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Evaluation of the residual solvent content of counterfeit tablets and capsules
Accepted Manuscript Title: Evaluation of the residual solvent content of counterfeit tablets and capsules Author: E. Deconinck M. Canfyn P.-Y. Sacr´e P. Courselle J.O. De Beer PII: DOI: Reference:
S0731-7085(13)00141-6 http://dx.doi.org/doi:10.1016/j.jpba.2013.03.023 PBA 9017
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
26-2-2013 29-3-2013 30-3-2013
Please cite this article as: E. Deconinck, M. Canfyn, P.-Y. Sacr´e, P. Courselle, J.O. De Beer, Evaluation of the residual solvent content of counterfeit tablets and capsules, Journal of Pharmaceutical and Biomedical Analysis (2013), http://dx.doi.org/10.1016/j.jpba.2013.03.023 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Evaluation of the residual solvent content of counterfeit tablets and
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capsules
3 E. Deconinck1,*, M. Canfyn1, P.-Y. Sacré1,2, P. Courselle1, J.O. De Beer1
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Division of food, medicines and consumer safety, Section Medicinal Products, Scientific Institute of
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Public Health (WIV-ISP)), J. Wytmansstraat 14, B-1050 Brussels, Belgium
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²Analytical Chemistry Laboratory, CIRM, University of Liège, Avenue de l’Hôpital 1, B36, B-
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4000 Liège, Belgium
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Highlights Counterfeit tablets and capsules were screened for residual solvents. Detected solvents were quantified. Counterfeit tablets and capsules have significantly higher concentrations of residual solvents than genuine ones. In some toxic amount of residual solvents were detected.
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Abstract:
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A group of counterfeit samples of Viagra® and Cialis® were screened for their residual solvent
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content and compared to the content of the genuine products.
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It was observed that all counterfeit samples had higher residual solvent contents compared to
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the genuine products. A more diverse range of residual solvents was found as well as higher
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concentrations. In general these concentrations did not exceed the international imposed
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maximum limits. Only in a few samples the limits were exceeded.
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A Projection Pursuit analysis revealed clusters of samples with similar residual solvent
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content, possibly enabling some future perspectives in forensic research.
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Keywords: Counterfeit Medicines, Gas Chromatography, Residual Solvents
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*corresponding author: [email protected] Tel. +32 2 642 51 36
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Fax. +32 2 642 53 27
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Introduction
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Counterfeit medicines, imitations and substandard medicines are a growing problem
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worldwide. The problem is situated both in developing countries as in industrialized regions.
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In the developing countries the problem concerns the whole medicine supply chain, especially
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essential medicines like antibiotics, anti-malaria products, etc. This is often due to the lack of
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effective enforcement agencies and the high prices of genuine medicines in these countries. In
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the industrialized world the problem concerns essentially life style drugs like the PDE-5
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inhibitors, anabolic hormones and slimming products, although sometimes counterfeited
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antibiotics or insulin are intercepted by the customs [1,2]. The growing threat of these
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products is mainly due to the extension of the internet, where about 50% of the medicines
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sold through internet sites disclosing their identity is estimated to be counterfeit [3].
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The World Health Organization (WHO) [4] defines a counterfeit drug as: “one which is
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deliberately and fraudulently mislabelled with respect to identity and/or source.
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Counterfeiting can apply to both branded and generic products and counterfeit products may
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include products with the correct ingredients or with the wrong ingredients, without the active
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ingredients, with insufficient active ingredient or with fake packaging.”
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The fight against the counterfeiting of medicines resulted in numerous articles where several
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analytical techniques have already been used for the detection of counterfeit medicines. These
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techniques are separated in two main groups: chromatographic and spectroscopic techniques.
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The chromatographic techniques are used for the separation, identification and quantification
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of the active substances. They may also contribute to the elucidation of the structure of new
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analogues. Commonly used chromatographic techniques contain cheap and easy ones such as
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thin layer chromatography (TLC) [5,6] but also more sophisticated and expensive ones: such
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as HPLC-UV [7-9], LC-MS [7-20] and LC-DAD-circular dichroïsm [18].
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The spectroscopic techniques are often preferred to chromatography for the identification of
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counterfeit drugs because of the fact that they are fast, need less (or no) sample preparation
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and some of them are non-destructive. Fourier-transformed Infrared spectroscopy (FT-IR)
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[16,17,21], NIR [5,21], Raman spectroscopy [21-23], X-ray diffraction (XRD) [24],
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colorimetry [25,26] and Nuclear Magnetic Resonance (NMR) [23,27] have demonstrated their
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usefulness to detect counterfeit or adulterated drugs.
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All the above mentioned references, including the definition of the WHO focus on the
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presence and sometimes the amounts of active ingredients. This means that the fact that
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counterfeit/imitation medicines are essentially produced by street laboratories and small
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companies not respecting the GMP norms is not taking into account. The fact is that next to
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the presence of wrong active ingredients, the absence of active ingredients or a wrong dosage,
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the risk for public health can also result from the presence of impurities or residues of
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synthesis of the excipients/active ingredients used.
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One of these potential groups of toxic impurities are the residual solvents. In 1997 the
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International Committee for Harmonisation issued a guideline for residual solvents [29]. The
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limits proposed in this guideline have been adopted by the United States Pharmacopoeia [30],
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the European Pharmacopoeia [31] and the Japanese Pharmacopoeia [32]. The guidelines
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divide the residual solvents into three classes. Class 1 consists of solvents that should be
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avoided in pharmaceutical preparations due to their high toxicity, class 2 are the solvents that
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should be limited and class 3 consists of the solvents with a relatively low toxicity. Following
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the European Pharmacopoeia [31] the class 1 solvents (e.g. benzene, carbon tetrachloride,…)
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have limits between 2 and 8 ppm. 1,1,1-trichloroethane is also part of the class 1 solvents due
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to its environmental hazard. The limit here is up to 1500 ppm. For the class 2 solvents (e.g.
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chloroform, methanol, hexane,…) the limits vary between 50 and 5000 ppm, while the class 3
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solvents (e.g. acetic acid, ethanol, formic acid,…) are limited to 5000 ppm.
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In this paper a set of 85 different counterfeit or imitation tablets and capsules of Viagra® and
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Cialis® were screened for the presence of residual solvents. When residual solvents were
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detected, they were quantified in order to show their compliance or non-compliance to the
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ICH guidelines.
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2.1. Samples
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All counterfeit and imitation samples were donated by the Federal Agency for Medicines and
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Health Products (FAMHP) in Belgium. Genuine samples of Viagra® were kindly provided by
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Pfizer SA/NV (Belgium). Eli Lilly SA/NV (Benelux) kindly provided genuine samples of
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Cialis®.
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2-propanol (HPLC grade), dichloromethane (HPLC grade), ethylacetate (pesti-S), acetone
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(HPLC-grade) ethanol absolute and acetonitrile (HPLC grade) were purchased from Biosolve
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(Valkenswaard, The Netherlands). Chloroform (for gas chromatography), benzene, carbon
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tetrachloride (CCl4) (for spectroscopy) and ethylbenzene (for gas chromatography) were
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purchased from Merck (Darmstadt, Germany) and Toluene (pesticide residues) and
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cyclohexane from VWR prolabo (Fontenay-Sous-Bois, France). These solvents were used as
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reference standards. Dimethyl Sulfoxide, used as solvent for the samples was purchased from
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Merck.
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2.3. Instrumental conditions
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The samples were injected on a GC-MS system using a G188A headspace sampler (Agilent
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Technologies, Palo Alto, USA). The analyses were performed on an Agilent 6890N gas
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chromatograph coupled to an Agilent 5973N mass selective detector. Full automation was
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achieved using Agilent MSD ChemStation data acquisition and data handling software. After
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incubation of the sample (5 ml in a 10 ml headspace vial) at 120°C for 15 min, during which
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it was shaken, 0.5 ml of the vapour phase was injected into the GC/MS system in a split
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injection mode (split ration 6.8:1). The temperatures of the headspace loop, the transfer line
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and the EPC volatiles interface were 135, 145 and 160°C respectively. The solvents were
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separated on a Phenomenex 624 capillary column (60 m x 0.32 mm; 1.8 µm film thickness)
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(Phenomenex, Torrance, USA). The oven temperature was programmed from 60°C (held for
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5 min) to 270°C at 25 °C/min. 270°C is held for 10 min. The total run time is 23.4 minutes.
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The temperatures of the injection port, the ion source, the quadrupole and the interface were
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set at 160, 230, 150 and 280°C, respectively. For the identification of the solvents present in
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the samples the mass spectrometer was operated in full scan mode. For quantification and
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validation the mass spectrometer was operated in SIM mode (100 ms dwell times).
