Evaluation and comparison of colorimetric, radiometric and high performance liquid chromatographic assays for aminopyrine-N-demethylation by rat liver microsomes.

Aminopyrine (DMAP) is metabolized by two successive N-demethylations to monomethyl-4-aminoantipyrine (MMAP) and 4-aminoantipyrine (AAP). Separation and quantification of DMAP and its metabolites in microsomal incubation mixtures by h.p.l.c. showed that other reactions also occur. In the radiometric method where [Me2-14C]DMAP is used as substrate, [14C]formaldehyde is formed during the N-demethylation. However since commercial [14C]DMAP, is not completely double labelled, and both MMAP and AAP are formed, it is impossible to calculate the formaldehyde formation accurately from the specific activity of [14C]DMAP. Moreover, it was shown that DMAP, AAP and particularly MMAP may all develop considerable colour intensity with the Nash reagent, which is used to determine formaldehyde. Despite this difficulty, DMAP may still be used as a model substrate in vitro, with the Nash assay being used to determine formaldehyde if low substrate concentrations and a short incubation time are used. Thus the interference of DMAP or its metabolites with the Nash assay is negligible. As the same Km and Vmax values were obtained from both the radiometric and the relatively precise colorimetric assay it is suggested that there is little wrong with either method, at least under our experimental conditions. The h.p.l.c. method however, underestimates formaldehyde formation, probably because metabolites other than MMAP and AAP are formed. The latter method however may be used to analyse the aminopyrine metabolism in more detail.