Quantification of metabolites by mass spectrometry in the absence of authentic reference standards or without a radiolabel is often called 'semi-quantitative', which acknowledges that mass spectrometric responses are not truly quantitative. For many researchers, it is tempting to pursue this practice of semi-quantification in early drug discovery and even preclinical development, when radiolabeled ADME studies are being deferred to later stages of drug development. The caveats of quantifying metabolites based on parent drug response are explored in this investigation. A set of 71 clinically relevant drugs/metabolites encompassing common biotransformation pathways, was subjected to flow injection analysis coupled with electrospray ionization mass spectrometry (ESI-MS). The results revealed a large variation in ESI response even for structurally similar parent drug/metabolite pairs. The ESI response of each metabolite was normalized to that of the parent drug, to generate an ESI relative response factor. Overall, relative response factors ranged from 0.014 (> 70-fold lower response than parent) to 8.6 (8.6-fold higher response than parent). Various 2D molecular descriptors were calculated that describe physicochemical, topological and structural properties for each drug/metabolite. The molecular descriptors, along with the ESI response factors were used in univariate analyses as well as a principal components analysis to ascertain which molecular descriptors best account for the observed discrepancies in drug/metabolite ESI response. This investigation has shown that the practice of using parent drug response to quantify metabolites should be used with caution.
- The American Society for Pharmacology and Experimental Therapeutics