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2.4. Sample preparations
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2.4.1. Preparation of internal standard solution
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A stock solution containing 1000 ppm of acetonitrile and cyclohexane in dimethylsulfoxide
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was prepared separately. 1 ml was diluted to 100,0 ml dimethylsulfoxide and used as internal
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standard. Cyclohexane was used for the quantification of CCl4, benzene, toluene and
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ethylbenzene, while acetonitrile was used as internal standard for the remaining solvents. The
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selection of the internal standards was based on an initial screening of counterfeit samples.
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Both internal standards were not detected in the samples or only as traces. Therefore the
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choice of the internal standards was considered legitimate.
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2.4.2. Preparation of standards One stock solution was prepared containing 1000 ppm of all the selected solvents, besides
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acetonitrile and cyclohexane, in dimethyl sulfoxide. Starting from the stock solutions,
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dilutions were prepared in the same solvent to obtain standards containing 10, 1 and 0.5 ppm
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of each solvent. 500 µl of the internal standard solution was added to 5 ml of the standard
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solutions to obtain concentrations of internal standard of about 1 ppm. The solutions were
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brought in vials and automatically sealed. Dimethyl sulfoxide was used as blank. The choice
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of the solvent was based on a preliminary screening of about 150 samples in which dimethyl
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sulfoxide could not be detected. Therefore it was assumed that dimethyl sulfoxide is not
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present in the samples.
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For the analysis of tablets, the tablets were broken in two before addition of 5 ml of
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dimethylsulfoxide. Tablets were broken because of the coating of some of the tablets,
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possibly preventing the recovery of the residual solvents. To this solution 500 µl of the
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internal standard solution was added. Capsules were opened before addition of the solvent.
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2.5. Projection Pursuit (PP)
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PP is a unsupervised projection and variable reduction technique that is able to project high
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dimensional data into a low dimensional space, defined by a few latent variables, called
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projection pursuit features (PPFs). Contrary to Principal Component Analysis, PPFs are
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obtained by maximising a projection index describing inhomogeneity of the data. In such way
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PP can reveal the data structure and groups of similar samples [32]. In this study the
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algorithm as described by Croux and Ruiz-Gazen was used with the yenyukov index as
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projection index [33].
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2.6. Calculations Data processing and Projection Pursuit (PP) were performed using Matlab version 8.0.0 (The
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Mathworks, Natick, MA, USA). The algorithm PP was part of the ChemoAC toolbox
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(Freeware, ChemoAC Consortium, Brussels, Belgium, version 4.0).
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3. Results
In first instance both the genuine as the counterfeit samples were screened for the presence of
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residual solvents using the above mentioned method in full scan mode. This screening
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revealed the presence of the twelve solvents, summed in section 2.2, in one or more
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counterfeit samples. Calibration lines were prepared for the twelve solvents and quantification
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was performed using the mass spectrometer in SIM mode. Table 1 presents the specific m/z
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ratios (instrumental parameters) monitored for each of the selected solvents as well as their
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retention times and the group start times (opening window) for the SIM mode. The specific
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m/z ratios used for quantification are indicated in italic. These specific ions were selected as
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being the ones with the highest abundance. The calibration lines were measured and the
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sample concentrations were calculated using ordinary least squares regression.
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3.1. Genuine samples
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Screening of the genuine samples of Viagra® revealed the presence of 2-butanone, ethyl
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acetate and toluene (Figure 1). These solvents could result from the synthesis pathway of
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sildenafil citrate, presented in figure 2, where toluene is used in one of the early steps in the
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synthesis, ethyl acetate in the cyclisation reaction and 2-butanone in the formation of the
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citrate salt [34]. All three solvents were below the quantification limits of the method and so
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well below the limits set by the international guidelines.
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For the genuine samples of Cialis® tetrahydrofuran was found as residual solvent. As
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illustrated in figure 3 tetrahydrofuran can be used as a solvent in which the reaction between
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D-tryptophan methyl amide with piperonal is effectuated to provide an intermediate that after
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reaction with chloroacetyl chloride cyclises to tadalafil [35]. Also here the solvent is well
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below the quantification limit of the method and so well below the international acceptance
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limit. Dimethyl sulphide and dimethyl disulphide were also detected. These are not
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considered as residual solvents and are probably some impurities resulting from the synthesis
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of tadalafil or impurities present in one of the excipients used in the tablets.
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44 counterfeit samples of Viagra® and 41 counterfeit samples of Cialis® were screened for the
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presence of the 10 residual solvents listed in table 1. Table 2 gives an overview of the
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quantification limits (LOQ) obtained with the selected method for each of the twelve solvents
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[36]. Full validation of the method used, is described in reference [36]. Tables 3 and 4 give an
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overview of the solvents detected in each sample above the quantification limits of the
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method.
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In the majority of the counterfeit samples the same solvents as observed in the genuines could
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be detected. In most cases these solvents were present in amounts lower than the LOQ.
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Exception to this general trend are the samples 13, 22, 26, 31 and 44 of the Viagra® like
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samples, where ethyl acetate was detected in amounts exceeding the LOQ. Though the
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observed amounts were still far below the international limit of 5000 ppm.
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In the counterfeit samples of Viagra® five other solvents could be detected (Table 3), ethanol,
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2-propanol, acetone, tetra chloromethane (CCl4) and dichloromethane (DCM). Ethanol, 2-
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propanol and acetone, although present in the majority of the samples, never exceeded the
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international limits of 5000 ppm (figure 4a). The same can be said of the samples, where
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DCM was detected. At contrary two samples (sample 23 and 24) contained CCl4 in a dose of
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respectively 7.8 and 47 ppm per tablet, exceeding largely the limit of 4 ppm (figure 4a). These
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samples can therefore be considered as a threat for public health, since CCl4 is a class I
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solvent, which should be avoided in pharmaceutical preparations due to toxicity and an
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important environmental hazard [30]. An explanation for the presence of these solvents in the
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tablets is not easy. Ethanol, 2-propanol and acetone could result from their use as reaction
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solvent for the synthesis of sildenafil or from a substitution of the expensive 2-butanone
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during the forming of the citrate salt of sildenafil [37]. Indeed several solvents can be used for
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these purposes, all with different yields [37]. DCM could probably also be used as reaction
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solvent, but more likely it was used during the coating of the tablets. The presence of CCl4 in
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some of the tablets could not be explained, probably CCl4 was used in an extraction step
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during synthesis of sildenafil. Though it should be kept in mind that most of these products
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are manufactured without taking into account the Good Manufacturing Procedures (GMP).
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Therefore these solvents can also be present due to contamination.
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For the Cialis® like samples seven solvents, not present in the genuines, could be detected in
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amounts exceeding the LOQ of the methods (table 4). The observed solvents were ethanol, 2-
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propanol, acetone, ethyl acetate, CCl4, chloroform and DCM. In general the observed solvents
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were present in amounts beneath the authorized limits (figure 4b). Only for sample 24 the
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limit of 60 ppm for chloroform was slightly exceeded, since it contained 63.9 ppm per tablet.
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Sample 37 contained 17.3 ppm CCl4 per tablet exceeding largely the limit of 4 ppm (figure
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4b). Again in can be said that sample 34 and 37 are a risk for public health, for the first
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sample the limit of a class II solvent is exceeded, a solvent that should be limited due its
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inherent toxicity and for the latter the limit for the class I solvent CCl4 is exceeded [30].
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The presence of DCM and chloroform can easily be explained since they can be used during
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the synthesis of tadalafil, as illustrated by figure 3. The presence of 2-propanol could be
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explained by the synthesis pathway for tadalafil as presented by Xiao et al. [38]. This pathway
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starts from L-tryptophan and propanol is used in a recrystallization step. Acetone and ethanol
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can possibly be used as reaction solvent in alternative synthesis pathways, in similarity with
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the sildenafil synthesis. The presence of ethyl acetate and CCl4 is quite difficult to explain. It
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could be that they were used in alternative synthetic pathways, that the solvents are resulting
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from secondary components, like the excipients used to manufacture the tablets or that they
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are just contaminations.
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In general it can be stated that counterfeit samples contain more residual solvents than the
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genuine products and also in higher amounts. The most frequently found solvents are
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respectively acetone, 2-propanol and ethanol. For the Viagra® like samples acetone was found
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in all samples in concentrations ranging from 0.7 to 336.5 ppm. This can be explained by the
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fact that acetone as reaction solvent for the forming of the citrate salt has a yield above 90%
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[37]. 2-Propanol was found in concentrations ranging from 2.0 to 2826.4 ppm and ethanol,
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that was only found in 12 samples was present in ranges from 3.8 to 858.6 ppm. Also with
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these solvent high yields for citrate salt forming can be obtained [37]. Also for the Cialis® like
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samples 2-propanol was found in 90% of the samples in concentrations ranging from 0.2 to
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2157.4 ppm. 22 samples contained acetone concentrations of 0.8 to 162.3 ppm and 13
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samples contained ethanol in concentrations ranging from 3.2 to 480.2 ppm. Table 3 and 4
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contain also the amount active ingredient in each of the samples. The variation of the amounts
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of the three most occurring solvents could not be related to the amounts of active components.
