Bioanalytical methods often involve the quantification of a parent compound to determine the pharmacokinetic properties of
a potential drug. In some cases, it may also be necessary to quantify a metabolism product in addition to the parent molecule
as these may be either active or toxic. Simultaneous determination can be challenging because of differences in chemical properties,
such as acidity, basicity, and polarity, which can significantly increase the difficulty in developing both the extraction
method as well as suitable chromatography. Developing chromatographic conditions involves not only separating the parent from
the metabolite, but also effectively separating both compounds from potential coeluted contaminants such as phospholipids
that may affect ionization efficiency and cause suppression or enhancement. The stabilities of the parent and metabolite also
must be considered, as any interconversion between the two during the bioanalytical procedure may lead to variability within
the results. In this article, we discuss the method development process required for the accurate quantification of both clopidogrel
and its acid metabolite with a lower limit of quantification (LLOQ) of 1 pg/mL in human plasma.
It is of great importance to integrate pharmacokinetic, pharmacodynamic, and toxicokinetic information in the process of drug
development. Serious decisions are often made based on bioanalytical data, so it is vitally important that these data are
precise and accurate. Before any bioanalytical method can be used for the analysis of samples acquired from dosed subjects,
it is necessary to validate the method according to the appropriate guidelines, set forth by the US Food and Drug Administration
(FDA), the European Medicines Agency (EMA), and others. Because of the strict requirements for method validation, it is essential
to be thorough during the method development process and to identify any potential issues before the validation process. The
best strategy for method development is to take a systematic approach with the three main aspects as follows: the extraction
method, the liquid chromatography (LC) method, and the detection method. For the development of a sensitive, robust, and accurate
method, all three portions are equally important.
As stated above, many bioanalytical methods require the quantification of more than one component. In many cases, the quantification
of one or more metabolism products is essential for obtaining accurate data regarding the drug's pharmacokinetic profile.
When an assay requires the quantification of multiple components, the development process becomes more difficult than it would
be for a single compound. For some assays, the relative concentration requirements of the parent and metabolite may be vastly
different because of the expected levels found in vivo. In this case, it may be best to optimize the method for the compound
requiring the lower limit of quantification (LLOQ), and then make any necessary modifications to accommodate the other components.
Clopidogrel (trade name Plavix, Figure 1), is a thienopyridine derivative antiplatelet prodrug used in the prevention of artherosclerotic
events. It is dosed in an inactive form and requires a hepatic biotransformation to yield the active thiol-metabolite, which
binds to cell receptor P2Y12, irreversibly inhibiting the platelet activation process (1). In addition to the active metabolite,
an inactive carboxylic acid metabolite is also formed. This acid metabolite accounts for the majority of circulating clopidogrel-related
material with very low levels of the active metabolite and unchanged prodrug present (2,3). Because of the reactivity of the
thiol metabolite, coupled with the low levels of the unchanged prodrug, most quantitative studies are based on the circulating
levels of the inactive metabolite. The ability to detect the low levels of unchanged prodrug will provide more accurate data
on the pharmacokinetics of clopidogrel, allowing improved evaluation of the bioavailability of new formulations.
Figure 1: Chemical structures of clopidogrel and clopidogrel carboxylic acid metabolite.
The finalized experimental conditions for the extraction procedure and ultrahigh-pressure liquid chromatography coupled with
tandem mass spectrometry (UHPLC–MS-MS) are described below.