A recurring topic in this column is structural characterization including the underpinnings of mass accuracy and resolution
and spectral interpretation.1–3 The need to determine the exact structure, especially of an unknown, is at the core of why we employ mass spectrometers
in all the variations and "flavours". Mass spectrometry (MS) has developed into a mainstream analytical technique in the pharmaceutical
industry where one of its major roles is identifying and characterizing new chemical entities by elucidating the structure
of unknown compounds.
Jean-Claude Wolff, a primary contributor to this discussion as a scientist with GSK (Medicines Research Centre, Stevenage,
Hertfordshire, UK) has found characterizing unknowns in potential counterfeit medicines both a challenging and excellent focus
for his talents.
I would not suggest that elucidating the structure of impurities is easy. My purpose is merely to show how combined mass spectrometric
techniques can gather the information necessary to elucidate the structure of an unknown compound. In so doing, I suggest
a pathway that leads to identifying the structure of the molecule analysed. As an example, I offer the exercise of determining
the substitute, active pharmaceutical ingredients in counterfeit medicines.
Often the only information available when considering a novel chemical entity might be that the new, unknown compound of interest
belongs to a particular class of compounds — in natural product research alkaloids, for instance. Additional information ("background"
information) becomes available after performing structure elucidation on impurities or degradants structurally related to
a pharmaceutically active compound. The World Health Organization (WHO) defines counterfeit medicines as:
Those medicines that are deliberately and fraudulently mislabelled with respect to identity and/or source. Counterfeiting
can apply to both branded and generic products. Counterfeit products may include products with the correct ingredients or
with the wrong ingredients, without active ingredients, with insufficient active ingredient, or with fake packaging.4
"I focus on products with the wrong active pharmaceutical ingredient whose identity I then determine using MS techniques.
My sole guidance is that a compound [to be a probable suspect] be readily available commercially and that it be cheap." These
are the key elements making the compound attractive to the counterfeiters. Note, however, that "substitute actives" are not
necessarily so common that they will be found in libraries. Hence, the reason for structure elucidation.
Counterfeits with an incorrect active ingredient are commonplace. A study of antibiotics and antimalarials by the German Pharma
Health Fund showed about 16% of cases investigated contained incorrect ingredients.5
The MS tools available to determine incorrect pharmaceutical ingredients are numerous: you can use a series of sample introduction
techniques such as direct inlet or probe, gas chromatography (GC) and liquid chromatography (LC). The choice of inlet technique
depends on a sample's characteristics. Inlet technique is also linked to the ionization method you choose — electron ionization
(EI), chemical ionization (CI), atmospheric pressure chemical ionization (APCI), electrospray ionization (ESI) — or the newer
techniques such as desorption electrospray ionization (DESI) or direct analysis in real time (DART). All of these techniques
have been reported in this column in recent years — though admittedly we treated GC techniques rather lightly, an imbalance
we shall soon redress.
Structural elucidation of unknowns (in this instance substitute, active pharmaceutical ingredients) demands using MS instruments
capable of tandem MS and accurate mass measurements to determine the elemental composition of an analyte. Here the choice
is large, ranging from ion trap to Fourier-transform (FT) ion cyclotron resonance via quadrupole time of flight (Q-TOF). Regardless
of the instrument or ionization technique used, however, you can elucidate an unknown compound's structure by using the arsenal
of MS techniques available. The goal here is simply to show a possible pathway and discuss its merits.
Structure elucidation of a substitute, active pharmaceutical ingredient in an antimalarial tablet: The most effective approach for identifying and characterizing counterfeit medicines is to analyse a suspect sample using
LC coupled to accurate-mass, tandem MS. This approach is more precise and quantitative than direct-inlet analysis (probe or
infusion) of a sample. LC provides distinct separation of the active or substitute-active pharmaceutical ingredient from the
excipients. Moreover, using LC reduces the potential for ion suppression in the ion source. Another advantage of introducing
a sample via LC is the ability to compare LC–UV and LC–MS profiles of a suspect sample with a genuine one.