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Probable explanations are the use of different qualities of primary substances by the different
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producers and the differences in production processes of the tablets (different excipients,
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different hygiene norms, different risks for contaminations, bad cleaning protocols,…).
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3.3. Projection Pursuit (PP)
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Projection pursuit (PP) was applied to both normalized data sets in order to differentiate
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different groups within the sets of counterfeit samples. Based on the three dimensional plots it
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was tried to define groups of samples belonging together. Figure 5a and 5b represent the score
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plots (PPF1, PPF2 and PPF3) for respectively the data set of the Viagra® like samples and the
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one of the Cialis® like samples.
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For the Viagra® like samples 8 clusters could be distinguished within the whole of the 44
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counterfeit samples screened. Cluster 1 consists of the samples containing high amounts of 2-
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propanol, high amounts of acetone and the highest amounts of dichloromethane in the data set
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(samples 8, 10 and 29). Cluster 2 contains the samples with high amounts of 2-propanol and
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relatively low amounts of acetone (samples 3, 16 and 34). Cluster 3 are the samples
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containing relatively high amounts of ethanol and containing ethyl acetate (samples 13, 22,
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24, 26 and 44). All of these samples contain relatively low amounts of 2-propanol and
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acetone. Clusters 4 consist of samples 28 and 38, both containing high amounts of 2-propanol
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and low amounts of DCM. Cluster 5 are samples with a low residual solvent content (samples
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4, 11 and 36). Cluster 6 are all samples containing low amounts of 2-propanol and acetone
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and no other residual solvents while cluster 7 contains samples with relatively high amounts
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of 2-propanol, low amounts of acetone and moderate or high amounts of DCM (samples 8,
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17, 20, 27 and 30. Cluster 8 are all samples containing low amounts of residual solvents and
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sample 18 is alone, since it is the sample with the lowest residual solvent content in the set.
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The score plot (figure 5b) for the Cialis® like samples shows eight clusters. Cluster one
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contains samples with high amounts of ethanol and low amounts of the other solvents
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(samples 1, 2, 30, 32 and 37). Cluster 2 consist of two samples with high 2-propanol and
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aceton levels (samples 19 and 20), while the two samples of cluster 3 contain relatively high
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amounts of 2-propanol and acetone as well as DCM (samples 25 and 26). Cluster 4
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reassembles the samples with the highest levels of 2-propanol (8, 12 and 38) and the two
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samples of cluster 5 contain only moderate amounts of ethanol and no other solvents (samples
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13 and 21). Cluster 6 which is the biggest cluster contains all the samples with relatively low
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amounts of 2-propanol, low amounts of acetone and no other solvents. The samples of cluster
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7 (samples 14 and 28) have low amounts of ethanol and 2-propanol and the samples of cluster
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8 are all the ones containing only low amounts of 2-propanol.
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From the analysis with PP it could clearly be observed that some of the samples could be
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grouped. Different hypothesis could be linked to the different clusters observed: e.g. the
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clustered samples have the same origin, are produced with the same quality of primary
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substances, are using active ingredients produced by the same synthetic path or active
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ingredients produced by the same company,…. In order to prove such hypothesis although,
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more information about the samples is necessary.
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A set of 44 counterfeit samples of Viagra® and 41 counterfeit samples of Cialis® were
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screened for the presence of residual solvents. The observed solvents were quantified and the
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results were compared with the ones obtained for the genuine products.
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As a first conclusion it was observed that counterfeit samples contain significant higher
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amounts of residual solvents than the genuine products, indicating that they are not produced
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following the same quality rules. Although it is clear that the counterfeit products contain
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more residual solvents both in diversity as in concentrations, the majority of the samples are
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conform with the international guidelines, formulating maximum doses of residual solvents in
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medicines. Over the two data sets only four samples were not conform. For the Viagra® like
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samples it were samples 23 and 24, both containing CCl4 in concentrations above the limit of
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4 ppm and for the Cialis® like samples, sample 24 contained to much DCM, while sample 37
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contained also more than 4 ppm CCl4.
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These results show that even if the identity and the dosage of the active ingredients is correct
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and therefore the counterfeit product can be considered as a limited risk for public health, the
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presence of other components as residual solvents can imply a serious threat to the patience
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health.
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Another important observation is that practically all samples contained 2-propanol and
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acetone. Both solvents could have different origins like alternative synthetic pathways for the
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active ingredients, originating from the excipients or used during the fabrication of the tablets
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or coating of the tablets.
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Based on the PP analysis it could be concluded that within both data sets different clusters
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could be observed. The clusters could be explained based on their residual solvent content,
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though for conclusions concerning the same origin, the same production processes or the use
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of the same primary substances, more information about the samples is necessary.
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In general it can be concluded that the screening for residual solvent is a good manner to
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distinguish counterfeit and genuine products, the more since almost no sample preparation is
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necessary.
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It can also be important to carry out the analysis for residual solvents in studies concerning
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the risk of counterfeit medicines for public health, since some contain very toxic solvents
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(CCl4).
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The use of residual solvent analysis could also be helpful in forensic research, since it should
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be possible to link the residual solvent content to synthetic pathways, production processes or
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even origin of the samples, enabling tracking of the companies implied in the production and
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distribution of counterfeit medicines.
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Hong, J. Youm, S.B. Han, D.J. Kim, J.H. Park, S.W. Kwon, Structure determination of new
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Isolation and identification of thiohomosildenafiland and thiosildenafil in health supplements,
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identification of a nitrosated prodrug of the PDE-5 inhibitor aildenafil in a dietary
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[18] B.J. Venhuis, G. Zomer, M.J. Vredenbregt, D. de Kaste, The identification of (-)-trans-
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tadalafil and sildenafil in counterfeit Cialis® and the optical purity of tadalafil stereoisomers,
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[19] A. Häberli, P. Girard, M.Y Low, X. Ge, Isolation and structure elucidation of an
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elucidation of a novel analogue of vardenafil included as an adulterant in a dietary supplement
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by liquid chromatography-electrospray ionization mass spectrometry, infrared spectroscopy
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Crommen, J.O. De Beer, Comparison and combination of spectroscopic techniques for the
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detection of counterfeit medicines. J. Pharm. Biomed. Anal. 53 (2010) 445-453
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counterfeit Viagra with Raman spectroscopy. J. Pharm. Biomed. Anal. 46 (2008) 303-309
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[23] S. Trefi, C. Routaboul, S. Hamieh, V. Gilard, M. Malet-Martino, R. Martino, Analysis of
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Raman spectroscopy, J. Pharm. Biomed. Anal. 47 (2008) 103-113
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[24] J.K. Maurin, F. Pluciński, A.P. Mazurek, Z. Fijałek, The usefulness of simple X-ray
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powder diffraction analysis for counterfeit control - the Viagra example, J. Pharm. Biomed.
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Anal. 43 (2007) 1514-1518
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citrate (Viagra) through ion-associate complex formation, J. AOAC Int. 92 (2009) 125-130
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[26] A.L. Rodomonte, M.C. Gaudiano, E. Antoniella, D. Lucente, V. Crusco, M. Bartolomei,
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P. Bertocchi, L. Manna, L. Valvo, N. Muleri, Counterfeit drugs detection by measurement of
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tablets and secondary packaging colour, J. Pharm. Biomed. Anal. 53 (2010) 215-220
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[27] I. Wawer, M. Pisklak, Z. Chilmonczyk, 1H, 13C, 15N NMR analysis of sildenafil base and
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citrate (Viagra) in solution, solid state and pharmaceutical dosage forms, J. Pharm. Biomed.
406
Anal. 38 (2005) 865-870
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[28] International Conference on Harmonisation (ICH) of Technical Requirements for the
408
Registration of Pharmaceuticals for Human Use, Q3C: impurities: Guidelines for Residual
409
Solvents, Step 4 (1997)
410
[29] United States Pharmacopoeia 35 (2010), United States Pharmacopeial Convention, Inc.,
411
Rockville, MD, USA
412
[30] European Pharmacopoeia 7.0 (2010), Council of Europe, Strasbourg, France
413
[31] Japanese Pharmacopoeia, 16th ed., Society of Japanese Pharmacopoeia, Tokyo (2011)
414
[32] D.L. Massart, B.G.M. Vandeginste, L.M.C. Buydens, S. De Jong, P.J. Lewi, J. Smeyers-
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Verbeke, Handbook of Chemometrics and Qualimetrics-Part A. Elsevier Science.
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Amsterdam, 1997.
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[33] C. Croux, C. Ruiz-Gazen (1996). A COMPSTAT : Proceedings in computational
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statistiques. Physica-Verlag. Heidelberg.
419
[34] M.M. Kichhoff, Promoting sustainability through green chemistry, Resour. Conserv.
