A method for the estimation of acetanilide, paracetamol and phenacetin in plasma and urine using mass fragmentography

Phenacetin, paracetamol and acetanilide can be determined in a plasma or urine sample by the use of deuterium labelled analogues. These are produced by reaction of hexadeuterioacetic anhydride with the appropriate aromatic amine. The -NHCOCD3 group is stable to hydrogen exchange below pH 8. The internal standard is added to the plasma or urine after enzymatic hydrolysis of the paracetamol conjugates and an ethyl acetate extract at pH 5 is evaporated under nitrogen and the residue derivatized with N,O-bis-(trimethylsilyl)-acetamide. An aliquot of this solution is injected into a g.c.m.s. system, and one ion characteristic of the material under study and the ion from the deuterium analogue (3 mass units greater) are monitored using a voltage switching technique. In the case of phenacetin, for example, ions at 251 and 254 are monitored. Calibration curves relating different weight ratios of the hydrogen and deuterium compounds to their respective signals from the gas chromatography mass spectrometer are used to calculate the amount of a compound in a particular sample. These methods have been developed to study the oxidation of acetanilide to paracetamol and the de-ethylation of phenacetin to paracetamol. Preliminary results from experiments with phenacetin will be discussed.