Fake pharmaceuticals: A review of current analytical approaches

Microchemical Journal 149 (2019) 104053

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Review article

Fake pharmaceuticals: A review of current analytical approaches☆ a

b,⁎

Paola Bottoni , Sergio Caroli a b

T

National Institute of Health (ISS), Viale Regina Elena, 00299–00161 Rome, Italy Italian Society of Pharmaceutical Medicine (SIMeF), Viale Abruzzi, 32-20131 Milan, Italy

ABSTRACT

Poor quality pharmaceuticals may have serious consequences for human health because of treatment failure, development of antimicrobial resistance and dramatic adverse drug reactions. This can significantly undermine consumer's confidence in healthcare systems and cause an increase in healthcare costs. The vast array of analytical approaches available nowadays makes an important contribution to distinguishing between genuine and fake medicines. The scientific literature published mostly over the last decade on this subject matter was searched and the support provided by modern analytical techniques in combating falsified drugs was discussed. This survey showed that chromatography–based techniques, often in combination with mass spectrometry, have the lion's share. In turn, also colorimetry, infrared spectroscopy and Raman spectroscopy appear to be rather popular approaches.

1. Introduction It is generally accepted by the scientific community that the definition of fake pharmaceuticals includes medicines with a false representation of their identity (such as name and fraudulent mislabeling with respect to content) and source (e.g., country of origin and marketing authorization holder), substandard pharmaceuticals (legitimate drugs not meeting quality specifications) and degraded medicines (authentic pharmaceuticals deteriorated because of improper storage or distribution). The pharmaceutical crime has indeed many facets, namely: the counterfeit can be a perfect imitation of the original pharmaceutical with the same Active Pharmaceutical Ingredients (APIs) at the right concentration in the same packaging; the medicine packaging is the same as for the original pharmaceutical, but it contains the APIs at concentrations different from those declared; the medicine looks like the original product, but it contains no APIs at all; the medicine contains ingredients different from those declared; the packaging of the product has been falsified. Although no accurate data are available, it is estimated that due to weak regulatory systems globally not less than 10% of pharmaceuticals may be falsified with 50% of falsified medicines coming from Internet purchases [1]. The problem concerning falsification of medicines is particularly acute not only in developing countries, but also in developed ones. It is generally recognized that falsified pharmaceuticals originates primarily from Hong Kong, Singapore, South Korea and Taiwan, i.e., the so-called Asian Dragons or Four Little Dragons [2,3]. When it comes to the harmonization of terminology to be used in

this context, it is apparent that there is some confusion because the concept of counterfeiting (frequently linked to that of intellectual property) is often mixed up with that of quality, safety and efficacy of medicinals, not to mention the fact that the term counterfeit is also used as a synonym of trademark infringement and false or substandard products. As of January 2018, WHO issued a key document to illustrate the basic concepts behind the organized crime of medicine counterfeiting [4]. The trade of Fake Medicines (FMs), meaning by this term both counterfeit, substandard and degraded drugs, is becoming more and more dramatic, especially so for the most popular brands and for drugs for which no prescription is required. The prime targets of criminal activities in industrialized countries are life-style expensive pharmaceuticals (hormones, steroids, anorectics, erectile dysfunction drugs and psychotropic drugs), whereas in developing countries life-saving medicines (antibiotics, antimalarials, antitubercolosis drugs and antiretroviral drugs) are the goal. The complexity of the import-export mechanisms at the global level as well as of the supply chain and storage of medications greatly facilitates the entry into the market of FMs and make the monitoring of drug quality an arduous task. Furthermore, it should be considered that medicines of good quality at the time of batch release may gradually get worse when they are marketed. As a consequence, FMs are never equivalent in terms of quality, safety and efficacy to properly managed original medicines. Even when FMs are of adequate quality and contain the correct amount of APIs, their production can never stick to the principles of Good Manufacturing Practice (GMP) that are mandatory for the

Selected papers from the XVI Hungarian – Italian Symposium on Spectrochemistry: Technological innovation for water science and sustainable aquatic biodiversity, 3-6th October 2018, Budapest. ⁎ Corresponding author. E-mail address: [email protected] (S. Caroli). ☆

https://doi.org/10.1016/j.microc.2019.104053 Received 14 February 2019; Received in revised form 29 June 2019; Accepted 29 June 2019 Available online 30 June 2019 0026-265X/ © 2019 Elsevier B.V. All rights reserved.

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pharmaceutical industry. Last but not least, the presence of residue and metabolites of authentic pharmaceuticals in environmental media, food commodities and workplace is well by itself a big challenge to human health and to the ability of the experimentalist to analyze them. In the case of FMs the problem turns to be even more serious as their actual composition in terms APIs and excipients may often be unknown. From this viewpoint, moreover, it should be stressed that there is in particular a keen need of quick inspection methods for ascertaining whether a drug is an FM. This paper is thus primarily intended to offer a wide selection of published material that can highlight the importance of modern analytical methods in contrasting the pharmaceutical crime. Without claiming to be exhaustive, the most fit-for-purpose analytical tools to face the situation depicted above are reviewed and a substantial number of papers illustrating the advances made in this field by current analytical approaches over the past decade or so are reported and commented. Furthermore, it must be clearly understood that publications dealing with dietary supplements are out of the scope of this survey.