420
Recycling 44 (2005) 237-243
421
[35] European patent: A process for the preparation of tadalafil, EP2181997 A1, publication
422
date 5 may 2010.
423
[36] E. Deconinck, M. Canfyn, P.-Y. Sacré, S. Baudewyns, P. Courselle, J.O. De Beer, A
424
validated GC-MS method for the determination and quantification of residual solvents in
425
counterfeit tablets and capsules, J. Pharm. Biomed. Anal. 70 (2012) 64-70
426
[37] European patent: Methods for the production of sildenafil bas and citrate salt, EP
427
1633364 A2, publication date 15 mars 2005.
428
[38] S. Xiao, X. Shi, J. Xing, J. Yan, S. Liu, W. Lu, Synthesis of tadalafil (Cialis) drim L-
429
tryptophan. Tetrahedron: Asymmetry 20 (2009) 2090-2096
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430 431
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Page 18 of 33
Figure captions
432
Figure 1: GC chromatogram of genuine Viagra® and Cialis® wit the residual solvents detected
433
Figure 2: synthesis pathway of sildenafil citrate
434
Figure 3 : synthesis pathway of tadalafil
435
Figure 4 : Graphical representation of the amounts residual solvents found in (a) the Viagra®
436
like samples and (b) the Cialis® like samples (The limits of the solvents that are not indicated
437
on the figures are 5000 ppm)
438
Figure 5: Score plot obtained with Projection Pursuit for (a) the Viagra® like samples and (b)
439
the Cialis® like samples
an
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ip t
431
440
M
441 442
te
445
Ac ce p
444
d
443
19
Page 19 of 33
1
Tables
2
Table 1: specific m/z ratios and retention times for the selected solvents. The m/z ratio
3
indicated in italic was used for the quantification.
Retention times5
times (min)
(min)
cr
Group start
45.0
-
2-propanol
41.0, 43.0, 45.0, 49.0
5.50
Acetone
41.0, 43.0, 45.0, 49.0
5.50
5.73
Dichloromethane
49.0
5.50
6.33
Ethylacetate
43.1
7.65
7.81
Chloroform
82.9
8.10
8.20
8.32
8.63
5.81
us
M
56.1, 116.9
5.14
Benzene
78.1
8.75
8.87
Toluene
te
Carbon tetrachloride
d
Ethanol
Specific m/z ratios
an
Solvent
ip t
4
91.1
10.70
10.86
106.0
12.20
12.30
Ac ce p
Ethylbenzene
1
Page 20 of 33
Table 2: Estimated values LOQ (SIM-mode, quantification) for the different solvents
2-propanol
0.001
Acetone
0.001
Ethylacetate
0.421
Chloroform
0.002
Carbon tetrachloride
0.001
Benzene
0.001
Toluene
0.002
Dichloromethane
0.002
Ethylbenzene
0.002
Ac ce p
te
d
6
cr
0.114
us
Ethanol
ip t
LOQ (ppm)
an
Component
M
5
2
Page 21 of 33
i cr us
17.5 10.8 2826.4 16.5 17.9 60.9 221.8 1115.8 4.2 2205.9 7.1 5.2 2.0 7.7 184.9 1112.7 333.7 23.2 563.8 22.9 4.8 116.5 140.6 384.3 3.6 414.1 926.5 1456.0 166.0
M
6.7 4.4 188.5 858.6 25.5 27.4 559.5 -
2-propanol (ppm)
ed
104.9 98.5 100.1 102.0 15.0 48.6 95.0 95.0 28.2 98.0 99.0 102.0 66.0 97.0 46.5 97.0 98.0 97.7 103.0 98.0 88.0 56.0 50.5 102.0 99.0 64.8 96.0 98.8 98.3 98.0
Ac
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Ethanol (ppm)
pt
Dosis (mg/comp)
ce
Sample nr.
an
Table 3: Results for the screening of 44 counterfeit samples of Viagra® Acetone (ppm)
3.3 265.5 6.8 84.8 55.0 30.8 20.7 124.1 44.1 220.5 88.3 2.0 3.4 4.5 20.5 7.7 4.9 16.1 217.0 22.3 0.7 7.4 63.8 5.3 15.3 59.4 18.9 16.3 70.0 19.8
Ethylacetate (ppm)
CCl4 (ppm) 1.8 10.6 8.9 -
Dichloromethane (ppm) 7.8 47.0 -
1.1 17.5 23.9 3.3 6.8 5.9 5.6 4.8 14.0 3.5
3 Page 22 of 33
i cr us 336.5 35.6 34.8 11.3 106.0 92.6 61.7 8.1 6.9 10.3 0.7 215.5 1.6 3.9
an
128.6 12.0 11.7 16.6 1161.8 23.9 5.1 777.0 18.4 91.1 15.6 21.0 3.3 4.1
M
3.8 6.8 139.7 4.2 805.6
ed
48.0 15.0 96.3 70.6 98.1 71.0 96.0 93.0 94.0 102.0 103.0 99.0 122.5 99.5
1.9 4.8
-
1.0 1.6 -
Ac
ce
pt
31 32 33 34 35 36 37 38 39 40 41 42 43 44
4 Page 23 of 33
i cr us
2-propanol (ppm)
17.5 48.4 14.3 0.2 433.8 4.5 722.9 4.4 0.1 68.5 1388.8 1.1 34.0 30.4 8.8 150.5 2157.4 2072.4 200.7 4.5 639.4 357.1 155.0 2.2 97.0 1.3
M
ed
Ethanol (ppm) 319.5 295.5 4.7 177.1 3.7 4.3 179.5 5.5 3.8 3.2 480.2
pt
Ac
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
dosis (mg/compr.) 12.2 12.2 17.9 18.7 18.9 19.8 19.9 19.0 20.0 19.9 19.6 19.4 20.1 21.5 21.5 20.4 19.7 20.4 19.8 9.6 20.3 19.8 20.2 17.4 17.3 20.1 20.0 21.0 17.9 14.2
ce
Sample nr.
an
Table 4: Results for the screening of 41 counterfeit samples of Cialis® Acetone (ppm)
2.3 2.5 30.4 54.0 3.5 103.0 0.8 9.7 162.3 155.2 13.0 17.6 4.9 49.5 20.6 7.3 1.8
Ethylacetate (ppm)
Chlorform (ppm) 4.4 2.3 11.4 6.0
63.9 -
CCl4 (ppm) -
Dichloromethane (ppm) 7.8 28.7 20.0 7.9 22.5 -
5 Page 24 of 33
i cr us 7.6 6.0 5.8 8.6 6.8
an
114.0 2.1 2.5 0.7 91.4 2.7 86.5 1670.8 1.7 0.5 8.8
M
327.4 403.2 -
ed
18.6 13.1 19.3 19.3 18.2 20.6 20.1 19.0 19.4 17.4 23.6
3.6 -
26.6 -
17.3 -
-
Ac
ce
pt
31 32 33 34 35 36 37 38 39 40 41
6 Page 25 of 33
cr us
Ethyl actetate
toluene
ed
M an
counterfeit
benzene
2-butanone
i
*Graphical Abstract
ce pt
genuine
Ac
44 counterfeit samples
Page 26 of 33
us
cr
i
Figure(s)
genuine Viagra®
an
500000
450000
M
400000
genuine Cialis®
ed
350000
50000
dimethyl disulfide
100000
toluene
150000
tetrahydrofuran
Ac
dimethylsulfide
200000
ethylacetate
ce
250000
2-butanone
pt
300000
0 5
6
7
8
9
10
11
12
Page 27 of 33
13
an
us
cr
ip t
Figure(s)
d
M
CDI, EtOAc
Ac c
ep te
Figure 2
Page 28 of 33
Figure 3
Ac c
ep te
d
M
an
us
cr
ip t
Figure(s)
Page 29 of 33
cr
i
Figure(s)
us
50 40 30
an
ppm
3.000
10 00 3
5
7
9
Ethanol (ppm) 2-propanol (ppm)
limit for CCl4 (4 ppm)
11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43
Acetone (ppm) Ethylacetate (ppm)
ed
1
M
20
2.500
CCl4 (ppm)
1.000
Ac
1.500
ce
ppm
pt
2.000
Limit for dichlormethane (600 ppm)
500
00 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
Page 30 of 33
70
2-propanol (ppm) Acetone (ppm) Ethylacetate (ppm)
M
20 2.000 10
Ethanol (ppm)
an
ppm
30
CCl4 (ppm)
limit for chloroform (60 ppm)
50
us
Chlorform (ppm)
60 2.500
40
cr
i
Figure(s)
limit for CCl4 (4 ppm)
00
Chlorform (ppm)
ed
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41
ce
ppm
pt
1.500
Ac
1.000
Limit dichlormethane (600 ppm)
500
00 1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41
Page 31 of 33
cr
i
Figure(s)
8 30
27 20 17
12 43 21 114 3934 40 7 15 41
29
IV
25
28 10
33 6 32 9
1.5
I
5 37
36 4 11
V
23
18
30 17
PPF3
0.5
29
27 20
36 12 9 37 2 28 4 1 14 39 34 345 33 42 11 19 21 63 0 742 1541 23 25 13 31 38
0
42 19
Ac
-0.5
24
16 35
-1
2
III 44
II
-1.5 24
10
8
1
13
16
VI
ce pt
38
VIII
ed
VII
M an
us
18
26
35
22 2
-3 -2
1
0
-1
-1
3 -2
0 1
Page 32 of 33 31 PPF2 PPF1
us
cr
i
Figure(s)
M an
27
4
23
1.5
IV
38
16
18
8
1
II
ed
19 0.5
V
21 13
0
III
2
I
-0.5
ce pt
20
15 31 29 35
6 27 23 18 31 29 35 1341 1 4 1 16 14 28 734 9 4570 5 10 36 33 39
11 41 3 4 16 15 14 2817 739 9 36 33 410 34 50
25
VII 14 28
VIII 17 9 7 36 33 39 34 4510 0
1
30 -1
37
Ac
PPF3
12
VI
26
32
-1.5
22 24
2 1 0 -1 -2 PPF2
-3
-4
-3
-2 PPF1
-1
0
Page 33 of 33
S0731-7085(13)00141-6 http://dx.doi.org/doi:10.1016/j.jpba.2013.03.023 PBA 9017
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
26-2-2013 29-3-2013 30-3-2013
Please cite this article as: E. Deconinck, M. Canfyn, P.-Y. Sacr´e, P. Courselle, J.O. De Beer, Evaluation of the residual solvent content of counterfeit tablets and capsules, Journal of Pharmaceutical and Biomedical Analysis (2013), http://dx.doi.org/10.1016/j.jpba.2013.03.023 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Evaluation of the residual solvent content of counterfeit tablets and
2
capsules
3 E. Deconinck1,*, M. Canfyn1, P.-Y. Sacré1,2, P. Courselle1, J.O. De Beer1
ip t
4 5 1
Division of food, medicines and consumer safety, Section Medicinal Products, Scientific Institute of
