Separation and measurement of clofibroyl coenzyme a and clofibric acid in rat liver after clofibrate administration by reversed-phase high-performance liquid chromatography with photodiode array detection

Add time:09/03/2019    Source:sciencedirect.com

A method to identify and quantitate clofibric acid and clofibroyl coenzyme A (CoA) products in rat liver was developed using reversed-phase high-performance liquid chromatography. The system was developed with baseline separation of clofibroyl-CoA from clofibric acid using isocratic elution, with a mobile phase consisting of 52% methanol and 28 mM potassium phosphate buffer (pH 4.2). With this high methanol concentration, the large amount of UV-absorbing materials present in the liver extracts were eluted earlier than the investigated compounds. Clofibroyl-CoA has a characteristic absorbance spectrum with distinct peaks at 260 and 230 nm, while clofibric acid showed only a distinct peak at 230 nm. Using an on-line photodiode array detector, the spectra could be recorded during analysis without interrupting the flow of the mobile phase. This spectral analysis improves identification possibilities and evaluation of the purity of the chromatographic peaks. In a perchloric extract of rat liver, the recovery of clofibric acid and clofibroyl-CoA added to the liver extract ranged from 70 to 80%. A linear relationship was observed between clofibric acid and clofibroyl-CoA concentration and the area of their peaks in the chromatogram. The detection limit of the method was lower than 5 pmol for both compounds when the absorbance was recorded at 230 nm. The method could be used without modification for the estimation of clofibroyl-CoA and clofibric acid in biological extracts.

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