increasingly raising public concern and attracting media attention, but the problem is yet to be significantly mitigated. In their opinion, the use of analytical approaches resorting to, e.g., interference pattern, encryption, spectroscopy and chromatographic techniques can help provide evidence of the authenticity of medicinals. On the other hand, it was stressed that only coordinated actions of national control and regulatory agencies, pharmaceutical industries and the public at large can play a significant role in curtailing criminal activities in this arena. In a review with 53 references R. Martino et al. discussed the advantages and limits of current analytical techniques to distinguish authentic pharmaceuticals from those suspected to be counterfeit [11]. The cheapest and easiest approaches, such as colorimetry and ThinLayer Chromatography (TLC), as well as the most sophisticated ones, namely Nuclear Magnetic Resonance (NMR), Vibrational Spectroscopy (VS) and Mass Spectrometry (MS) – often in combination with chromatography, were critically assessed using as an example artemisinin derivatives, i.e., pharmaceuticals frequently counterfeited. In particular, it should be noted that the coupling of NMR with Liquid Chromatography (LC) allows structural problems in complex mixtures to be solved. On the other hand, this approach is not comparable to other LChyphenated techniques because of its lower sensitivity and the need for expensive deuterated solvents and solvent suppression of the residual protonated solvents, to say nothing of the compatibility of the volume of the NMR flow cell with that necessary for the chromatographic run. Furthermore, a significant drawback is the high costs of purchase and service not easily affordable in LMICs. An interesting survey on the extent of drug counterfeiting in Peru over the period from 2005 to 2008 was carried out by the National Quality Control Center [12]. The percentage of FMs relative to the total number of pharmaceuticals assessed was found to be in the range of 3–9.2% and consisted mainly of medicinals for the nervous system, the musculo-skeletal system and the alimentary tract and metabolism as well as of anti-infectives for systemic use. Forgey could be primarily ascribed to manufacturers different than those indicated as well as lack of APIs. The formulations most typically counterfeited were tablets, injectables and capsules. H. Kaur et al. published a review with 46 references on poor quality antimalarials, a major health problem in Africa, Asia and Latin America [13]. These FMs are known to cause treatment failure, economic loss and drug resistance. Methods available to determine the quality of antimalarials were thoroughly examined. The role of packaging to provide protection against counterfeiting of pharmaceuticals was illustrated by Shah et al. [14]. The pros and cons of approaches such as Radio Frequency Identification (RFID), barcodes, holograms and sealing tapes were highlighted and the possible development of devises resorting to the principles of nanotechnology were predicted. In a review with 50 references by D. Bansal et al. current anticounterfeiting tactics and their adoption in different countries were discussed [15]. The best approach to minimize this problem was said to be technological protection based on, e.g., RFID and 2D barcodes. A review with 45 references on technologies available for the detection of FMs was prepared by S. Kovacs et al. [16]. Over 40 technologies from plain checklists for packaging to sophisticated instrumental techniques were surveyed and scored from 0 to 8 on the basis of their suitability for use in LMICs by taking into account the need for electricity, sample preparation, reagents, portability, training required and throughput, those with higher scores being considered to be the best for the purpose. In particular, twelve technologies were deemed portable and therefore potentially useful in the field. As no single technology can meet all of the requirements necessary to identify FMs, the categorization proposed in this paper can greatly help select the best combination of approaches to adequately solve a given problem. The merits and drawbacks of chromatographic and spectroscopic methods to distinguish genuine products from FMs were outlined in a review with 125 references [17]. It was concluded that use of proper

2. General aspects Examples of FMs can be found even in the past. The cases of fake antimalarials (cinchona bark in the 17th century and of fake quinine in the 19th century) with their dramatic consequences provide striking evidence of a problem that started long ago and constantly grew ever since [5]. The consequences of the trade of FMs on decision makers, consumers, health care providers and manufacturers of pharmaceuticals were examined in a review with 55 references by A. K. Deisingh [6]. The most fit-for purpose analytical techniques (near-infrared spectroscopy, Raman spectroscopy, isotopic characterization, tensiography, chromatography and mass spectrometry) were surveyed along with the efficacy of some anti-counterfeiting measures (holograms, tracers, taggants and electronic tracking). Stability of economy and social security both at the national and international level are seriously threatened by counterfeit goods of all kinds [7]. They constitute not only consumer fraud, but also – and most importantly – a source of revenues for organized crime and terrorism. M. H. Anisfeld reported that West Africa and South America are among the countries most plagued by counterfeit pharmaceuticals [8]. In particular, the dramatically growing market penetration by FMs was thoroughly discussed in a paper by P. E. Chaudhry and S. A. Stumpf [9]. According to these Authors the gravity of this situation should be ascribed to policy makers who failed at the early stage to note the warning signs of this phenomenon and to take timely measures to combat its consequences. It was concluded that education campaigns, authentication technology and internationally harmonized laws deployed by both public authorities and industrial enterprises are mandatory to recover lost ground. The key risks associated with the illegal production and sale of pharmaceuticals along with the legal means of combating the falsification of drugs in the EU were also recently analyzed by A. Hall and G. A. Antonopoulos with particular regard to regulations of ten Member States and their national criminal laws [1]. The causes at the root of the production and sale of FMs were highlighted. A review with 194 references was published by W. L. Hamilton et al. This dealt with public health interventions undertaken to counter the circulation of FMs in Low and Middle Income Countries (LMICs) [2]. Fit-for-purpose medicine regulatory agencies and efficient Pharmacovigilance (PV) systems were considered essential to provide multiple barriers of protection from FMs in low-resource settings. The rapidly growing sale of FMs and its worldwide consequences on public health and pharmaceutical business were discussed in a review with 40 references by B. Kumar and A. Baldi [10]. According to these Authors, this widespread hugely profitable illicit market is nowadays 2

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analytical information for forensic purposes is still open to dispute.

