Comparison of genuine, generic, and counterfeit Cialis tablets

Comparison of genuine, generic, and counterfeit Cialis tablets using vibrational spectroscopy and statistical methods (2021)
Dita Spálovská, Tomáš Pekárekb, Martin Kuchař ,Vladimír Setnička



Abstract

The dubious online market in phosphodiesterase type 5 inhibitors is growing on a global scale. Counterfeit medical products can represent health issues for the user and cause medical mistrust. Within this work, genuine Cialis containing the active pharmaceutical ingredient (API) tadalafil, its generics available in the Czech Republic, and the Cialis tablets from questionable online pharmacies were analyzed. The methods of infra-red and Raman spectroscopy were used for the identification of the counterfeit tablets and for the verification of their API and excipients. All 9 tablets from online pharmacies were counterfeit with 2 of them even containing a different API (sildenafil, vardenafil). In addition, Raman mapping was used to determine the API and excipients’ distribution and, in combination with multivariate data analysis, to separate similar tablets in clusters and to identify the outliers. Scanning electron microscopy of the samples revealed that the process of a wet granulation of micronized API was used during the formulation of the tablets. This comprehensive approach of analysis can be used for advanced exploration of the dubious samples of various medical products.




1. Introduction

According to the OECD [1] and World Health Organization (WHO) [2], medicines for erectile dysfunction treatment are among the most frequently counterfeited. The main representatives of these lifestyle drugs, which act as inhibitors of phosphodiesterase type 5 (PDE5), are primarily Viagra with sildenafil citrate as its active pharmaceutical ingredient (API), Cialis with tadalafil as its API, Levitra with vardenafil, and Spedra with avanafil. The advantage of Cialis over other PDE5 inhibitors is in the duration of action in the human organism – it has a terminal half-time of approximately 17.5 h in comparison to approximately 4–6 h for Viagra, Levitra, and Spedra. The increasing phenomenon of counterfeit medicines is caused mostly by illegal online pharmacies where everyone can buy those prescription-only medicines without the embarrassment of consulting a doctor. The second advantage of ordering those counterfeits via online pharmacies for patients is a lower price than the price of the genuine medicine in a regular pharmacy but in comparison with the generic equivalent tablets, the price is similar. However, their unknown origin and composition may cause a potential health risk [3].

Substandard and counterfeit medical products, as defined by the WHO in 2017 [4], have an impact not only on the individual’s health but also on the economy, families, national health systems as well as on pharmaceutical companies. Moreover, the poor quality of such medical products often caused by not following good manufacturing practices (GMP) could arouse mistrust for medicines in general. A comprehensive analysis of these dubious medical products is highly desirable as well as education of the general public about the dangers of purchasing medicines online.

In general, experts in the pharmaceutical field in cooperation with the WHO and national institutes of health are suggesting simple and also sophisticated forensic chemistry techniques to detect, screen, and analyze counterfeit medical products [5,6]. Beside visual inspection [7], the methods of thin-layer chromatography (TLC) [8], colorimetry [9], chromatographic techniques [10,11], nuclear magnetic resonance (NMR) [12,13], X-ray fluorescence spectrometry [14], X-ray powder diffraction (XRD) analysis [15] and infra-red (IR) and Raman spectroscopy [16–22] are highly recommended. Several analytical methods have also been used to distinguish between counterfeit medical products of PDE5 inhibitors and genuine products. Vibrational spectroscopy is one of those with the highest application potential in pharmacy for its reliability, speed, minimal (or no) need for sample preparation, and non-destructiveness [23,24]. Another promising tool for the analysis of counterfeit tablets is Raman mapping, which enables a detailed analysis of the tablet composition and spatial distribution [25]. Moreover, in combination with multivariate data analysis, it could help to reveal more significant modifications during the counterfeiting of tablets [26,27]. Furthermore, when deciding which analytical method to choose (Fig. 1), vibrational spectroscopy combined with statistical methods is unique among separation methods and NMR in its capability of components and particle size distribution, which is highly desirable for the pharmaceutical industry. This method has already been used for the analysis of the counterfeit Viagra tablet core [28,29] and provided information on the particle distribution, size as well as excipients used and even for a simple analysis of two counterfeit Cialis tables [30]. However, there is still a lack of detailed studies on other PDE5 inhibitors using Raman mapping.