cr
6
Public Health (WIV-ISP)), J. Wytmansstraat 14, B-1050 Brussels, Belgium
8
²Analytical Chemistry Laboratory, CIRM, University of Liège, Avenue de l’Hôpital 1, B36, B-
9
4000 Liège, Belgium
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10
d
M
Highlights Counterfeit tablets and capsules were screened for residual solvents. Detected solvents were quantified. Counterfeit tablets and capsules have significantly higher concentrations of residual solvents than genuine ones. In some toxic amount of residual solvents were detected.
te
11 12 13 14 15 16 17
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7
18
Ac ce p
19 20
Abstract:
21
A group of counterfeit samples of Viagra® and Cialis® were screened for their residual solvent
22
content and compared to the content of the genuine products.
23
It was observed that all counterfeit samples had higher residual solvent contents compared to
24
the genuine products. A more diverse range of residual solvents was found as well as higher
25
concentrations. In general these concentrations did not exceed the international imposed
26
maximum limits. Only in a few samples the limits were exceeded.
27
A Projection Pursuit analysis revealed clusters of samples with similar residual solvent
28
content, possibly enabling some future perspectives in forensic research.
29 1
Page 1 of 33
30
Keywords: Counterfeit Medicines, Gas Chromatography, Residual Solvents
31
*corresponding author: [email protected] Tel. +32 2 642 51 36
33
Fax. +32 2 642 53 27
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2
Page 2 of 33
Introduction
36
Counterfeit medicines, imitations and substandard medicines are a growing problem
37
worldwide. The problem is situated both in developing countries as in industrialized regions.
38
In the developing countries the problem concerns the whole medicine supply chain, especially
39
essential medicines like antibiotics, anti-malaria products, etc. This is often due to the lack of
40
effective enforcement agencies and the high prices of genuine medicines in these countries. In
41
the industrialized world the problem concerns essentially life style drugs like the PDE-5
42
inhibitors, anabolic hormones and slimming products, although sometimes counterfeited
43
antibiotics or insulin are intercepted by the customs [1,2]. The growing threat of these
44
products is mainly due to the extension of the internet, where about 50% of the medicines
45
sold through internet sites disclosing their identity is estimated to be counterfeit [3].
46
The World Health Organization (WHO) [4] defines a counterfeit drug as: “one which is
47
deliberately and fraudulently mislabelled with respect to identity and/or source.
48
Counterfeiting can apply to both branded and generic products and counterfeit products may
49
include products with the correct ingredients or with the wrong ingredients, without the active
50
ingredients, with insufficient active ingredient or with fake packaging.”
51
The fight against the counterfeiting of medicines resulted in numerous articles where several
52
analytical techniques have already been used for the detection of counterfeit medicines. These
53
techniques are separated in two main groups: chromatographic and spectroscopic techniques.
54
The chromatographic techniques are used for the separation, identification and quantification
55
of the active substances. They may also contribute to the elucidation of the structure of new
56
analogues. Commonly used chromatographic techniques contain cheap and easy ones such as
57
thin layer chromatography (TLC) [5,6] but also more sophisticated and expensive ones: such
58
as HPLC-UV [7-9], LC-MS [7-20] and LC-DAD-circular dichroïsm [18].
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3
Page 3 of 33
The spectroscopic techniques are often preferred to chromatography for the identification of
60
counterfeit drugs because of the fact that they are fast, need less (or no) sample preparation
61
and some of them are non-destructive. Fourier-transformed Infrared spectroscopy (FT-IR)
62
[16,17,21], NIR [5,21], Raman spectroscopy [21-23], X-ray diffraction (XRD) [24],
63
colorimetry [25,26] and Nuclear Magnetic Resonance (NMR) [23,27] have demonstrated their
64
usefulness to detect counterfeit or adulterated drugs.
65
All the above mentioned references, including the definition of the WHO focus on the
66
presence and sometimes the amounts of active ingredients. This means that the fact that
67
counterfeit/imitation medicines are essentially produced by street laboratories and small
68
companies not respecting the GMP norms is not taking into account. The fact is that next to
69
the presence of wrong active ingredients, the absence of active ingredients or a wrong dosage,
70
the risk for public health can also result from the presence of impurities or residues of
71
synthesis of the excipients/active ingredients used.
72
One of these potential groups of toxic impurities are the residual solvents. In 1997 the
73
International Committee for Harmonisation issued a guideline for residual solvents [29]. The
74
limits proposed in this guideline have been adopted by the United States Pharmacopoeia [30],
75
the European Pharmacopoeia [31] and the Japanese Pharmacopoeia [32]. The guidelines
76
divide the residual solvents into three classes. Class 1 consists of solvents that should be
77
avoided in pharmaceutical preparations due to their high toxicity, class 2 are the solvents that
78
should be limited and class 3 consists of the solvents with a relatively low toxicity. Following
79
the European Pharmacopoeia [31] the class 1 solvents (e.g. benzene, carbon tetrachloride,…)
80
have limits between 2 and 8 ppm. 1,1,1-trichloroethane is also part of the class 1 solvents due
81
to its environmental hazard. The limit here is up to 1500 ppm. For the class 2 solvents (e.g.
82
chloroform, methanol, hexane,…) the limits vary between 50 and 5000 ppm, while the class 3
83
solvents (e.g. acetic acid, ethanol, formic acid,…) are limited to 5000 ppm.
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4
Page 4 of 33
In this paper a set of 85 different counterfeit or imitation tablets and capsules of Viagra® and
85
Cialis® were screened for the presence of residual solvents. When residual solvents were
86
detected, they were quantified in order to show their compliance or non-compliance to the
87
ICH guidelines.
ip t
84
88 2. Methods and materials
90
2.1. Samples
cr
89
All counterfeit and imitation samples were donated by the Federal Agency for Medicines and
92
Health Products (FAMHP) in Belgium. Genuine samples of Viagra® were kindly provided by
93
Pfizer SA/NV (Belgium). Eli Lilly SA/NV (Benelux) kindly provided genuine samples of
94
Cialis®.
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2-propanol (HPLC grade), dichloromethane (HPLC grade), ethylacetate (pesti-S), acetone
98
(HPLC-grade) ethanol absolute and acetonitrile (HPLC grade) were purchased from Biosolve
99
(Valkenswaard, The Netherlands). Chloroform (for gas chromatography), benzene, carbon
100
tetrachloride (CCl4) (for spectroscopy) and ethylbenzene (for gas chromatography) were
101
purchased from Merck (Darmstadt, Germany) and Toluene (pesticide residues) and
102
cyclohexane from VWR prolabo (Fontenay-Sous-Bois, France). These solvents were used as
103
reference standards. Dimethyl Sulfoxide, used as solvent for the samples was purchased from
104
Merck.
