Abstract
Introduction
Medicine counterfeiting is a phenomenon that now concerns all parts of the world and all types of pharmaceutical products (1, 2). While this counterfeiting is used to touch mostly lifestyle products, nowadays life-saving medicines are also widely targeted. This can be explained by their generally high price and, therefore, the important profit margin obtained by the counterfeiters. The quality of the medicine counterfeits (referred to here as “counterfeits” or “counterfeit”) is extremely variable; therefore, they represent a danger to the patients. Counterfeits can indeed contain toxic compounds or impurities, the wrong active ingredient, the right ingredients but in different quantities – potentially triggering drug resistance – or no active ingredient at all, which, for some diseases, can lead to death. Even when the chemical composition is correct and “only” the packaging has been counterfeited or manipulated, the danger is still there since no one can guarantee that the correct storage conditions (e.g., in case a cold chain is needed) have been respected (3, 4). Studies have demonstrated that organized crime is behind this lucrative traffic. Dismantling counterfeiting activities is a real challenge, since it has become a structured trade with manufacturers, distributors, wholesalers, and local sellers (5-8). Therefore, a fast answer is required in order to increase the chances to pursue the criminals. Providing adequate analytical tools can help to efficiently fight this phenomenon. The chemical and packaging analysis of suspected counterfeits requires a good strategy and efficient technologies in order to speed up the investigations. Many articles have been published concerning the chemical analysis of bulk medicines, mainly using spectroscopic (9-12) and chromatographic (13, 14) methods, in the lab or with handheld devices (15-18). A few papers are available that deal with the packaging analysis of counterfeit medicines (19, 20). In this article, an analytical strategy based on a mobile lab is presented, which enables the analysis of both the bulk medicine and its packaging as a first screening tool. The counterfeits can then be confirmed and their chemical composition studied in a lab in a second phase, in order to determine the danger encountered by the patients and to reveal potential links with former counterfeits based on the analytical data.
Material and methods
Analytical strategy
The strategy presented here for the analysis of counterfeits is innovative, and is based on the concept of a mobile lab, which is used for the first screening of counterfeits. This is followed by a forensic lab analysis.
The mobile lab can be deployed on the field; for example, at Customs, or at affiliates that are not equipped by a lab. The instruments can be used by nonspecialists, but trained operators. The aim of this set-up is to enable a first rapid screening of the samples, without any physical references, since the instruments possess integrated databases. Several handheld instruments are available on the market, and can be used either for the analysis of the packaging of the products, or for the analysis of the bulk medicine. Based on spectroscopic methods, the technology proposed is nondestructive; nevertheless, boxes, blisters, and vials should be opened to perform the analyses.
In the forensic-dedicated lab, the counterfeit cases are officially confirmed by specialists, and their exact chemical composition is determined in order to evaluate the danger to the patients. The possible links can be revealed between counterfeits for forensic intelligence purposes. The packaging analysis is completed by a comparison with a retained sample of the same batch in order to compare the samples one by one.
The packaging analysis and the chemical analysis of the bulk product are led in parallel in both the mobile and forensic labs. The whole concept is presented in Figure 1, which shows several analytical methods that are proposed as an example to illustrate the strategy. This concept can be applied to various types of medicines: solid products, such as capsules, tablets and powders; or liquids containing either small molecules or bigger molecules, such as proteins.
Innovative strategy proposed for the analysis of suspected counterfeits of medicines. CZE = capillary zone electrophoresis; GC = gas chromatography; HPLC = high performance liquid chromatography; IR = infrared spectroscopy; MS = mass spectrometry; NIR = near-infrared spectroscopy; NMR = nuclear magnetic resonance; SEM EDX = scanning electron microscopy energy dispersive x-ray spectroscopy; UV = ultraviolet spectrophotometry; XRF = x-ray fluorescence spectroscopy.
Instruments of the mobile lab
Packaging investigation
Microscopes can be used, for instance, to observe potential defects on the packaging. An instrument example is the Dino-Lite Premier handheld digital microscope, using the DinoCapture 2.0 software. Pictures can be recorded with a zoom going until x200 and a resolution of 5 megapixels.
For instance, x-ray fluorescence spectroscopy (XRF) enables the composition of the glass and the aluminum caps of the primary packaging (e.g., vials) to be checked. The examples presented in this paper were performed with the XRF handheld device from Thermo Scientific, the Niton® XL3t 950 GOLDD+. The packaging items are placed in a large sampling accessory that protects the operators from the x-rays and, thanks to a camera, allows the correct positioning of the samples. The device possesses an Ag anode (6-50 kV, 0-200 μA max) and a geometrically optimized large area drift detector (GOLDD). The whole range of elements from Mg element (magnesium) to U element is looked for during each analysis.
An infrared (IR) spectrometer with an attenuated total reflection (ATR) module is an interesting tool for the analysis of the boxes and the instruction leaflets. Examples will be presented of measurements taken with the TruDefender FTX (Thermo Scientific), a handheld Fourier Transform IR spectrometer equipped with a diamond ATR, providing a resolution of 4 cm-1 and a range of 4000 cm-1 to 650 cm-1.
