Analysis and purification of acyl coenzyme A thioesters by reversed-phase ion-pair liquid chromatography.

A rapid method is described for the analysis of mixtures of short-chain acyl coenzyme A thioesters by reversed-phase ion-pair chromatography on LiChrosorb RP-8 and μBondapak C₁₈ columns. The technique is applicable to separation of CoASH, acetyl-CoA propionyl-CoA, and 3-hydroxy-3-methylglutaryl-CoA, as well as dicarboxylic acids and several nucleotides commonly used as cofactors for biosynthetic reactions. The method was utilized on a preparative scale for purification of 3-hydroxy-3-ethylglutaryl-CoA from CoASH and 3-hydroxy-3-ethylglutaric acid. The counterion employed was tetrabutylammonium (phosphate), pH 5.5, in various methanol:water mixtures. Elution profiles and retention values of compounds were influenced by the concentration of counterion and mass of injected sample. Tetrabutylammonium ions could be removed from effluent by ionexchange chromatography on Amberlite IR-120 resin.