approach already in use for this purpose. Over these last two decades counterfeit artesunate with no or subtherapeutic amounts of the active antimalarial ingredient has been a serious public health problem in South East Asia because of untreated malaria [22]. With the support of the International Criminal Police Organization (INTERPOL) and the Western Pacific World Health Organization (WHO) Regional Office, a total of 391 samples of genuine and counterfeit artesunate from Cambodia, Lao PDR, Myanmar (Burma), Thai/Myanmar border and Vietnam were collected and analyzed by HPLC and/or MS. A fair share of them was strongly suspected to be a hoax as sixteen different fake packaging hologram types could be uncovered. Analytical results actually showed that one half of the samples tested contained no or insufficient artesunate (lower than 12 mg per tablet), whereas genuine tablets should contain 50 mg. A number of banned active ingredients could also be identified, e.g., metamizole, safrole (a carcinogen) and raw stuff for manufacturing ecstasy (methylenedioxymethamphetamine). On the basis of chemical, mineralogical, biological and packaging data solid evidence was gained supportive of the fact that a certain portion of the FMs might have been manufactured in southeast People's Republic of China. The quality of antimalarials available from retail outlets across mainland Tanzania was assessed [23]. Antimalarial formulations for a total of 1080 products were collected including sulfadoxine/pyrimethamine, sulfamethoxypyrazine/pyrimethamine, amodiaquine, quinine and artemisinin derivatives and the dissolution profile of 304 subsamples thereof was elucidated, whereas the amount of the APIs was determined by HPLC. The test results indicated that over 12% of the samples were of low quality. In particular, 24% of the quinine tablets exceed the tolerance limits set for dissolution and quantification determinations. All products were substandard as they contained amounts of the stated APIs lower than prescribed. S. Tomić et al. tested samples of medicines for erectile dysfunction (sildenafil, tadalafil and vardenafil) by HPLC to check the actual sildenafil and tadalafil content [24]. The large majority of the samples were found not to contain the APIs within the acceptable −5 to +5% range of deviation from the declared content. Although most samples could not be said to be fake with absolute certainty, there was a reasonable doubt that these products were counterfeit. Additional laboratory tests were thought to be necessary. Popular anthelminthics were collected from 137 private drug stores in Cambodia and their authenticity was investigated by HPLC at the National Health Product Quality Control Centre [25]. All of them were purchased in open packs or containers and in most cases they had been manufactured abroad and had no registration. More than 4% of the products investigated were identified as FMs. The authenticity of 82 anti-obesity medicines purchased from internet pharmacies and pharmaceutical suppliers accessible from Japan was investigated through a collaborative effort between manufacturers and regulatory authorities [26]. 82 samples were purchased from 36 internet sites. About one half of the sites contacted had no physical address and no package insert was available for 45% of the samples. HPLC was used to assess quality of the products a good deal of which were found to be counterfeit and unapproved anti-obesity medicines. As no satisfactory method is available for the simultaneous determination of the two APIs of the fixed-dose combination artesunate (AS) - amodiaquine (AQ) (a widely used treatment for uncomplicated falciparum malaria), a Reversed Phase (RP) HPLC for their quantitative determination was developed [27]. An end-capped octadecylsilyl silica gel column was employed characterized by a binary gradient with the aqueous phase containing potassium dihydrogen phosphate and acetonitrile. Validation of the method in terms of specificity, linearity, accuracy and precision was done in compliance with the International Conference on Harmonization (ICH) guidelines and the potential interferences of excipients and degradation products on the analytical performance were assessed. An interlaboratory study with the participation of seven African National Quality Control Laboratories and the

3. Capillary electrophoresis (CE) A CE method was developed and validated to analyze four different phytopharmaceutical dosage forms (two different ground herbal blends with their relevant infusions, a capsule and a tincture) used for weight control programs in order to detect the possible presence of adulterants [18]. Under optimized experimental conditions ephedrine, norephedrine, caffeine and furosemide could be quantifierd in a number of samples and were baseline separated in less than 7 min with migration times of 2.65, 2.90, 3.75 and 6.58 min, respectively. The concentration range for linearity was 1–1000 μg ml−1, while the Limits of Detection (LoD) and Limits of Quantification (LoQ) were (in μg ml−1), respectively 0.42 and 1.40 for ephedrine, 0.47 and 1.40 for norephedrine, 0.12 and 0.48 for caffeine and 0.22 and 0.73 for furosemide. Measurement repeatability never exceeded 1.43% and intermediate precision RSD was always better than 3.06%. Results showed that the potential adulterants did not interfere with the common constituents of the samples. 4. Colorimetry A semiquantitative, quick, low-cost and easy-to use colorimetricbased method for the detection of counterfeit artesunate was devised [19]. This system relies on paper microfluidics with a better performance than conventional microfluidics. Artesunate tablets could be assayed within minutes by comparing the yellow color which develops on a paper chip to a color-coded key chart included in the kit. The method can be further improved by using an iPhone camera with a color analyzer to measure the intensity of the color on the paper test as there is a linear relationship between the said color intensity and the concentration of artesunate in the sample in the range of up to 20 mg/ mL. The LoD for artesunate was 0.98 mg/mL. An approach similar to the one mentioned above was reported by Marya Lieberman and her team who proposed the so-called Paper Analytical Devices (PADs) to solve a host of analytical problems in low resource settings [20]. Paper was printed with hydrophobic ink to create millifluidic structures such as solution channels, reagent storage areas and mixing zones. This enabled the user to automate operations that would normally be carried out with glassware in a laboratory setting. The PADs have 12 vertical lanes with embedded chemical reagents. By swiping a pharmaceutical across the lanes and then dipping the PADs in water, the reagents react with the various compounds of the drug and generate characteristic color tones in the vertical lanes, as a rule at or above the swipe. The set of color tones thus generated are distinctive of the genuine pharmaceutical and are considerably different from those of a fake version of the same drug. 5. High-performance liquid chromatography (HPLC) As natural herbal medicines can frequently be adulterated with undeclared synthetic drugs, an HPLC-Electrospray Ionization (ESI)tandem Mass Spectrometry (MS-MS) method was developed that could allow the most common synthetic adulterants in herbal remedies to be detected [21]. This study included eighty different drugs from various pharmacological classes (analgesics, antibiotics, antidiabetic agents, antiepileptics, aphrodisiacs, hormones and anabolic drugs, psychotropic drugs and weight reducing compounds). After a simple methanol extraction, the chromatographic separation was carried out in a gradient of acetonitrile - 10 mM ammonium formate buffer at pH 3.0. The LoD were found to range from 5 pg to 1 ng. The recovery rates of drugs spiked to drug-free herbal medicines were in the interval 63–100%. Sildenafil, tadalafil, testosterone and glibenclamide were some of the undeclared drugs identified in herbal remedies. The proposed technique was said to be a valuable extension of the standard GC–MS screening 3

