A simple method for determination of choline (Ch) and acetylcholine (ACh) in rat brain regions using high-performance liquid chromatography with electrochemical detection (HPLC-ED).

Abstract

A simple method for the determination of choline (Ch) and acetylcholine (ACh) in rat brain regions by application of high performance liquid chromatography with electrochemical detection (HPLC-ED) is described. Separation of endogenous Ch, ACh, and ethylhomocholine (EHC) as internal standard on a chemcosorb 3C-18 column is followed by electrochemical detection of H2O2 derived from the compounds. H2O2 was enzymatically produced in the reaction coil (10 m). The assay limit for quantitation was 10 .apprx. 30 pmole for both compounds. The brain tissue was homogenized with 15%-1N formic acid in acetone and centrifuged. The supernatant was washed with ether once and then blown off using N2 gas before an aqueous solution was immediately applied for analysis. Recovery rates were 96.1 .+-. 1.4% for Ch and 95.6 .+-. 2.2% for ACh. The levels of Ch and ACh in rat brain regions after sacrifice either by decapitation or microwave irradiation (10 kW, 2450 MHz, Magnetic-field type) were compared with those previously reported.