Application of Mass Spectrometry to Support Verification and Characterization of Counterfeit Pharmaceuticals - - Spectroscopy
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Application of Mass Spectrometry to Support Verification and Characterization of Counterfeit Pharmaceuticals

The production and sale of counterfeit drugs has risen sharply in recent years. The World Health Organization (WHO) estimates that counterfeit medicines account for approximately 1% of sales in developed countries and well over 10% in developing countries. These substandard versions of medications not only represent a significant safety threat to patients, but also challenge the credibility of the pharmaceutical industry and its ability to provide patients with safe and effective products. These counterfeit products may contain either the incorrect dose or none of the intended active compound. In some cases, these counterfeit medicines contain different active drug components and, in the worst cases, may even contain toxic substances. Because of these concerns, the pharmaceutical industry vigilantly monitors the global market for counterfeit products. When a suspected counterfeit product is detected at Bristol-Myers Squibb, it is fully characterized to assess the potential risks to patient safety. Liquid chromatography coupled to mass spectrometry (LC–MS), with accurate-mass capability, is a powerful tool for investigating counterfeit pharmaceuticals because it allows the rapid assignment of the molecular weight and formula of each component, which can then be used to search the literature or internet for a potential match. This approach is frequently successful because counterfeiters often formulate their products from commonly available, relatively simple materials, rather than novel products.

Figure 1: Decision-tree diagram of the general strategy for counterfeit product analysis.
The World Health Organization (WHO) defines counterfeit drugs as drugs that are "deliberately mislabeled with respect to identity and/or source. Counterfeiting can apply to both branded and generic products with counterfeit products including drugs with the correct ingredients or with the wrong ingredients; without active ingredients, with insufficient active ingredient or with fake packaging" (1). Counterfeit pharmaceutical products have been detected since approximately 1990 (2). Since then, the number of cases investigated by the US Food and Drug Administration (FDA) quadrupled to an average of about 20 per year in 2001 and 2002 (3). In an effort to ensure patient safety and brand integrity, the pharmaceutical industry vigilantly monitors the global market for counterfeit products. Counterfeit products at Bristol-Myers Squibb are fully characterized to assess the risks to patient health and safety.

Figure 2: LC–UV chromatogram (210 nm) of product A tablet extract.
The first step in detecting a counterfeit medicine is to conduct a visual inspection of its physical characteristics as well as the appearance of the accompanying packaging materials. The effectiveness of a visual inspection may have limited success given the increased sophistication adopted by counterfeiters. An even greater challenge than identifying a pharmaceutical product as a counterfeit is the identification and quantitation of all components present in the material. This process is necessary to make an accurate assessment of toxicology and patient risk. Identification and quantitation of components in a counterfeit product requires chemical analysis. Throughout the industry, various modern analytical techniques have been applied for the characterization of counterfeit pharmaceuticals, including thin-layer chromatography (TLC) (4), gas chromatography (GC) (5), high performance liquid chromatography (HPLC) (6), Raman and near infrared (NIR) spectroscopy (7–11), mass spectrometry (MS) (12,13), and nuclear magnetic resonance (NMR) spectroscopy (14,15). Each of these techniques is capable of providing rich analytical data to assist in the characterization of counterfeit medicines. In practice, two or more of these techniques will be used orthogonally because the ideal goal of these investigations is to achieve absolute identification and quantitation of all components that are present (16).

Figure 3: Negative-ion ESI mass spectrum of component 1 observed in product A.
To enable the complete characterization of detected counterfeit medicines, a general strategy has been developed that uses vibrational spectroscopy (Raman and NIR), liquid chromatography coupled to mass spectrometry (LC–UV-MS), and NMR as an orthogonal structural confirmational technique. An overview of this strategy is depicted in Figure 1. A visual inspection followed by analysis with Raman or NIR spectroscopy is used as an initial screen to identify product authenticity. If the product is deemed authentic based on a comparison of the collected spectra of the possible counterfeit product to library spectra acquired on the authentic product, then the investigation is complete. If the drug product is deemed a counterfeit based on this initial screen, then the material is subjected to low-resolution LC–UV-MS to assess the number of components present and assign molecular weights to all components observed in the LC–UV and MS total ion chromatograms (TIC). The initial screen by LC–UV-MS is performed on systems that are equipped and maintained for open-access usage. Use of the open-access systems improves the efficiency of the analysis process since these systems are preconfigured and available to run samples. If low-resolution MS analysis provides adequate ionization of the counterfeit components, then the sample is analyzed on a high-resolution instrument for accurate-mass measurement and assignment of molecular formulas. To achieve a narrow list of possible formulas, the elemental composition search is restricted with respect to included elements and the allowed deviation of calculated masses from the measured mass. Initially, the search is restricted to formulas that contain only C, H, N, and O. The allowed deviation from the measured mass is set to a maximum of 1.25 mmu. The isotopic distribution is also evaluated to check for the presence of elements other than C, H, N, and O that should be included in the element set.

Figure 4: Accurate mass, positive-ion ESI mass spectrum of component 2 observed in product A.
As an alternative, the sample can also be analyzed initially on an accurate-mass instrument, thereby bypassing the need for the low-resolution screen. If the low-resolution LC–MS analysis indicates a single component, or if the response in the mass spectrometer is found to be poor by electrospray ionization (ESI) then the sample is analyzed by NMR next. For samples that fail to ionize by ESI, and were observed to be relatively pure by NMR, desorption chemical ionization (DCI) is attempted to obtain a molecular weight and elemental composition for the unknown component. Quantitation of components is performed following identification of the components. For counterfeit products containing a single component, such as aspirin, LC–UV can be utilized to quantify the analyte by comparison with an authentic standard. Quantitative NMR (qNMR) is also used to determine the level of analytes in counterfeit products (17). For more-complex mixtures, a combination of techniques may be required to identify and quantify all of the individual components present in the counterfeit medicine. For example, techniques such as LC–UV-MS coupled with NMR spectroscopy (qualitative and quantitative), LC–UV-MS coupled with Raman or NIR spectroscopy, or these techniques coupled as needed with preparative LC isolation, X-ray diffractometry, or ICP-MS for metals analysis (18,19) can be used.

Figure 5: Positive- and negative-ion ESI total ion chromatograms of the product B extract.
The strategy described above has been used to complete numerous counterfeit investigations at Bristol-Myers Squibb. In general, LC–MS has proven to be a powerful tool for the characterization of counterfeit products. The ability to rapidly assign a molecular weight and often a single formula for each counterfeit component affords the opportunity to expedite the putative identification using literature or internet searching. This process could be likened to electron ionization (EI) library searching in its ability to quickly filter potential candidates. Investigations using this approach are generally successful because counterfeiters often produce their products from commonly available, relatively simple materials, not novel products. A series of case studies are presented here that demonstrate the general strategy for conducting investigations of counterfeit materials.

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