Specific detection of acetyl-coenzyme A by reversed-phase ion-pair high-performance liquid chromatography with an immobilized enzyme reactor.
A selective chromatographic detection system for the determination of acetyl-coenzyme A (CoA) is reported. The short-chain acyl-CoA thioesters were separated by reversed-phase ion-pair high-performance liquid chromatography (HPLC), and then acetyl-CoA was selectively detected on-line with an immobilized enzyme reactor (IMER) as a post-column reactor. Thio-CoA liberated enzymatically from acetyl-CoA was determined spectrophotometrically after reaction with Ellman's reagent in the reagent stream. The IMER with phosphotransacetylase had a substrate specificity sufficient to determine acetyl-CoA and was active and stable in the mobile phase containing methanol and the ion-pair reagent. The calibration graph was linear between 0.2 and 10 nmol, with a detection limit of 0.05 nmol. This HPLC system with detection by IMER allows the selective identification and determination of acetyl-CoA in a mixture of acetoacetyl-CoA and 3-hydroxy-3-methylglutaryl-CoA, which are difficult to separate with ion-pair HPLC.
Yamato
S
S
Department of Analytical Chemistry, Niigata College of Pharmacy, Japan.
Nakajima
M
M
Wakabayashi
H
H
Shimada
K
K
D000105:Acetyl Coenzyme A / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D004800:Enzymes, Immobilized / Q000378:metabolism; D007477:Ions; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D004800:Enzymes, Immobilized; D007477:Ions; D000105:Acetyl Coenzyme A