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2.3. Instrumental conditions
107
The samples were injected on a GC-MS system using a G188A headspace sampler (Agilent
108
Technologies, Palo Alto, USA). The analyses were performed on an Agilent 6890N gas
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chromatograph coupled to an Agilent 5973N mass selective detector. Full automation was
110
achieved using Agilent MSD ChemStation data acquisition and data handling software. After
111
incubation of the sample (5 ml in a 10 ml headspace vial) at 120°C for 15 min, during which
112
it was shaken, 0.5 ml of the vapour phase was injected into the GC/MS system in a split
113
injection mode (split ration 6.8:1). The temperatures of the headspace loop, the transfer line
114
and the EPC volatiles interface were 135, 145 and 160°C respectively. The solvents were
115
separated on a Phenomenex 624 capillary column (60 m x 0.32 mm; 1.8 µm film thickness)
116
(Phenomenex, Torrance, USA). The oven temperature was programmed from 60°C (held for
117
5 min) to 270°C at 25 °C/min. 270°C is held for 10 min. The total run time is 23.4 minutes.
118
The temperatures of the injection port, the ion source, the quadrupole and the interface were
119
set at 160, 230, 150 and 280°C, respectively. For the identification of the solvents present in
120
the samples the mass spectrometer was operated in full scan mode. For quantification and
121
validation the mass spectrometer was operated in SIM mode (100 ms dwell times).
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2.4. Sample preparations
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2.4.1. Preparation of internal standard solution
126
A stock solution containing 1000 ppm of acetonitrile and cyclohexane in dimethylsulfoxide
127
was prepared separately. 1 ml was diluted to 100,0 ml dimethylsulfoxide and used as internal
128
standard. Cyclohexane was used for the quantification of CCl4, benzene, toluene and
129
ethylbenzene, while acetonitrile was used as internal standard for the remaining solvents. The
130
selection of the internal standards was based on an initial screening of counterfeit samples.
131
Both internal standards were not detected in the samples or only as traces. Therefore the
132
choice of the internal standards was considered legitimate.
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134
2.4.2. Preparation of standards One stock solution was prepared containing 1000 ppm of all the selected solvents, besides
136
acetonitrile and cyclohexane, in dimethyl sulfoxide. Starting from the stock solutions,
137
dilutions were prepared in the same solvent to obtain standards containing 10, 1 and 0.5 ppm
138
of each solvent. 500 µl of the internal standard solution was added to 5 ml of the standard
139
solutions to obtain concentrations of internal standard of about 1 ppm. The solutions were
140
brought in vials and automatically sealed. Dimethyl sulfoxide was used as blank. The choice
141
of the solvent was based on a preliminary screening of about 150 samples in which dimethyl
142
sulfoxide could not be detected. Therefore it was assumed that dimethyl sulfoxide is not
143
present in the samples.
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For the analysis of tablets, the tablets were broken in two before addition of 5 ml of
147
dimethylsulfoxide. Tablets were broken because of the coating of some of the tablets,
148
possibly preventing the recovery of the residual solvents. To this solution 500 µl of the
149
internal standard solution was added. Capsules were opened before addition of the solvent.
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2.5. Projection Pursuit (PP)
152
PP is a unsupervised projection and variable reduction technique that is able to project high
153
dimensional data into a low dimensional space, defined by a few latent variables, called
154
projection pursuit features (PPFs). Contrary to Principal Component Analysis, PPFs are
155
obtained by maximising a projection index describing inhomogeneity of the data. In such way
156
PP can reveal the data structure and groups of similar samples [32]. In this study the
157
algorithm as described by Croux and Ruiz-Gazen was used with the yenyukov index as
158
projection index [33].
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159 160
2.6. Calculations Data processing and Projection Pursuit (PP) were performed using Matlab version 8.0.0 (The
162
Mathworks, Natick, MA, USA). The algorithm PP was part of the ChemoAC toolbox
163
(Freeware, ChemoAC Consortium, Brussels, Belgium, version 4.0).
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3. Results
In first instance both the genuine as the counterfeit samples were screened for the presence of
167
residual solvents using the above mentioned method in full scan mode. This screening
168
revealed the presence of the twelve solvents, summed in section 2.2, in one or more
169
counterfeit samples. Calibration lines were prepared for the twelve solvents and quantification
170
was performed using the mass spectrometer in SIM mode. Table 1 presents the specific m/z
171
ratios (instrumental parameters) monitored for each of the selected solvents as well as their
172
retention times and the group start times (opening window) for the SIM mode. The specific
173
m/z ratios used for quantification are indicated in italic. These specific ions were selected as
174
being the ones with the highest abundance. The calibration lines were measured and the
175
sample concentrations were calculated using ordinary least squares regression.
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3.1. Genuine samples
178
Screening of the genuine samples of Viagra® revealed the presence of 2-butanone, ethyl
179
acetate and toluene (Figure 1). These solvents could result from the synthesis pathway of
180
sildenafil citrate, presented in figure 2, where toluene is used in one of the early steps in the
181
synthesis, ethyl acetate in the cyclisation reaction and 2-butanone in the formation of the
182
citrate salt [34]. All three solvents were below the quantification limits of the method and so
183
well below the limits set by the international guidelines.
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For the genuine samples of Cialis® tetrahydrofuran was found as residual solvent. As
185
illustrated in figure 3 tetrahydrofuran can be used as a solvent in which the reaction between
186
D-tryptophan methyl amide with piperonal is effectuated to provide an intermediate that after
187
reaction with chloroacetyl chloride cyclises to tadalafil [35]. Also here the solvent is well
188
below the quantification limit of the method and so well below the international acceptance
189
limit. Dimethyl sulphide and dimethyl disulphide were also detected. These are not
190
considered as residual solvents and are probably some impurities resulting from the synthesis
191
of tadalafil or impurities present in one of the excipients used in the tablets.
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192 3.2. Counterfeit samples
194
44 counterfeit samples of Viagra® and 41 counterfeit samples of Cialis® were screened for the
195
presence of the 10 residual solvents listed in table 1. Table 2 gives an overview of the
196
quantification limits (LOQ) obtained with the selected method for each of the twelve solvents
197
[36]. Full validation of the method used, is described in reference [36]. Tables 3 and 4 give an
198
overview of the solvents detected in each sample above the quantification limits of the
199
method.
200
In the majority of the counterfeit samples the same solvents as observed in the genuines could
201
be detected. In most cases these solvents were present in amounts lower than the LOQ.
202
Exception to this general trend are the samples 13, 22, 26, 31 and 44 of the Viagra® like
203
samples, where ethyl acetate was detected in amounts exceeding the LOQ. Though the
204
observed amounts were still far below the international limit of 5000 ppm.
205
In the counterfeit samples of Viagra® five other solvents could be detected (Table 3), ethanol,
206
2-propanol, acetone, tetra chloromethane (CCl4) and dichloromethane (DCM). Ethanol, 2-
207
propanol and acetone, although present in the majority of the samples, never exceeded the
208
international limits of 5000 ppm (figure 4a). The same can be said of the samples, where
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DCM was detected. At contrary two samples (sample 23 and 24) contained CCl4 in a dose of
210
respectively 7.8 and 47 ppm per tablet, exceeding largely the limit of 4 ppm (figure 4a). These
211
samples can therefore be considered as a threat for public health, since CCl4 is a class I
212
solvent, which should be avoided in pharmaceutical preparations due to toxicity and an
213
important environmental hazard [30]. An explanation for the presence of these solvents in the
214
tablets is not easy. Ethanol, 2-propanol and acetone could result from their use as reaction
215
solvent for the synthesis of sildenafil or from a substitution of the expensive 2-butanone
216
during the forming of the citrate salt of sildenafil [37]. Indeed several solvents can be used for
217
these purposes, all with different yields [37]. DCM could probably also be used as reaction
218
solvent, but more likely it was used during the coating of the tablets. The presence of CCl4 in
219
some of the tablets could not be explained, probably CCl4 was used in an extraction step
220
during synthesis of sildenafil. Though it should be kept in mind that most of these products
221
are manufactured without taking into account the Good Manufacturing Procedures (GMP).
222
Therefore these solvents can also be present due to contamination.
223
For the Cialis® like samples seven solvents, not present in the genuines, could be detected in
224
amounts exceeding the LOQ of the methods (table 4). The observed solvents were ethanol, 2-
225
propanol, acetone, ethyl acetate, CCl4, chloroform and DCM. In general the observed solvents
226
were present in amounts beneath the authorized limits (figure 4b). Only for sample 24 the
227
limit of 60 ppm for chloroform was slightly exceeded, since it contained 63.9 ppm per tablet.
