Development and validation of a highly sensitive LC-MS/MS method for simultaneous quantitation of acetyl-CoA and malonyl-CoA in animal tissues.

A highly sensitive and specific LC-MS/MS method was developed for simultaneous estimation of acetyl co-enzyme A (ACoA) and malonyl co-enzyme A (MCoA) in surrogate matrix using n-propionyl co-enzyme A as an internal standard (IS). LC-MS/MS was operated under the multiple reaction-monitoring mode using the electrospray ionization technique. Simple acidification followed by dilution using an assay buffer process was used to extract ACoA, MCoA and IS from surrogate matrix and tissue samples. The total run time was 3 min and the elution of both analytes (ACoA, MCoA) and IS occurred at 1.28 min; this was achieved with a mobile phase consisting of 5 mM ammonium formate (pH 7.5)-acetonitrile (30:70, v/v) delivered at a flow rate of 1 mL/min on a monolithic RP-18e column. A linear response function was established for the range of concentrations 1.09-2187 and 1.09-2193 ng/mL for ACoA and MCoA, respectively. The intra- and inter-day precision values for ACoA and MCoA met the acceptance as per FDA guidelines. ACoA and MCoA were stable in a battery of stability studies viz. bench-top, auto-sampler and long-term. The developed assay was used to quantitate ACoA and MCoA levels in various tissues of rat.