Identification of amperozide metabolites in urine from rats, rabbits, dogs and man by Frit‐FAB LC/MS using deuterated solvents to gain additional structural information

A general procedure for screening of amperozide, N ‐ethyl‐4‐[4,4‐bis( p ‐fluorophenylbutyl)]‐1‐piperazine carboxamide, labelled with 3 H and 14 C was developed. Urine extracts were first fractionated by preparative reversedphase chromatography with an acetonitrile gradient elution. The collected fractions were finally analysed using a methanol gradient on packed capillary LC columns with an internal diameter of 0.32 or 0.5 mm connected to the Frit‐FAB probe of a Jeol SX‐102 mass spectrometer for structural analysis. A micro‐gradient system dedicated for the use of deuterated solvents was constructed from two six‐port switching valves to reduce the consumption of the eluents. The number of hydrogens bound to heteroatoms (OH, NH) was determined by comparing the spectra recorded from mobile phases using water and deuterium oxide. The mass spectra recorded during elution with deuterated solvents was also useful for the interpretation of the fragmentation pattern of standard compounds and unknown metabolites. The technique proved especially useful to differentiate between hydroxylation and N ‐oxidation which gave the same increase in molecular mass by 16 u but a difference in the number of exchangeable protons. Metabolites formed by oxidative N ‐dealkylation of amperozide either at the basic nitrogen or at the N ‐ethylcarboxamide nitrogen were identified. Additional metabolites derived from deethylated amperozide inovlving N ‐oxidation of the basic nitrogen of the piperazine ring and/or hydroxylation of the piperazine ring were identified. Metabolites formed by oxidative N ‐dealkylation and opening of the piperazine ring were also identified.