Chemical analysis of the bulk product
This type of handheld IR spectrometer using an ATR module also can be used for the analysis of the bulk product, either solid (e.g., capsules, tablets, or powders) or liquid. For the liquids, a drop of the product can be placed on the ATR window and the dried residue then measured.
A handheld Raman spectrometer is particularly useful for the analysis of tablets, capsules, and powders. The TruScan RM Analyzer from Thermo Scientific®, a direct dispersive Raman using a 785 nm laser excitation wavelength, was used for the presented example. Its spectral resolution varies between 8 and 10.5 cm-1 on average, and its Raman shift range is between 250 and 2,875 cm-1.
Complementary to the Raman technology, near-infrared (NIR) spectroscopy can be used for the analysis of solid samples. Two devices can be quoted for this purpose. The SCiO device from ConsumerPhysics Inc® is a low-cost sensor using a short wavelength NIR range (700–1100 nm) and Bluetooth wireless technology to communicate with a smartphone and the “SCiO Lab” application. The spectrometer possesses an embedded light source and a silicon detector. The second NIR handheld device, the microPHAZIR RX Analyzer from Thermo Scientific, works with a normal laptop and is made of a tungsten lamp source and a cooled InGaAs detector. The reflectance signal covers a spectral range of 1600–2400 nm.
Lab instruments
Part of the technology used in a forensic lab is similar to that used for a mobile lab. However, the instruments are benchtop and are not handheld. Further methods can be used for the confirmation of the counterfeits and the determination of the chemical composition of the confirmed cases.
For example, capillary zone electrophoresis can enable the identification of the protein that was previously detected by IR spectroscopy. The protein content of a biologic sample can be provided by ultraviolet (UV) spectrophotometry. The use of high performance liquid chromatography (HPLC) devices is another standard method for the identification and quantification of both solid and liquid samples, which is especially valuable for low-concentration products.
If a counterfeit has been confirmed, further techniques are needed to determine the unknown compounds present in the product in order to evaluate the toxicity of the sample. Many technologies are available for that purpose; for example, scanning electron microscopy with energy dispersive x-ray spectroscopy (SEM EDX), mass spectrometry (MS), and nuclear magnetic resonance (NMR) technologies. The results presented for the SEM EDX were acquired with a SEM Quanta FEG 250 instrument from FEI using 60 Pa and 15 kV.
Results
Mobile lab
The evaluation of the suspected counterfeit usually starts with a global observation of its primary packaging (i.e., vials, syringes, or blisters), and its secondary packaging (i.e., boxes and instruction leaflets). The microscope allows inspection if the defects are visible; if the boxes have been manipulated (e.g., by adding glue or erasing variable data); if the covert features are different than they should be; or if the font and colors are incorrect. “Bad quality” counterfeits are already detectable using a simple digital microscope.
XRF spectroscopy can help to investigate the packaging in more depth; for example, regarding the chemical composition of the glass of the primary packaging (i.e., vial or syringe). The composition of the glass can vary a lot, and XRF allows the detection of many of its elements, such as Si, AI, and Ca (21, 22). The advantage of this method is that it is able to independently detect if the vial (or syringe) itself is fake, separate from the chemical composition of the bulk product inside. The information on both the outside and the inside of the product can then be gathered to provide a consolidated analytical result.
The vial caps, which are mainly made of aluminum, possess elements that can also greatly vary between the genuine products and the counterfeits, as observed in Table I. In this table, the elemental composition obtained by XRF for five counterfeits, and the corresponding references, has been presented. The counterfeits were identified based on the differences of content in Pb, Cu, Fe, Mn, and Si elements.
XRF results obtained for the quantitative analyis of caps of five counterfeits and the references (averaged values) in ppm
LOD = limit of detection.
The possible components that are present in paper, and especially in inks, are numerous and easily analyzed by IR spectroscopy (23, 24). IR spectra of the carton of the boxes, the paper of the leaflets, and the ink present on both types of support packaging, can be acquired by ATR and then compared to the reference spectra previously recorded in the database of the device.
Handheld IR instruments also perform very well for the analysis of solid-state medicines, such as capsules and tablets, and for liquid samples. The ATR module, for example, allows the analysis of dried residues of protein-based medicines. The resulting spectra are compared with a reference spectrum and are representative of the excipient profile and the presence of a protein (Fig. 2) and then can be compared with a reference spectrum. If no protein is present or if the excipients differ, the sample already can be detected as counterfeit. Even genuine samples that have been strongly diluted with water by the counterfeiters are detected by ATR, since the resulting spectra present less signal.
Overlaid IR spectra of a protein-based medicine, a protein and the sugar used as main excipient in the studied product. IR = infrared spectroscopy.