Our intent was to combine the common methods of IR and Raman spectroscopy and explore the possibilities of an innovative approach for reliable Raman imaging of the Cialis tablets in connection with the statistical method based on using the principal component analysis (PCA) and soft independent modeling of class analogy (SIMCA). The aim was to distinguish between the counterfeit medical products and the genuine or generic medicines, to examine their quality, to identify the APIs and the excipients used, to determine the particle size distribution, and to inspect the dubious online market in Cialis which is a prescription-only medical product in the Czech Republic. Furthermore, we analyzed the samples using scanning electron microscopy (SEM) to confirm the mechanisms of their formulation and to verify the API distribution from Raman mapping.




4. Conclusion

In this study, we have focused on an analysis of genuine Cialis, its generics available in the Czech Republic, and the Cialis tablets purchased from dubious online pharmacies. The aim was to investigate the online market with Cialis, detect the counterfeit tablets, identify the APIs and excipients used in those tablets and compare them with the genuine medicines also containing 20 mg of tadalafil as an API.

Our comprehensive approach embraced the methods of IR and Raman spectroscopy, SEM, and Raman mapping along with the multicomponent statistical method PCA to explore each tablet. All the Cialis tablets from dubious online pharmacies were counterfeit while two (tablets A and B) of the nine samples even contained a different API (sildenafil and vardenafil). Those tablets were excluded from further PCA. Even if the IR and Raman spectra of the ground tablets were very similar to genuine Cialis in four cases, Raman mapping provided reliable discrimination of all counterfeit tablets. Besides, PCA supported by SIMCA applied to the Raman mapping data allowed separation of the individual clusters of the generics and counterfeit Cialis tablets according to their different chemical profiles and distinguished successfully between the genuine, generic, and counterfeit Cialis samples. Using SEM, the wet granulation of micronized tadalafil was identified as the formulation method for the counterfeit tablets.

Routine analysis is usually not able to detect well-made counterfeit medical products. The proposed combination of several analytical methods together with statistical evaluation is much more reliable. Moreover, the information obtained by Raman mapping is important not only for the identification of the counterfeit but also for determining the formulation process used, the distribution, and the particle size or clusters of API and excipients. This information can be especially valuable for generic pharmaceutical companies looking to draw ideas for their manufacturing process. In addition, the knowledge of particle distribution can help to identify unauthorized products.

The proposed combination of different methods for the analysis of the dubious medical products resulted in a comprehensive approach for gaining maximum information about the samples.

 

[MDavidW76]

Well, picked up my “generics” locally, 10mg Cialis… worked like a charm this morning, almost too well… looked like a freaking double stuffed sausage (I know, probably TMI).

 

[madman]

Forgot to post this up when I started the thread LOL!




3. Results and discussion

3.1. Visual inception of the tablets

From all the studied counterfeit tablets (Fig. 2, b and Supplementary material – Fig. S1, b), only tablets A and C corresponded to genuine Cialis (Fig. 2, Fig. S1, a) with the almond shape, size and imprint. However, their colour shade was slightly different. The counterfeit tablets E and H were of a similar shape to genuine;the size and imprint were different. The counterfeit tablets B, D, F, Gand I were of an elliptical shape. The generics of Cialis (Figs. 2, S1, c)were of an elliptical or circular shape.




3.2. Infra-red and Raman spectroscopy

Based on the Raman and IR spectroscopy analysis (Fig. S2), the API and the main excipients were identified in all the counterfeit tablets (Table 1) by comparing the spectra of the tablets with the spectra of the individual substances measured previously in-house by employing the in-house built spectral library. Comparisons were carried out by assigning the individual vibrational bands in the Raman spectra of the tablets to the vibrational bands of API and each excipient (demonstrated in Fig. S3). Those excipients were subsequently verified by Raman mapping. Instead of the API tadalafil, sildenafil (both citrate and base) and vardenafil were found in the counterfeit tablets A and B, respectively. For this reason, those tablets were excluded from further statistical analyses. The most similar counterfeit tablets to genuine Cialis according to the Ramanand IR spectra include tablets F, G, H and I with the same composition differing in a maximum of one component. Using Raman mapping, also a small amount of additional excipients could be identified: amberlite in the counterfeit tablets D and E, and CaCO3 inthe counterfeit tablets A, D and E. In the core of the tablets A and D, an identical component has not been identified yet.




3.3. Raman mapping

The Raman mapping provided a powerful method for analysing the counterfeit Cialis tablets and their discrimination from genuine Cialis. Even if the composition of the counterfeit tablet was very similar to genuine Cialis, the Raman maps of those tablets were different. In Fig. 3, there is an example of the Raman spectra of genuine Cialis and the counterfeit tablets G (very similar composition to the genuine) and C (only similar composition to the genuine). The difference between genuine Cialis and the counterfeit tablet G was noticeable at a glance due to the different spatial distribution of the API and the main excipients (MCC and lactose monohydrate). Raman maps of the rest of the counterfeit Cialis tablets can be found in Supplementary material (Fig. S4). In comparison with the consistent distribution of the particles and good homogeneity of the genuine Cialis tablet, the API in the counterfeit tablets was in separated clusters with lactose. Using Raman mapping, a small amount of CaCO3 was found in the counterfeit tablets A, D and E and a negligible amount of amberlite in tablets D and E. One unknown substance in a very small amount was found in tablets A and D and has not yet been identified.




