Metabolite impurities are byproducts formed in the body after a drug substance is ingested. During metabolism, the API and
drug product in the body are exposed to various enzymes, from which metabolite impurities can be formed (26–34). Drug metabolism
is traditionally divided into two phases: metabolic (i.e., hepatic) clearance and the Phase I and Phase II process. The division
is based on the observation that a drug substance first undergoes oxidative attack (e.g., benzene to phenol), and the newly
introduced hydroxyl function will undergo glucouronidation (e.g., phenol to phenyl glucouronic acid). Some metabolites are
formed as impurities during the development of a process. Control of these process-related metabolite impurities in the final
API may not be necessary if control of other metabolites has already occurred and taken into consideration. Tightening the
limits, therefore, may not be needed.
Examples are asenapine N-oxide, asenapine desmethyl, and ciprofoxacin ethyl diamino impurity, which are formed as process impurities, but are also
metabolites of the same process (see Figure 5). It put forth a question whether limiting such a metabolite impurity in the
final API is still required.
Figure 5: Process impurities, thermal decomposition impurities, and metabolites of asenapine. CAS No. is Chemical Abstracts
Select analytical methodologies
The development of a new drug mandates that meaningful and reliable analytical data be generated at various steps of drug
development. The drug also should exhibit excellent stability throughout its shelf-life. To meet these requirements, methodologies
need to be developed that are sensitive enough to measure low levels of impurities. This need has led to analytical methods
that are suitable for determining trace and ultra-trace levels (i.e., submicrogram) quantities of various chemical entities
(35–39). Various methods are available for monitoring impurities.
. Various spectroscopic methods can be used for characterization of impurities, such as UV-visible spectroscopy, FTIR spectroscopy,
NMR spectroscopy, and mass spectrometry (MS).
. Various separation methods can be used, including thin-layer chromatography (TLC), gas chromatography (GC), HPLC, capillary
electrophoresis (CE), and supercritical fluid chromatography (SFC). A review of these methods is provided in the literature
(39). CE is an electrophoretic method that is frequently lumped with chromatographic methods because it shares many of the
common requirements of chromatography. A broad range of compounds can be resolved using TLC by using different plates and
mobile phases. GC is a useful technique for quantification. It can provide the desired resolution, selectivity, and ease of
quantification. This technique is useful for organic volatile impurities. SFC offers some of the advantages of GC in terms
of detection and HPLC in terms of separation.
The following hyphenated methods can be used effectively to monitor impurities: GC–MS; liquid chromatography (LC)–MS; LC–diode-array
detection (DAD)–MS; LC–NMR; LC–DAD–NMR–MS; and LC–MS–MS.
. It is often necessary to isolate impurities because the instrumental methods are not available or further confirmation
is needed. The following methods have been used for isolation of impurities: solid-phase extraction, liquid–liquid extraction,
accelerated solvent extraction, supercritical fluid extraction, column chromatography, flash chromatography, TLC, HPLC, CE,