Metabolite identification metabolic stability studies

Among the high-throughput assays, metabolic stability studies have evolved as a possible starting point for metabolite identification. Traditional metabolic stability... 

Until very recently, metabolic stability screening and metabolite identification (metabolite ID) have been sequential processes that is, the metabolic stability assays are typically performed hrst to identify rapidly metabolized compounds and a follow-up metabolite ID study is performed next, typically at a 10-fold higher substrate concentration to ensure generation of sufficiently high-quality MS/MS information to support structure elucidation. 

Triple quadrupole mass spectrometers can perform tandem mass scan experiments in various modes including product ion (MS/MS), precursor ion, and neutral loss scan and SRM experiments, but they cannot be used for sequential MS" experiments. The high sensitivity and specificity, in the SRM mode, have made triple quadrupole mass spectrometers a logical choice for metabolic stability experiments performed at relevant substrate concentrations. Despite the sensitivity of the triple quadrupole mass spectrometers, when an NCE or an NCE series exhibit unacceptable PK properties, metabolite identification studies are often initiated as follow-up studies in a separate set of experiments using incubation concentrations higher than the Km of an NCE. Incubations at higher concentrations are required because conventional metabolite identification experiments required operation of the triple quadrupole mass spectrometer in the full-scan mode, which results in poor duty cycle and diminished sensitivity [287,288],... 

At the early stage of compound selection and optimization for deciding a clinical candidate, drug metabolism studies are often conducted in vitro with liver fractions and in vivo with rats using nonlabeled compounds to define metabolic stability, soft-spot identification, CYP inhibition/induction potential, bioactivation or toxic metabolite formation, major in vitro metabolic pathways, and the limited in vitro interspecies comparison. Mass balance (or ADME) studies with collection of plasma in animals and humans using a... 

Proper sample preparation and analysis are essential in all studies of xenobiotic metabolism. With known standards of the free metabolites and often with just a radioactive unknown, it is customary to test their stability under conditions of analysis. The same caution is not always possible with conjugates, but much can be done to improve the validity of conjugate identification. Not only will greater caution in sample handling improve the chances of correctly identifying conjugates, it will also reduce the endless hours of time spent by metabolism chemists in inventing bizarre pathways to justify the formation of metabolites which never existed in the first place. 

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