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French Agency for Medicines was conducted to test the suitability of this approach for official controls. It was concluded that this method had much potential for use by official laboratories for routine control, market survey and the identification of possible substandard pharmaceuticals.

identified, as it was the case for a good deal of the studies reported in other parts of this review. Further examples of this are given below. The MS ionization method known as Direct Analysis in Real Time (DART) allows solid drugs to be rapidly screened at almost constant temperature and under atmospheric pressure with a high throughput [33]. As mass misidentification is minimized, this analytical approach lends itself well for identifying FMs. The analytical potential of MS-based techniques to distinguish between genuine medications and FMs was illustrated by H. M. Shackman et al. [34]. A validated forensic method for confirming alprazolam tablets based on DART-Time-Of-Flight (TOF) MS was developed by W. C. Samms et al. [35]. This approach featured minimal sample preparation, direct analysis in the atmospheric sample gap and rapid identification of the analytes of interest. High resolution and mass accuracy with unique ion fragments and chlorine isotopic ratios allowed optimal selectivity to be achieved. Tableted pharmaceuticals such as glycin, nootropyl, anaprilin, mexidol and biseptol were assayed by DART-MS for the identification of the APIs [36]. The applicability of this analytical approach to fast screening of FMs was outlined. In a paper by M. J. Culzoni et al. the capabilities of high resolution ambient MS techniques to verify the authenticity of pharmaceuticals were discussed [37]. With their high throughput, minimal sample preparation and great selectivity, MS techniques allow investigation of pharmaceuticals with minimal or no sample preparation, thus facilitating the identification of FMs. The screening properties of Solvent Assisted Ionization (SAI)-MS as well as the analytical potential of Liquid Chromatography (LC)–SAI-MS were illustrated [38]. As an example of this versatility, samples of suspect fexofenadine hydrochloride and hydroxychloroquine were assayed and the results compared to those obtained for their legal counterparts.

6. High-performance thin-layer chromatography (HPTLC) HPTLC and HPLC-based methods were set up to carry out fingerprint analysis aimed at ascertaining identity, stability and concentrations of traditional Chinese herbal medicine [28]. In particular, these methods lend themselves to assessing the authentication of various species of ginseng, the stability of ginseng preparations and the batchto-batch consistency of extracts of total glycosides of peony used as a raw material and in finished products. Moreover, the representative HPLC fingerprints of immature fruits of Terminalia chebula and the consistent quality of total flavonoids of commercial extracts of Ginkgo biloba leaves were documented along with the detection of adulterations. Quantitative HPTLC methods with automated densitometric detection were developed to assay pharmaceuticals vs. the nominal label values [29]. The methods are based on already existing Thin Layer Chromatography (TLC) and HPTLC screening methods intended for use in countries with limited resources in support of regulatory action to detect FMs. Quantification of aciclovir, albendazole, amoxicillin and amodiaquin + artesunate combined tablets were carried out. Their precision, accuracy, sample peak purity and identification ability were assessed. A limited number of inexpensive and relatively innocuous reagents were employed. Results were satisfactory for all of the above compounds with the exception of amoxicillin. 7. Infrared spectroscopy (IRS) and near-infrared spectroscopy (NIRS) Fourier-Transform (FT)-IRS imaging with high spatial resolution and Desorption Electrospray Ionization (DESI)-linear ion-trap MS were used to characterize FMs [30]. Both methods were applied to counterfeit artesunate antimalarial tablets. The distribution of all components in the tablets could be obtained with inherent chemical specificity and rapid acquisition of data by FT-IRS in the non-destructive microAttenuated Total Reflection (ATR) mode and their identities were confirmed by the high–sensitivity DESI-MS approach. The two orthogonal surface-characterization methods require no sample preparation and can work in the open air. The Local Straight-Line Screening (LSLS) approach was adopted for the qualitative and quantitative IR analysis of possibly adulterated synthetic drugs in suspected herbal medicines [31]. The method required no sample pretreatment. Its applicability was tested in the case of Sibutramine Hydrochloride (SH), an anti-obesity medicine which is often adulterated in herbal medicines-based diet pills. It was found that accurate discrimination and determination of SH in the said pills could be achieved. Counterfeit and original artesunate antimalarial tablets were determined by NIRS [32]. The presence or absence of spectral signatures related to artesunate were tested by multivariate classification models and found to influence this discriminatory ability to a certain extent. The method was field-portable and required little training after calibration, this being an essential prerequiste for non-time consuming and reliable detection of FMs.

