Analysis of acetaminophen metabolites in urine by high-performance liquid chromatography with UV and amperometric detection.

Acetaminophen and several of its metabolites are separated isocratically on a reversed-phase (C18) column using a mobile phase of 7% methanol and 0.75% glacial acetic acid in 0.1 M KH2PO4. Metabolites that can be separated include the sulfate, glucuronide, cysteine, and mercapturic acid conjugates of acetaminophen, as well as 3-hydroxyacetaminophen, 3-methoxyacetaminophen, and 3-methylthioacetaminophen. Although all of the metabolites can be detected by UV spectrophotometry, the sensitivity limits of detection are improved significantly for acetaminophen and all of the metabolites except the sulfate and glucuronide, by amperometric detection (electrochemical) of the same sample as it elutes from the UV detector. Minimal detectable limits (signal-to-noise ratio greater than or equal to 2) for acetaminophen and its metabolites, other than the glucuronide and sulfate conjugates, were in the order of 1--2 ng injected on column using UV detection at 248 nm, and 0.1--0.5 ng using electrochemical detection at + 0.60 V with reference to an Ag/AgCl standard electrode.