HPLC detection of choline and acetylcholine in serum and urine by an immobilized enzyme reactor followed by chemiluminescence detection.

A method using HPLC has been developed for the detection of choline (Ch) and acetylcholine (ACh) using an immobilized enzyme reactor which converts Ch and ACh into hydrogen peroxide and betaïne. The formed H(2)O(2) is quantified by means of a solid-state peroxyoxalate chemiluminescence detector based on an immobilized fluorophore and addition of oxalate from a solid bed. The conditions necessary for chemiluminescence detection are obtained by using a make-up flow of acetonitrile after the enzyme reactor. Precipitation problems due to the poor solubility of salts in the final acetonitrile-water mixture are circumvented by adding a crown ether to the make-up flow. The reproducibility of the method was calculated to be 3.4-3.7% RSD. Detection limits are in the sub-picomole range and a linear range of at least three orders of magnitude is found. Measurements in urine and serum reveal no matrix effects.