Analysis of counterfeit Cialis® tablets using Raman microscopy and multivariate curve resolution
Highlights
► Counterfeit Cialis tablets were analyzed using Raman microscopy and multivariate curve resolution (MCR). ► Excipients and API can be resolved by this technique and chemical maps were obtained to show the spatial distribution of components. ► MCR reveals subtle differences between genuine and counterfeit tablets. ► Raman microscopy combined with MCR provides chemical insights to the analysis of counterfeit medicines.
Introduction
Counterfeit products have been present since ancient times; the famous Greek mathematician Archimedes proved that a gold crown made for King Hiero II contained elements other than gold. Counterfeiting remains a serious problem in the modern society and counterfeit products can be found in a wide range of consumer goods, including clothing, media products, food and even automobiles. It has been reported that even the National Aeronautic and Space Association (NASA) may have purchased counterfeit materials [1]. Counterfeit products have detrimental effects on the economy and the Organisation for Economic Co-operation and Development (OECD) has estimated that the monetary value of the international trade in counterfeit products approached 200 billion in 2005 [2].
The pharmaceutical industry has been highly targeted by counterfeiters in recent years. The World Health Organization (WHO) defines counterfeit medications as follows [3]: “Counterfeit medicines are medicines that are deliberately and fraudulently mislabeled with respect to identity and/or source. The use of counterfeit medicines can result in treatment failure or even death. Counterfeit medicines may include products with the correct ingredients or with the wrong ingredients, without active ingredients, with insufficient or too much active ingredient, or with fake packaging”. Counterfeit drugs are part of the broader phenomenon of substandard medicines. The WHO defines substandard medicines as follows: “Substandard medicines (also called out of specification (OOS) medicines) are genuine medicines produced by manufacturers authorized by the National Medicines Regulatory Authority (NMRA) which do not meet quality specifications set for them by national standards”. The difference between counterfeit and substandard medicines is that the former are produced by unauthorized manufacturers who intend to deliberately mislead the consumer with respect to the origin of the product [3]. Besides the economic consequences of counterfeit products, counterfeit medicines can negatively impact the health of the consumer with the worst possible outcome being death. For example, diethylene glycol (DEG) contamination of paracetamol syrup was responsible for approximately 192,000 deaths in 2002 in China [4].
The seriousness of the counterfeit problem has motivated analytical and pharmaceutical scientists to develop and implement a variety of analytical techniques for the detection and analysis of counterfeit medicines. Among them, separation techniques such as thin layer chromatography (TLC) [5], high performance liquid chromatography (HPLC) [6], [7] and capillary electrophoresis (CE) [8] are still the more common methods, but spectroscopic and imaging methods such as direct ionization mass spectrometry [9], near-infrared (NIR) spectroscopy [10], [11], Raman spectroscopy [12], [13], and nuclear magnetic resonance (NMR) spectroscopy [14] have been developed as they provide information on the molecular structural level and spatial distribution of the active pharmaceutical ingredients (API) and excipients. Raman spectroscopy has gained popularity for counterfeit analysis recently [12], [13] because minimal or no sample preparation is required and even closely related molecules typically display spectral uniqueness. Raman spectroscopy is particularly suitable for analyzing solid dosage forms and can be used to detect different solid state forms of an API such as polymorphs [15], hydrate versus anhydrate [16] or amorphous versus crystalline drug [17]. Raman spectroscopy can be combined with microscopy to analyze microscopic features of a sample. A small but highly focused laser spot can be generated with the use of a high power objective and used to probe local microscopic features of a sample surface. Subsequently, a chemical image or map of the sample can be obtained when a large number of these spots are sampled and the spatial distribution of the chemical components can be generated and studied. Such an approach is especially useful and applicable to the analysis of tablets [18].
Over the past decade, the field of chemometrics has advanced tremendously [19] and numerous applications have been demonstrated [19], [20], [21], [22]. Multivariate curve resolution (MCR) is a chemometric method that involves resolving a multicomponent system into individual pure components. In general, the process of MCR includes the decomposition of a data matrix that contains the response of a multicomponent system into a product of two matrices that contain pure component information in the row and column directions of the original data matrix [23]:where D is the original data matrix, C and ST are the pure component matrices containing information in the row and column directions respectively, and E is the error matrix containing information irrelevant to the system. When MCR is being applied to spectroscopic data, C and ST are usually referred to as the relative concentrations and spectral profiles of the pure components respectively. In order to obtain the resolved components, a number of algorithms are available. One of these algorithms is alternating least squares (ALS). This is an iterative least square process used to minimize the least square errors of the C and ST matrices simultaneously. This process continues until a pre-defined convergence criterion is reached. No prior information is needed for MCR-ALS, only an initial estimate of either C or ST is required to start the calculation. Constraints are often implemented into the calculation to better describe the chemical system and avoid ambiguous results.
The objective of this study was to combine Raman microscopy and multivariate curve resolution for the analysis of and comparison between genuine and counterfeit Cialis® tablets. Cialis is a phosphodiesterase 5 (PDE5) inhibitor used to treat erectile dysfunction. Counterfeit Cialis tablets have been studied by Raman and NMR spectroscopy [24], HPLC and NIR spectroscopy [25], and mass spectrometry [26]. In this research, detection and analysis of the excipients used in the counterfeit tablets was of interest, emphasis will be placed on the resolving and the spatial distribution of the excipients and API.
Section snippets
Materials
Genuine Cialis® tablets (20 mg) (Eli Lilly, Indianapolis, IN) were purchased from the Purdue University Pharmacy (West Lafayette, IN). Counterfeit Cialis® tablets were obtained from Eli Lilly and Company. There are 11 excipients and one active pharmaceutical ingredient (API) present in the real tablets. The API in Cialis® is tadalafil while 11 excipients are lactose monohydrate (LMH), croscarmellose sodium (CMS), hydroxypropylcellulose (HPC), microcrystalline cellulose (MCC), sodium lauryl
Results and discussion
Genuine and two different counterfeit Cialis® tablets (Samples A and B) were analyzed. The photographs of these tablets are shown in Fig. 1. The genuine tablet is yellow with an almond shape and has the “C20” logo inscribed on one side of the tablet. The counterfeit tablets look very similar (size, shape, color, inscription) to the genuine tablets; it is rather difficult to distinguish between them using only appearance. However, the chemical details of these tablets were revealed by Raman
Conclusions
Raman microscopy combined with MCR-ALS was used to successfully analyze genuine and counterfeit Cialis® tablets. For the genuine tablets, two excipients and the API were resolved. The homogeneous nature of the genuine tablet most likely explains why only the three major components could be resolved. For counterfeit sample A, seven excipients, the API, and an unknown compound were resolved. Two of the excipients present are not found in the genuine tablets. The API content was more than four
Acknowledgments
This work was supported by a grant from the Lilly Endowment Inc. to the College of Pharmacy and a research fellowship from United States Pharmacopeia to K.K.
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