Liquid-liquid extraction coupled with LC/MS/MS for monitoring of malonyl-CoA in rat brain tissue |
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Authors: | Joelle M Onorato Luping Chen Petia Shipkova Zhengping Ma Anthony V Azzara James J Devenny Ningning Liang Tasir S Haque Dong Cheng |
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Institution: | (1) Departments of Bioanalytical and Discovery Analytical Science, Pharmaceutical R&D, Bristol-Myers Squibb Co., P.O. Box 5400, Princeton, NJ 08543-5400, USA;(2) Discovery Biology, Pharmaceutical R&D, Bristol-Myers Squibb Co., Princeton, NJ 08543-5400, USA;(3) Discovery Chemistry, Pharmaceutical R&D, Bristol-Myers Squibb Co., Princeton, NJ 08543-5400, USA |
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Abstract: | The formation of malonyl-CoA is catalyzed by acetyl-CoA carboxylase (ACC), the rate-limiting enzyme of de novo fatty acid
synthesis. Monitoring the changes of malonyl-CoA concentration 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-CoA levels in rat brain tissue. The assay involved
removal of tissue lipids by liquid-liquid extraction followed 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-CoA
in 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-CoA levels. |
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