3.4. Principal component analysis

The data from the Raman maps processed with the DCLS method were used for PCA to provide a reliable comparison of the genuine, generic and counterfeit tablets. First, the PCA explained variance plot (Supplementary material, Fig. S5) gave us information on how many PCs should be used for further evaluation. The first and second PC explain 80% and 9% of variance, respectively. In general, 89% of total variability are enough for our purpose. The loading plots of PC-1 and PC-2 (Supplementary material, Fig. S6) showed the most relevant variables for the variance explanation, in this case it means mostly the bands reflecting the API and main excipients: lactose monohydrate and MCC. Positive and negative correlation between bands of PC-1 showed that the content of MCC depended on the content of lactose. If the content of MCC grew, the content of lactose decreased. In the PCA score plot (Fig. 4), each score represented one averaged spectrum and the scores located near each other meant that the spectra have a similar pattern while the distant scores suggest differences [35]. This helps to reveal the similar patterns, clusters and also outliers. The scores of genuine Cialis could be found in the middle of the four quadrants. The cluster of the generics Rakifre and Gerocilan was separated above, along PC-2. The scores of Tadalafil Mylan could be found close to it. The cluster of two generics (Tadalafil Accord and Zenavil) together with the counterfeit tablet F was separated along the PC-1 towards the negative values. The last cluster consisted of the counterfeit tablets D, E, G, H and I and was shifted to the more positive values of PC-1 and negative values of PC2 in relation to genuine Cialis. The most outlying were the scores of generic Tadilec to separated mostly along PC-1 towards the negative values. This separation was caused by the presence of the different excipients hypromellose (HPMC) and mannitol instead of MCC and lactose.

According to the PCA, the most similar to genuine Cialis were the counterfeit tablets D and E. In general, the counterfeit tablets were more like Cialis than generics as was expected since they try to mimic the genuine tablet. Clusters differences of generic Cialis tablets from original Cialis show the effort of generic pharma companies to produce bioequivalent products by patent non-infringing formulations. On the other hand, the overlap of counterfeit clusters with genuine Cialis is significant. Therefore, the SIMCA being a supervised method was applied on the data to analyse possible differences of counterfeits and Cialis. The SIMCA compares the distances of the individual cluster centres of tablet’s spectra. These determined distances of the cluster centres from each other therefore show the similarity of individual tablets. If the distances are small, the models are close together or even overlap. Large distances between models indicate that they are different.




3.5. Soft independent modelling of class analogies

SIMCA was used as a dependent method on the previous PCA models of the individual tablets to provide a quantitative classification of those models. The result is a matrix with a unit diagonal (Table 2) based on the measurement of the distances between the individual PCA models to find the similarities between them. Lower distances (values in units, dozens) mean greater mutual similarity, higher distances (hundreds, thousands) then lesser similarity of thePCA models. In our case, it was obvious that the distances of the counterfeit tablets F, D, G, E, H, I were low, specifically in dozens, reflecting their mutual similarity. To compare, the distances of the generics were in thousands as generic pharma companies try to set themselves apart from the original. For Cialis, the distances from the other models were very different. The lowest distances were observed for the counterfeit medicines F, D, G, E, H, I. However, even such small distances (shown in Table 2) enabled us to differentiate counterfeits from genuine Cialis. The highest distance of the Cialis PCA model was observed in comparison to the generic Tadilec to PCA model.




3.6. Scanning electron microscope

To gain more information about the tablets, SEM was used allowing us to verify that crystalline and micronized tadalafil was incorporated into genuine Cialis by the method of a wet granulation[36] resulting in tablets with good homogeneity. In homogeneous blends can evince discrepancy in the content of the API and possibly affect the quality of the final product. The wet granulation is based on the production of the granules by wet massing of the excipients and API with granulation liquid with or without binder and subsequent drying of the mass [37]. Based on the results from Raman mapping and SEM, formulations of the counterfeit Cialis tablets were probably also made by a wet granulation from micronized tadalafil with particle size of ~2 µm as well and the API was mostly in the bounded clusters with lactose (Fig. 5). This formulation is evident due to the API particles being stuck to larger lactose particles and the crystals not having regular and sharp shapes.