Information obtained from metabolite profiling includes the number,
relative concentrations, and structures of metabolites present in a biological
sample. LC/MS/MS is the most used analytical technique for sensitive
metabolite detection and structural elucidation (see Chapter 11). However,
the sensitivity and response of an LC/MS instrument depend on metabolite
structure and biological matrix. Therefore, metabolite quantification by
LC/MS without metabolite standards may not be reliable. On-line UV
detection is useful for semiquantification of high concentrations of metabolites
when the parent drug has a good UV chromophore. In the drug discovery stage
when radiolabeled compounds are not available, LC/UV/MS is the most often
used analytical technique for profiling in vitro and in vivo metabolites.
Quantitative metabolite profiling in the late discovery and development stages,
especiallyin vivo metabolite profiling, is mainly accomplished using radi-
olabeled materials. Liquid radiochromatographic techniques, including online
radioflow detection, and off-line microplate scintillation counting (Zhu et al.,
2005a, 2005b), provide sensitive and accurate measurement of the radiolabeled
drug and its metabolites. LC/radiodetection/MS is the choice for metabolite
profiling in radiolabeled ADME studies. Application of radiochromatographic
techniques to metabolite profiling is described in Chapter 10.
7.3.5 Special Considerations
7.3.5.1 Terms Involved in Metabolite Safety Testing
Exposure This term refers to the systemic exposure to drug-related material.
It can be expressed as the plasma AUC of administered drug or metabolite
within a dosage interval, typically assessed at steady state. The FDA
metabolite safety draft guidance also uses the percent of the dose as the
measurement of exposure to a metabolite.
Major Metabolites The MIST document defines a major metabolite ‘‘as one
that accounts for 25% or more of the exposure to circulating drug-related
material.’’ The FDA draft guidance defines it as ‘‘a metabolite in humans that
accounts for plasma levels greater than 10% percent of the administered dose
or systemic exposure, whichever is less.’’ The ‘‘major metabolite’’ concept used
in the FDA draft guidance refers to two types of metabolites: (1) a circulating
metabolite that accounts for 10% or more of the total drug-related
components in the plasma, and (2) a metabolite that accounts for 10% or
more of the administered dose.
Unique Human Metabolites The FDA draft guidance document defines the
term as ‘‘a metabolite only in humans.’’ The MIST document refers to a unique
human metabolite as a metabolite identified in the plasma of humans but not in
animals. However, it is very rare to find a truly unique human metabolite that
is not formed in any animal species (Davis-Bruno and Atrakchi, 2006).
214 REGULATORY CONSIDERATIONS OF DRUG METABOLISM AND DRUG