method for the determination of malonyl-CoAlevels in rat brain tissue. The assay involved removal of tissue lipids by liquid-liquid extractionfollowed by LC/MS/MS analysis of the aqueous layer for malonyl-CoA. The method was sensitive enough (limit of quantitation = 50 ng/mL, or approximately 0.018 nmol/g brain tissue) to determine malonyl-CoAin individual rat brain preparations. The assay performance was sufficiently rugged to support drug discovery screening efforts and provided an additional analytical tool for monitoring brain malonyl-CoAlevels.