228
Sample 37 contained 17.3 ppm CCl4 per tablet exceeding largely the limit of 4 ppm (figure
229
4b). Again in can be said that sample 34 and 37 are a risk for public health, for the first
230
sample the limit of a class II solvent is exceeded, a solvent that should be limited due its
231
inherent toxicity and for the latter the limit for the class I solvent CCl4 is exceeded [30].
232
The presence of DCM and chloroform can easily be explained since they can be used during
233
the synthesis of tadalafil, as illustrated by figure 3. The presence of 2-propanol could be
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explained by the synthesis pathway for tadalafil as presented by Xiao et al. [38]. This pathway
235
starts from L-tryptophan and propanol is used in a recrystallization step. Acetone and ethanol
236
can possibly be used as reaction solvent in alternative synthesis pathways, in similarity with
237
the sildenafil synthesis. The presence of ethyl acetate and CCl4 is quite difficult to explain. It
238
could be that they were used in alternative synthetic pathways, that the solvents are resulting
239
from secondary components, like the excipients used to manufacture the tablets or that they
240
are just contaminations.
241
In general it can be stated that counterfeit samples contain more residual solvents than the
242
genuine products and also in higher amounts. The most frequently found solvents are
243
respectively acetone, 2-propanol and ethanol. For the Viagra® like samples acetone was found
244
in all samples in concentrations ranging from 0.7 to 336.5 ppm. This can be explained by the
245
fact that acetone as reaction solvent for the forming of the citrate salt has a yield above 90%
246
[37]. 2-Propanol was found in concentrations ranging from 2.0 to 2826.4 ppm and ethanol,
247
that was only found in 12 samples was present in ranges from 3.8 to 858.6 ppm. Also with
248
these solvent high yields for citrate salt forming can be obtained [37]. Also for the Cialis® like
249
samples 2-propanol was found in 90% of the samples in concentrations ranging from 0.2 to
250
2157.4 ppm. 22 samples contained acetone concentrations of 0.8 to 162.3 ppm and 13
251
samples contained ethanol in concentrations ranging from 3.2 to 480.2 ppm. Table 3 and 4
252
contain also the amount active ingredient in each of the samples. The variation of the amounts
253
of the three most occurring solvents could not be related to the amounts of active components.
254
Probable explanations are the use of different qualities of primary substances by the different
255
producers and the differences in production processes of the tablets (different excipients,
256
different hygiene norms, different risks for contaminations, bad cleaning protocols,…).
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257 258
3.3. Projection Pursuit (PP)
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Projection pursuit (PP) was applied to both normalized data sets in order to differentiate
260
different groups within the sets of counterfeit samples. Based on the three dimensional plots it
261
was tried to define groups of samples belonging together. Figure 5a and 5b represent the score
262
plots (PPF1, PPF2 and PPF3) for respectively the data set of the Viagra® like samples and the
263
one of the Cialis® like samples.
264
For the Viagra® like samples 8 clusters could be distinguished within the whole of the 44
265
counterfeit samples screened. Cluster 1 consists of the samples containing high amounts of 2-
266
propanol, high amounts of acetone and the highest amounts of dichloromethane in the data set
267
(samples 8, 10 and 29). Cluster 2 contains the samples with high amounts of 2-propanol and
268
relatively low amounts of acetone (samples 3, 16 and 34). Cluster 3 are the samples
269
containing relatively high amounts of ethanol and containing ethyl acetate (samples 13, 22,
270
24, 26 and 44). All of these samples contain relatively low amounts of 2-propanol and
271
acetone. Clusters 4 consist of samples 28 and 38, both containing high amounts of 2-propanol
272
and low amounts of DCM. Cluster 5 are samples with a low residual solvent content (samples
273
4, 11 and 36). Cluster 6 are all samples containing low amounts of 2-propanol and acetone
274
and no other residual solvents while cluster 7 contains samples with relatively high amounts
275
of 2-propanol, low amounts of acetone and moderate or high amounts of DCM (samples 8,
276
17, 20, 27 and 30. Cluster 8 are all samples containing low amounts of residual solvents and
277
sample 18 is alone, since it is the sample with the lowest residual solvent content in the set.
278
The score plot (figure 5b) for the Cialis® like samples shows eight clusters. Cluster one
279
contains samples with high amounts of ethanol and low amounts of the other solvents
280
(samples 1, 2, 30, 32 and 37). Cluster 2 consist of two samples with high 2-propanol and
281
aceton levels (samples 19 and 20), while the two samples of cluster 3 contain relatively high
282
amounts of 2-propanol and acetone as well as DCM (samples 25 and 26). Cluster 4
283
reassembles the samples with the highest levels of 2-propanol (8, 12 and 38) and the two
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samples of cluster 5 contain only moderate amounts of ethanol and no other solvents (samples
285
13 and 21). Cluster 6 which is the biggest cluster contains all the samples with relatively low
286
amounts of 2-propanol, low amounts of acetone and no other solvents. The samples of cluster
287
7 (samples 14 and 28) have low amounts of ethanol and 2-propanol and the samples of cluster
288
8 are all the ones containing only low amounts of 2-propanol.
289
From the analysis with PP it could clearly be observed that some of the samples could be
290
grouped. Different hypothesis could be linked to the different clusters observed: e.g. the
291
clustered samples have the same origin, are produced with the same quality of primary
292
substances, are using active ingredients produced by the same synthetic path or active
293
ingredients produced by the same company,…. In order to prove such hypothesis although,
294
more information about the samples is necessary.
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A set of 44 counterfeit samples of Viagra® and 41 counterfeit samples of Cialis® were
298
screened for the presence of residual solvents. The observed solvents were quantified and the
299
results were compared with the ones obtained for the genuine products.
300
As a first conclusion it was observed that counterfeit samples contain significant higher
301
amounts of residual solvents than the genuine products, indicating that they are not produced
302
following the same quality rules. Although it is clear that the counterfeit products contain
303
more residual solvents both in diversity as in concentrations, the majority of the samples are
304
conform with the international guidelines, formulating maximum doses of residual solvents in
305
medicines. Over the two data sets only four samples were not conform. For the Viagra® like
306
samples it were samples 23 and 24, both containing CCl4 in concentrations above the limit of
307
4 ppm and for the Cialis® like samples, sample 24 contained to much DCM, while sample 37
308
contained also more than 4 ppm CCl4.
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These results show that even if the identity and the dosage of the active ingredients is correct
310
and therefore the counterfeit product can be considered as a limited risk for public health, the
311
presence of other components as residual solvents can imply a serious threat to the patience
312
health.
313
Another important observation is that practically all samples contained 2-propanol and
314
acetone. Both solvents could have different origins like alternative synthetic pathways for the
315
active ingredients, originating from the excipients or used during the fabrication of the tablets
316
or coating of the tablets.
317
Based on the PP analysis it could be concluded that within both data sets different clusters
318
could be observed. The clusters could be explained based on their residual solvent content,
319
though for conclusions concerning the same origin, the same production processes or the use
320
of the same primary substances, more information about the samples is necessary.
321
In general it can be concluded that the screening for residual solvent is a good manner to
322
distinguish counterfeit and genuine products, the more since almost no sample preparation is
323
necessary.
324
It can also be important to carry out the analysis for residual solvents in studies concerning
325
the risk of counterfeit medicines for public health, since some contain very toxic solvents
326
(CCl4).
327
The use of residual solvent analysis could also be helpful in forensic research, since it should
328
be possible to link the residual solvent content to synthetic pathways, production processes or
329
even origin of the samples, enabling tracking of the companies implied in the production and
330
distribution of counterfeit medicines.
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Recycling 44 (2005) 237-243
421
[35] European patent: A process for the preparation of tadalafil, EP2181997 A1, publication
422
date 5 may 2010.
423
[36] E. Deconinck, M. Canfyn, P.-Y. Sacré, S. Baudewyns, P. Courselle, J.O. De Beer, A
424
validated GC-MS method for the determination and quantification of residual solvents in
425
counterfeit tablets and capsules, J. Pharm. Biomed. Anal. 70 (2012) 64-70
426
[37] European patent: Methods for the production of sildenafil bas and citrate salt, EP
427
1633364 A2, publication date 15 mars 2005.
428
[38] S. Xiao, X. Shi, J. Xing, J. Yan, S. Liu, W. Lu, Synthesis of tadalafil (Cialis) drim L-
429
tryptophan. Tetrahedron: Asymmetry 20 (2009) 2090-2096
Ac ce p
te
d
M
an
us
cr
ip t
407
430 431
18
Page 18 of 33
Figure captions
432
Figure 1: GC chromatogram of genuine Viagra® and Cialis® wit the residual solvents detected
433
Figure 2: synthesis pathway of sildenafil citrate
434
Figure 3 : synthesis pathway of tadalafil
435
Figure 4 : Graphical representation of the amounts residual solvents found in (a) the Viagra®
436
like samples and (b) the Cialis® like samples (The limits of the solvents that are not indicated
437
on the figures are 5000 ppm)
438
Figure 5: Score plot obtained with Projection Pursuit for (a) the Viagra® like samples and (b)
439
the Cialis® like samples
an
us
cr
ip t
431
440
M
441 442
te
445
Ac ce p
444
d
443
19
Page 19 of 33
1
Tables
2
Table 1: specific m/z ratios and retention times for the selected solvents. The m/z ratio
3
indicated in italic was used for the quantification.