Genuine tablets, capsules, and powders have been measured with a handheld Raman device, creating a huge database of reference spectra (16). Spectra of suspected counterfeits can then be compared with the recorded data, based on a multivariate test of equivalence. Above a certain limit, the spectrum of the suspected counterfeit is considered consistent with the reference spectra, and the sample is then identified as genuine. In contrast, below this limit the sample is detected as counterfeit. In Figure 3, the spectrum of a genuine tablet has been overlaid with the spectrum of a counterfeit. Both spectra share bands in common, which is representative of the active ingredient, but still can be differentiated thanks to the excipients' composition. The counterfeit could be easily detected by the instrument, since the computed value was significantly above the limit.
Overlaid Raman spectra of a genuine tablet and a counterfeit. Common bands, attributed to the active ingredient, can be observed on both spectra. However, the excipients' profile of the counterfeit presents differences, which enables to distinguish it from the reference.
Raman analyses of solid-state samples can be completed by NIR spectroscopy since both techniques are complementary. While Raman is usually adequate to detect changes in the active ingredients, NIR is very sensitive to differences in the excipients' composition and in the physical characteristics of the sample. NIR methods require the use of chemometric tools in order to effectively compare the spectrum of a suspected counterfeit with a database of genuine products. The methods developed on the two handheld NIR for the analysis of capsules and tablets were validated and the tested counterfeits could all be rejected by the chemometric models (25).
All the combined data, provided by the different handheld devices, can then be gathered to provide a first screening of counterfeits, based on both the packaging and the bulk medicine analyses.
Forensic lab
Once a counterfeit has been detected by a handheld device, it can be confirmed by specialists using lab instrumentation. Benchtop devices usually tend to present more sensitivity and robustness. For certain types of products, technology is needed that is not available in a handheld mode. This is the case for the analysis of protein-based medicines, for example. Handheld IR spectrometers can be deployed first in order to detect counterfeits devoid of proteins, those with the wrong excipients, or those made of genuine samples that were strongly diluted with water. However, in order to confirm the identity of the protein present in the sample, another method, such as capillary electrophoresis, HPLC, or liquid chromatography-MS (LC-MS), should be used. UV can then be used in order to provide the content of protein in the sample, and to make sure the protein has not been diluted.
The authenticity of a product can only be assured by the combined analysis of the bulk medicine and its packaging. The packaging items should then be investigated by comparing all the characteristics of the suspected counterfeits and of the retained sample of the same batch.
For confirmed counterfeits of the bulk medicine, further analyses are then required in a lab in order to determine the chemical composition of the counterfeit. This step is important since it enables the determination of the level of toxicity of the counterfeit and, therefore, the level of danger encountered by the patients. Numerous techniques are available that provide the chemical composition of a sample, and the analytical strategy highly depends on the galenic form of the counterfeit and on the compounds detected at each analytical step. In the present example (Fig. 4), SEM EDX was used to identify the salty residues obtained after drying the sample. The elemental composition provided by the instrument enabled the detection of the presence of NaCI salts. LC-MS is the gold standard technique to identify unknown proteins; and MS techniques in general (26), as well as NMR (27), provide valuable qualitative and quantitative information about unknown compounds. Additionally, NMR allows the detection of traces, and therefore impurities. The provided list of methods is not exhaustive, and several technologies have proven efficient for the analysis of the composition of counterfeits.
SEM picture and EDX spectrum of the solid residue obtained for the counterfeit of a protein-based medicine. Only a salt (sodium chloride) has been detected. EDX = energy dispersive x-ray; SEM = scanning electron microscopy.
Relevant data acquired during the chemical analysis of the bulk of the counterfeit and the packaging investigation can be recorded in a database and then compared with the data acquired from former counterfeits. Analytical data reveal relevant links between the counterfeits, based on the trace and the counterfeit itself, and therefore provide additional information from a forensic intelligence perspective. For instance, two counterfeit vials presenting the same - but incorrect - chemical composition, the same vial cap composition, and a similar glass composition, present common characteristics, and, therefore, potentially a common origin. This enables links between the counterfeits to be detected at the bulk medicine and/or packaging levels. Combined with seizure information, such as the country and date of seizure, such information can even lead to the detection and analysis of counterfeit networks (8).
Conclusion
Counterfeit medicines represent danger for patients, not only because of the harmful compounds they might contain, but also because of the deplorable manufacturing and/or storage conditions in which they are handled. This situation requires quick action, for example, at the analytical level, in order to detect the counterfeits as soon as possible and trigger rapid investigation of each case. The presented strategy is based on the concept of (i) a mobile lab, composed of handheld spectrometers and microscopes; and (ii) a forensic-dedicated lab, containing various analytical benchtop instruments. The advantage of this combined set-up is to enable rapid screening of counterfeits in the field in order to hasten the investigation, and to allow more counterfeits to be detected. In dedicated forensic labs, counterfeits can be identified; their exact chemical composition can be detected in order to determine the danger to which the patients are exposed, and to determine the links between counterfeit cases based on chemical and packaging characteristics of the traces. The support of the analytics, an increasing awareness of the problem, and the innovative technologies to protect the integrity of the medicines can all help fight against this criminal phenomenon.