9. Nuclear magnetic resonance (NMR) spectroscopy The merits and disadvantages of NMR to distinguish genuine products from counterfeit ones were set forth and compared to those of other analytical approaches in a review by R. Martino et al. already reported under the chapter General aspects (please see above) [11]. A survey with 17 references authored by H.-J. Xi and L. Jin summarized the current status of the use of NMR for the assessment of FMs [39]. Further development and applications of NMR-based methods in this investigation field were said to be necessary to enhance their capabilities to fight pharmaceutical crime. The use of 1H NMR spectroscopy to analyze formulations containing anabolic steroids and check their authenticity was proposed [40]. Testosterone propionate, methyltestosterone, acetate methenolone, oxandrolone, nandrolone decanoate and stanozolol were selected to test the method in 16 samples of anabolic drugs used in injectable tablet and capsule forms. A 600 MHz spectrometer with deuterated chloroform containing 0.03% tetramethylsilane as the solvent was employed. Dimethyl sulfone served as the internal standard. Four out of the 16 analyzed samples were found not to contain the active principle. The method afforded working concentration ranges of 1.0–16.0 mg/mL, intra-day and inter-day precision RSD values of 1.86–4.22% and 2.17–4.75%, respectively, recovery values of 95.2–104.0% and LoD and LoQ of 0.18–0.61 and 1.30–2.19 mg/mL, respectively. Authentic and counterfeit Viagra were differentiated by 1H spinlattice NMR relaxometry, vibrational spectroscopy and Atomic Force Microscopy (AFM) [41]. The frequency range selected for the relaxation studies covered the range 4 kHz - 40 MHz. Results showed that for fake Viagra the relaxation process is bi-exponential over the whole frequency range, while for the original product the process is always single exponential. As a consequence, it was concluded that falsified Viagra can be identified simply on the basis of a qualitative analysis of the

8. Mass spectrometry (MS) A powerful tool for investigating FMs is MS in combination with a chromatographic technique where the latter allo the separation of the chemical species to be measured and the former their quantification. Such hyphenated approaches are frequently used when FMs are to be 4

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relaxation data. Quantitative analysis of the relaxation data were also carried out to thoroughly assess the relaxation processes of authentic and falsified Viagra. On the other hand, vibrational spectroscopy was found not to differentiate between original and fake products and AFM was prone to generate erroneous conclusions. The well-known scandal of adulterated heparin in 2008 prompted analysts to develop new quantitative methods capable of distinguishing pure porcine heparin from porcine heparin blended with bovine species. A good example of this investigative effort is the paper by Y. B. Monakhova and B. W. K. Diehla who reported the use of 1H NMR spectroscopy in combination with multivariate modelling statistical methods to achieve the said goal [42]. Partial least squares regression and ridge regression were found to be the best statistical approaches for assessing heparin falsification in terms of its animal origin. The LoD and the root mean square error of validation were below 2% and 1%, respectively. This method turned out to be simple, cheap and ideal for screening heparin authenticity and purity. The growing popularity of Chinese herbal medicines calls for a critical analysis of safety issues. 1H NMR, Diffusion Ordered Spectroscopy (DOSY) and DOSY-COSY 1H NMR were used to analyze various herbal medicines marketed as natural slimming products [43]. Both active and inactive ingredients could be determined in these products. Two out of the 20 formulations under test were strictly herbal and four had a composition corresponding to ingredients declared while all of the others were adulterated. Sibutramine alone (4.4–30.5 mg/ capsule), sibutramine (5.0–19.6 mg/capsule or tablet) in combination with phenolphthalein (4.4–66.1 mg/capsule) and synephrine (19.5 mg/ capsule) were detected. In some cases also methylsynephrine, vitaberin, sugars and vitamins could be quantified.

Ghana, India, Kenya, Nigeria, Tanzania and Uganda were tested by semi-quantitative TLC, disintegration tests, Raman spectrometry and NIRS to measure the concentration of the APIs and excipients and assess their quality [48]. Results showed that 13%, 15%, 41% and 47% of the samples failed disintegration tests, TLC, NIRS and Raman spectroscopy, respectively. Although data obtained by NIRS and Raman spectroscopy could be considered equivalent to those of TLC in terms of time, cost and reliability, the need of this last for a climate controlled setting and trained laboratory operators makes it less competitive than the first two in developing countries. The concentration of APIs in medicinals obtained from two selected urban pharmacies in India was ascertained by semi-quantitative TLC and disintegration tests and their compliance with internationally acceptable standards was assessed [49]. Results showed that 5% to 12% of the samples analyzed were substandard thus pointing to a certain spatial and product heterogeneity between the pharmacies under test. It could be concluded that the substandard drug problem in India was driven by groups of manufacturers and pharmacies which succeeded in illegally selling their products thanks to an inadequately regulated control system. Eight different types of medicines including antimalarials, antibiotics and anti-mycobacterials selected from the WHO-approved medicine list were purchased by covert shoppers in eleven pharmacies in Africa, three pharmacies in India and five pharmacies in mid-income countries and subjected to semi-quantitative TLC, disintegration and Raman spectrometry tests for product authentication [50]. 15% of the samples did not pass at least one test and were therefore considered to be substandard. A weak correlation was found between poor quality and signals such as the price lower by 13.6–18.7%, the look of the pharmacy and the non-innovator brand. On the other hand, none of those signals would unequivocally identify FMs. The quick determination and quantification of sibutramine hydrochloride in adulterated herbal slimming products was achieved by a simple TLC-image analysis method [51]. A silica gel 60 F254 TLC plate was used for the chromatographic separation. The mobile phase was toluene-n-hexane-diethylamine (9:1:0.3, v/v/v) and the spot detection was done by the Dragendorff reagent. Good linear relationship of the polynomial regression data was obtained in the concentration range of 1–6 μg/spot for the calibration plots with LoD and LoQ of 190 and 634 ng/spot, respectively. Specificity, precision, accuracy and robustness of the method were all satisfactory. An investigation on the quality of two main firstline anti-tuberculosis medications, i.e., isoniazid and rifampicin, was conducted on 713 treatment packs procured from selected private sector pharmacies in 19 cities in Angola, Brazil, China, Democratic Republic of Congo, Egypt, Ethiopia, Ghana, India, Kenya, Nigeria, Russia, Rwanda, Thailand, Turkey, Uganda, United Republic of Tanzania and Zambia [52]. The amounts of APIs were determined by TLC and disintegration testing. It was ascertained that 9.1% of the samples failed basic quality testing by both methods with a failure rate of 16.6% being found in Africa. This fact is probably at the root of anti-tuberculosis drug resistance in LMIC. Four rapid TLC screening methods contained in a Compendium of methods to be used in LMIC for checking the authenticity of, e.g., acetaminophen, acetylsalicylic acid, ibuprofen, and chlorpheniramine maleate, were transformed into HPTLC-densitometry methods characterized by efficiency, selectivity and resolution better than the said TLC approaches [53]. Detection, identification and quantification were achieved by using Merck HPTLC silica gel 60 F 254 glass plates, automated standards and sample application in combination with automated densitometry, thus overcoming the limits of manual application and visual zone comparison. If required, full validation of the transferred methods can be done according to the ICH guidelines or by means of interlaboratory studies. This approach was further improved by K. Lianza and J. Sherma from the same institution of the team of the preceding investigation (Lafayette College, Easton, PA, USA) and applied to the assessment of