Retention times5
times (min)
(min)
cr
Group start
45.0
-
2-propanol
41.0, 43.0, 45.0, 49.0
5.50
Acetone
41.0, 43.0, 45.0, 49.0
5.50
5.73
Dichloromethane
49.0
5.50
6.33
Ethylacetate
43.1
7.65
7.81
Chloroform
82.9
8.10
8.20
8.32
8.63
5.81
us
M
56.1, 116.9
5.14
Benzene
78.1
8.75
8.87
Toluene
te
Carbon tetrachloride
d
Ethanol
Specific m/z ratios
an
Solvent
ip t
4
91.1
10.70
10.86
106.0
12.20
12.30
Ac ce p
Ethylbenzene
1
Page 20 of 33
Table 2: Estimated values LOQ (SIM-mode, quantification) for the different solvents
2-propanol
0.001
Acetone
0.001
Ethylacetate
0.421
Chloroform
0.002
Carbon tetrachloride
0.001
Benzene
0.001
Toluene
0.002
Dichloromethane
0.002
Ethylbenzene
0.002
Ac ce p
te
d
6
cr
0.114
us
Ethanol
ip t
LOQ (ppm)
an
Component
M
5
2
Page 21 of 33
i cr us
17.5 10.8 2826.4 16.5 17.9 60.9 221.8 1115.8 4.2 2205.9 7.1 5.2 2.0 7.7 184.9 1112.7 333.7 23.2 563.8 22.9 4.8 116.5 140.6 384.3 3.6 414.1 926.5 1456.0 166.0
M
6.7 4.4 188.5 858.6 25.5 27.4 559.5 -
2-propanol (ppm)
ed
104.9 98.5 100.1 102.0 15.0 48.6 95.0 95.0 28.2 98.0 99.0 102.0 66.0 97.0 46.5 97.0 98.0 97.7 103.0 98.0 88.0 56.0 50.5 102.0 99.0 64.8 96.0 98.8 98.3 98.0
Ac
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Ethanol (ppm)
pt
Dosis (mg/comp)
ce
Sample nr.
an
Table 3: Results for the screening of 44 counterfeit samples of Viagra® Acetone (ppm)
3.3 265.5 6.8 84.8 55.0 30.8 20.7 124.1 44.1 220.5 88.3 2.0 3.4 4.5 20.5 7.7 4.9 16.1 217.0 22.3 0.7 7.4 63.8 5.3 15.3 59.4 18.9 16.3 70.0 19.8
Ethylacetate (ppm)
CCl4 (ppm) 1.8 10.6 8.9 -
Dichloromethane (ppm) 7.8 47.0 -
1.1 17.5 23.9 3.3 6.8 5.9 5.6 4.8 14.0 3.5
3 Page 22 of 33
i cr us 336.5 35.6 34.8 11.3 106.0 92.6 61.7 8.1 6.9 10.3 0.7 215.5 1.6 3.9
an
128.6 12.0 11.7 16.6 1161.8 23.9 5.1 777.0 18.4 91.1 15.6 21.0 3.3 4.1
M
3.8 6.8 139.7 4.2 805.6
ed
48.0 15.0 96.3 70.6 98.1 71.0 96.0 93.0 94.0 102.0 103.0 99.0 122.5 99.5
1.9 4.8
-
1.0 1.6 -
Ac
ce
pt
31 32 33 34 35 36 37 38 39 40 41 42 43 44
4 Page 23 of 33
i cr us
2-propanol (ppm)
17.5 48.4 14.3 0.2 433.8 4.5 722.9 4.4 0.1 68.5 1388.8 1.1 34.0 30.4 8.8 150.5 2157.4 2072.4 200.7 4.5 639.4 357.1 155.0 2.2 97.0 1.3
M
ed
Ethanol (ppm) 319.5 295.5 4.7 177.1 3.7 4.3 179.5 5.5 3.8 3.2 480.2
pt
Ac
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
dosis (mg/compr.) 12.2 12.2 17.9 18.7 18.9 19.8 19.9 19.0 20.0 19.9 19.6 19.4 20.1 21.5 21.5 20.4 19.7 20.4 19.8 9.6 20.3 19.8 20.2 17.4 17.3 20.1 20.0 21.0 17.9 14.2
ce
Sample nr.
an
Table 4: Results for the screening of 41 counterfeit samples of Cialis® Acetone (ppm)
2.3 2.5 30.4 54.0 3.5 103.0 0.8 9.7 162.3 155.2 13.0 17.6 4.9 49.5 20.6 7.3 1.8
Ethylacetate (ppm)
Chlorform (ppm) 4.4 2.3 11.4 6.0
63.9 -
CCl4 (ppm) -
Dichloromethane (ppm) 7.8 28.7 20.0 7.9 22.5 -
5 Page 24 of 33
i cr us 7.6 6.0 5.8 8.6 6.8
an
114.0 2.1 2.5 0.7 91.4 2.7 86.5 1670.8 1.7 0.5 8.8
M
327.4 403.2 -
ed
18.6 13.1 19.3 19.3 18.2 20.6 20.1 19.0 19.4 17.4 23.6
3.6 -
26.6 -
17.3 -
-
Ac
ce
pt
31 32 33 34 35 36 37 38 39 40 41
6 Page 25 of 33
cr us
Ethyl actetate
toluene
ed
M an
counterfeit
benzene
2-butanone
i
*Graphical Abstract
ce pt
genuine
Ac
44 counterfeit samples
Page 26 of 33
us
cr
i
Figure(s)
genuine Viagra®
an
500000
450000
M
400000
genuine Cialis®
ed
350000
50000
dimethyl disulfide
100000
toluene
150000
tetrahydrofuran
Ac
dimethylsulfide
200000
ethylacetate
ce
250000
2-butanone
pt
300000
0 5
6
7
8
9
10
11
12
Page 27 of 33
13
an
us
cr
ip t
Figure(s)
d
M
CDI, EtOAc
Ac c
ep te
Figure 2
Page 28 of 33
Figure 3
Ac c
ep te
d
M
an
us
cr
ip t
Figure(s)
Page 29 of 33
cr
i
Figure(s)
us
50 40 30
an
ppm
3.000
10 00 3
5
7
9
Ethanol (ppm) 2-propanol (ppm)
limit for CCl4 (4 ppm)
11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43
Acetone (ppm) Ethylacetate (ppm)
ed
1
M
20
2.500
CCl4 (ppm)
1.000
Ac
1.500
ce
ppm
pt
2.000
Limit for dichlormethane (600 ppm)
500
00 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
Page 30 of 33
70
2-propanol (ppm) Acetone (ppm) Ethylacetate (ppm)
M
20 2.000 10
Ethanol (ppm)
an
ppm
30
CCl4 (ppm)
limit for chloroform (60 ppm)
50
us
Chlorform (ppm)
60 2.500
40
cr
i
Figure(s)
limit for CCl4 (4 ppm)
00
Chlorform (ppm)
ed
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41
ce
ppm
pt
1.500
Ac
1.000
Limit dichlormethane (600 ppm)
500
00 1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41
Page 31 of 33
cr
i
Figure(s)
8 30
27 20 17
12 43 21 114 3934 40 7 15 41
29
IV
25
28 10
33 6 32 9
1.5
I
5 37
36 4 11
V
23
18
30 17
PPF3
0.5
29
27 20
36 12 9 37 2 28 4 1 14 39 34 345 33 42 11 19 21 63 0 742 1541 23 25 13 31 38
0
42 19
Ac
-0.5
24
16 35
-1
2
III 44
II
-1.5 24
10
8
1
13
16
VI
ce pt
38
VIII
ed
VII
M an
us
18
26
35
22 2
-3 -2
1
0
-1
-1
3 -2
0 1
Page 32 of 33 31 PPF2 PPF1
us
cr
i
Figure(s)
M an
27
4
23
1.5
IV
38
16
18
8
1
II
ed
19 0.5
V
21 13
0
III
2
I
-0.5
ce pt
20
15 31 29 35
6 27 23 18 31 29 35 1341 1 4 1 16 14 28 734 9 4570 5 10 36 33 39
11 41 3 4 16 15 14 2817 739 9 36 33 410 34 50
25
VII 14 28
VIII 17 9 7 36 33 39 34 4510 0
1
30 -1
37
Ac
PPF3
12
VI
26
32
-1.5
22 24
2 1 0 -1 -2 PPF2
-3
-4
-3
-2 PPF1
-1
0
Page 33 of 33