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A sensitive and selective method for the simultaneous determination of acetylcholine (ACh) and choline (Ch) is reported. ACh and Ch were separated on a reversed-phase column, passed through an immobilized enzymes (acetylcholine esterase and choline oxidase) column, and converted to hydrogen peroxide. The generated hydrogen peroxide was detected by the peroxyoxalate chemiluminescence reaction. The linear determination ranges were from 10 pmol to 10 nmol. The detection limit for both cholines was 1 pmol.
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