10. Raman spectroscopy Qualitative analysis of fake Levitra tablets was carried out and the associated risk to potential users was assessed [44]. The products under test were sold without outer packages and patient information leaflets. NIRS and Raman spectroscopy revealed that the API of Levitra (vardenafil) was not contained in the tablets whereas trace amounts of sildenafil (the API of Viagra) were. The chemical characterization of authentic and fake artesunate antimalarial tablets was performed by a compact and robust portable Raman spectrometer [45]. This new, rapid, and dependable method showed much potential for the in situ chemical identification of counterfeit tablets. A portable Raman spectroscopy device equipped with a novel identification system for the on-site preliminary screening of the suspected FMs was described that combined a modified Local Straight-Line Screening (LSLS) algorithm with Principal Component Analysis (PCA) to allow FMs to be distinguished from genuine pharmaceuticals [46]. Low False Positive Rate (FPR) and low False Negative Rate (FNR) were established. Seven kinds (40 batches) of hypoglycemic tablets and 12 excipients used in popular pharmaceuticals were assayed by this approach with total sensitivity, specificity and accuracy of 96.8%, 97.5% and 96.3%, respectively. 11. Thin layer chromatography (TLC) Semi-quantitative TLC and dissolution testing were shown to reliably detect APIs in FMs and to allow their concentrations to be compared with internationally acceptable standards [47]. An array of antimalarial medications obtained from private pharmacies in major cities of six African countries were tested by the said methods. It could be thus ascertained that about one third of all samples failed either or both tests. Moreover, one third of the medications were artemisinin monotherapies explicitly discouraged by the WHO due to the fact that malarial parasites develop resistance to the drug. Antimalarial, antibiotic and antimycobacterial medicine sampled in 5

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genuity of mebendazole, diphenhydramine hydrochloride, amodiaquine and artesunate (already contained in the Compendium) as well as of amitriptyline hydrochloride (for which there was no Compendium) [54]. Peak identity and purity checks by spectral comparison were important additions to the previous model. Another step forward in this investigation area was taken by N. Popovic and J. Sherma, also from the Lafayette College who applied the same methodology to diazepam and amodiaquine [55].

[8] M.H. Anisfeld, Counterfeit pharmaceuticals and the International Pharmaceutical Federation (FIP) working group on counterfeit medicines, J. Pharm. Pract. 19 (2006) 178–181. [9] P.E. Chaudhry, S.A. Stumpf, The challenge of curbing counterfeit prescription drug growth: preventing the perfect storm, Business Horiz 56 (2013) 189–197. [10] B. Kumar, A. Baldi, The challenge of counterfeit drugs: a comprehensive review on prevalence, detection and preventive measures, Cur. Drug Saf. 11 (2016) 112–120. [11] R. Martino, M. Malet-Martino, V. Gilard, S. Balayssac, Counterfeit drugs: analytical techniques for their identification, Anal. Bioanal. Chem. 398 (2010) 77–92. [12] L.E.M. Exebio, J. Rodríguez, F. Sayritupac, Counterfeit pharmaceuticals in Peru, Rev. Panam. Salud Pub. 27 (2010) 138–143. [13] H. Kaur, M.D. Green, D.M. Hostetler, F.M. Fernández, P.N. Newton, Antimalarial drug quality: methods to detect suspect drugs, Therapy 7 (2010) 49–57. [14] R.Y. Shah, P.N. Prajapati, Y.K. Agrawal, Anticounterfeit packaging technologies, J. Adv. Pharm. Technol. Res. 1 (2010) 368–373. [15] D. Bansal, S. Malla, K. Gudala, P. Tiwari, Anti-counterfeit technologies: a pharmaceutical industry perspective, Sci. Pharm. 81 (2013) 1–13. [16] S. Kovacs, S.E. Hawes, S.N. Maley, E. Mosites, L. Wong, A. Stergachis, Technologies for detecting falsified and substandard drugs in low and middle-income countries, PLoS One 9 (2014) e90601. [17] K. Dégardin, Y. Roggo, P. Margot, Understanding and fighting the medicine counterfeit market, J. Pharm. Biomed. Anal. 87 (2014) 167–175. [18] V. Cianchino, G. Acosta, C. Ortega, L.D. Martínez, M.R. Gomez, Analysis of potential adulteration in herbal medicines and dietary supplements for the weight control by capillary electrophoresis, Food Chem. 108 (2008) 1075–1081. [19] M.T. Koesdjojo, Y. Wu, A. Boonloed, E.M. Dunfield, V.T. Remcho, Low-cost, highspeed identification of counterfeit antimalarial drugs on paper, Talanta 130 (2014) 122–127. [20] A.A. Weaver, M. Lieberman, Paper test cards for presumptive testing of very low quality antimalarial medications, Am. J. Trop. Med. Hyg. 92 (2015) 17–23. [21] M.J. Bogusz, H. Hassan, E. Al-Enazi, Z. Ibrahim, M. Al-Tufail, Application of LC–ESI–MS–MS for detection of synthetic adulterants in herbal remedies, J. Pharmac. Biomed. Anal. 41 (2006) 554–564. [22] P.N. Newton, F.M. Fernández, A. Plançon, D.C. Mildenhal, M.D. Green, L. Ziyong, E.M. Christophel, S. Phanouvong, S. Howells, E. McIntosh, P. Laurin, N. Blum, C.Y. Hampton, K. Faure, L. Nyadong, C.W.R. Soong, B. Santoso, W. Zhiguang, J. Newton, K. Palmer, A collaborative epidemiological investigation into the criminal fake artesunate trade in South East Asia, PLoS Med. 5 (2008) 0209–0219. [23] H. Kaur, C. Goodman, E. Thompson, A nationwide survey of the quality of antimalarials in retail outlets in Tanzania, PLoS One 3 (2008) e3403. [24] S. Tomić, N. Milčić, M. Sokolić, A. Ilić Martinac, Identification of counterfeit medicines for erectile dysfunction from an illegal supply chain, Arhiv Higij. Rada Toksikol. 61 (2010) 69–75. [25] M.H. Khan, J. Okumura, T. Sovannarith, N. Nivanna, M. Akazawa, K. Kimura, Prevalence of counterfeit anthelminthic medicines: a cross-sectional survey in Cambodia, Tropical Med. Int. Health 15 (2010) 639–644. [26] M.H. Khan, T. Tanimoto, Y. Nakanishi, N. Yoshida, H. Tsuboi, K. Kimura, Public health concerns for anti-obesity medicines imported for personal use through the internet: a cross-sectional study, Brit. Med. J. 2 (2012) e000854. [27] Y. Le Vaillant, C. Brenier, Y. Grange, A. Nicolas, P.A. Bonnet, L.R. Massing-Bias, P. Rakotomanga, B. Koumaré, A. Mahly, M. Absi, M. Ciss, M.H. Loueslati, D. Chauvey, Simultaneous determination of artesunate and amodiaquine in fixeddose combination by a RP-HPLC method with double UV detection: implementation in interlaboratory study involving seven African National Quality Control Laboratories, Chromatographia 75 (2012) 308–314. [28] P. Xie, S. Chen, Y. Liang, X. Wang, R. Tiana, R. Uptond, Chromatographic fingerprint analysis—a rational approach for quality assessment of traditional Chinese herbal medicine, J. Chromatog. A 1112 (2006) 171–180. [29] M. Nguyen, J. Sherma, Development of quantitative HPTLC-densitometry methods for analysis of fake and substandard pharmaceutical products following a model approach for transfer of TLC screening methods: albendazole, amodiaquin + artesunate, aciclovir and amoxicillin, J. Liquid Chromatog. Related Technol. 37 (2014) 2956–2970. [30] C. Ricci, L. Nyadong, F.M. Fernández, P.N. Newton, S.G. Kazarian, Combined Fourier-transform infrared imaging and desorption electrospray-ionization linear ion-trap mass spectrometry for analysis of counterfeit antimalarial tablets patient information leaflets, Anal. Bioanal. Chem. 387 (2007) 551–559. [31] F. Lu, S. Li, J. Le, G. Chen, Y. Cao, Y. Qi, Y. Chai, Y. Wu, A new method for testing synthetic drugs adulterated in herbal medicines based on infrared spectroscopy, Anal. Chim. Acta 589 (2007) 200–207. [32] F.E. Dowell, E.B. Maghirang, F.M. Fernández, P.N. Newton, M.D. Green, Detecting counterfeit antimalarial tablets by near-infrared spectroscopy, J. Pharm. Biomed. Anal. 48 (2008) 1011–1014. [33] N.J. White, P.N. Newton, Characterization of solid counterfeit drug samples by desorption electrospray ionization and direct-analysis-in-real-time coupled to timeof-flight mass spectrometry, Chem. Med. Chem 1 (2006) 702–705. [34] H.M. Shackman, M.B. Peddicord, M.S. Bolgar, S.A. Miller, C. Pathirana, Application of mass spectrometry to support verification and characterization of counterfeit pharmaceuticals, LC GC Special Issues 12 (2014) 8–15. [35] W.C. Samms, Y.J. Jiang, M.D. Dixon, S. Houck, A. Mozayani, Analysis of alprazolam by DART-TOF mass spectrometry in counterfeit and routine drug identification cases, J. Forensic Sci. 56 (2011) 993–998. [36] E.S. Chernetsova, P.O. Bochkov, G.V. Zatonskii, R.A. Abramovich, New approach to detecting counterfeit drugs in tablets by DART mass spectrometry, Pharm. Chem. J. 45 (2011) 306. [37] M.J. Culzoni, P. Dwivedi, M.D. Green, P. Newton, F.M. Fernández, Ambient mass

12. Conclusions Nowadays trade of FMs is overtaking narcotics and prostitution as the world's largest market for criminal traffickers. This bleak situation can cause far-reaching health consequences. It is therefore not surprising that a number of initiatives have been taken in recent years to fight the pharmaceutical crime. In 2006, e.g., B. A. Liang supported the view that to fully address this issue close cooperation between public and private agencies as well as with entities across country lines were essential [56]. In 2011 the plurilateral Anti-Counterfeiting Trade Agreement (ACTA) was signed by Australia, Canada, the EU, Japan, Mexico, Morocco, New Zealand, Singapore, South Korea Switzerland and the USA in order to set internationally shared standards for intellectual property rights and to counter more efficiently the production and marketing of FMs [57]. However, a noticeable drawback of ACTA was its potentially negative impact on global access to genuine pharmaceuticals beyond current international law. At that time a global policy framework based on public-private partnership models was also proposed with centralized surveillance to enable cooperation and coordination in this field [58]. In this respect, a major cornerstone to tackle pharmaceutical crime in the Member States of the European Union is Directive 2011/62/EU [59]. Its effective implementation depends on the actual cooperative efforts of all stakeholders, managers of pharmaceutical companies included. Another important provision of law in this field was adopted in 2012 by the US Food and Drug Administration (FDA) to strengthen the agency's ability to safeguard public health by enhancing the safety of the drug supply chain [60]. From a general viewpoint, the information reported above provides unequivocal evidence that internationally harmonized strategies are necessary to establish adequate protection from FMs along with the political will to support their actual implementation. In turn, achieving this goal definitely requires novel technologies for drug analysis to enable quick, accurate and versatile identification of FMs. Technology can in fact protect consumers and generate reliable estimates of the magnitude of the problem [61]. This is all the more true in the case of LMIC where making analytical techniques more accessible is invaluable to gauge trade in FMs. References [1] A. Hall, G.A. Antonopoulos, Fake Meds Online: The Internet and the Transnational Market in Illicit Pharmaceuticals, 144 Palgrave Macmillan. London, 2016. [2] W.L. Hamilton, C. Doyle, M. Halliwell-Ewen, G. Lambert, Public health interventions to protect against falsified medicines: a systematic review of international, national and local policies, Health Policy Plan. 31 (2016) 1448–1466. [3] G.M.L. Nayyar, J.G. Breman, P.N. Newton, J. Herrington, Poor-quality antimalarial drugs in southeast Asia and sub-Saharan Africa, Lancet Infect. Dis. 12 (2012) 488–496. [4] WHO, Substandard and falsified medical products, January (2018) 31 https:// www.who.int/news-room/fact-sheets/detail/substandard-and-falsified-medicalproducts. [5] F.M. Fernandez, D. Hostetler, K. Powell, H. Kaur, M.D. Green, D.C. Mildenhall, P.N. Newton, Poor quality drugs: grand challenges in high throughput detection, countrywide sampling, and forensics in developing countries, Analyst 136 (2011) 3073–3082. [6] A.K. Deisingh, Pharmaceutical counterfeiting, Analyst 130 (2005) 271–279. [7] M.M. Houck, Counterfeit Goods, Encyclopedia of Forensic Sciences, Facts on File Science Library, Second edition, Infobase Publishing, 2012, pp. 409–411.

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P. Bottoni and S. Caroli

[38] [39] [40] [41]

[42] [43] [44] [45]

[46] [47]

[48] [49]

spectrometry technologies for the detection of falsified drugs, Med. Chem. Commun. 5 (2014) 9–19. L. Hassan, C.N. McEwen, Solvent assisted ionization mass spectrometry for screening and quantification of counterfeit drugs, Spectroscopy Special Issues 13 (2015) 8-3. H.-J. Xi, L. Jin, Application of NMR in pharmaceutical analysis, Chinese J. Pharm. Biotechnol. 20 (2013) 453–457. M.V.D.M. Ribeiro, N. Boralle, H.R. Pezza, L. Pezza, Authenticity assessment of anabolic androgenic steroids in counterfeit drugs by 1H NMR, Anal. Methods 10 (2018) 1140–1150. S. Wilczyńki, M. Petelenz, M. Florek-Wojciechowska, S. Kulesza, S. Brym, B. Błońska-Fajfrowska, D. Kruk, Verification of the authenticity of drugs by means of NMR relaxometry-Viagra® as an example, J. Pharmac. Biomed. Anal. 135 (2017) 199–205. Y.B. Monakhova, B.W.K. Diehla, Combining 1H NMR spectroscopy and multivariate regression techniques to quantitatively determine falsification of porcine heparin with bovine species, J. Pharmac. Biomed. Anal. 115 (2015) 543–551. J. Vaysse, S. Balayssac, V. Gilard, D. Desoubdzanne, M. Malet-Martino, R. Martino, Analysis of adulterated herbal medicines and dietary supplements marketed for weight loss by DOSY 1H-NMR, Food Additiv. Contam. A 27 (2010) 903–916. E. Tajanko, J. Sein Anand, K. Roszkowska-Kranc, B. Kozłowska-Boszko, Z. Chodorowski, R. P. Korolkiewicz, Drug counterfeiting - the risk to the public health, Przegl. Lek., 64 (2007), 357–359. C. Ricci, L. Nyadong, F. Yang, F.M. Fernández, C.D. Brown, P.N. Newton, S.G. Kazarian, Assessment of hand-held Raman instrumentation for in situ screening for potentially counterfeit artesunate antimalarial tablets by FT-Raman spectroscopy and direct ionization mass spectrometry, Anal. Chim. Acta 623 (2008) 178–186. F. Lu, X. Weng, Y. Chai, Y. Yang, Y. Yu, G. Duan, A novel identification system for counterfeit drugs based on portable Raman spectroscopy, Chemometr. Intel. Lab. Sys. 127 (2013) 63–69. P. Phattanawasin, U. Sotanaphun, T. Sukwattanasinit, J. Akkarawaranthorn, S. Kitchaiya, Quantitative determination of sibutramine in adulterated herbal slimming formulations by TLC-image analysis method, Forensic Sci. Int. 219 (2012) 96–100. R. Bate, P. Coticelli, R. Tren, A. Attaran, Antimalarial drug quality in the most severely malarious parts of Africa - a six country study, PLoS One 3 (2008) e2132. R. Bate, R. Tren, K. Hess, L. Mooney, K. Porter, Pilot study comparing technologies to test for substandard drugs in field settings, Afr. J. Pharm. Pharmacol 3 (2009)

165–170. [50] R. Bate, R. Tren, L. Mooney, K. Hess, B. Mitra, B. Debroy, A. Attaran, Pilot study of essential drug quality in two major cities in India, PLoS One 4 (2009) e6003. [51] R. Bate, G.Z. Jin, A. Mathur, Does price reveal poor-quality drugs? Evidence from 17 countries, J. Health Econ. 30 (2011) 1150–1163. [52] R. Bate, P. Jensen, K. Hess, L. Mooney, J. Milligan, Substandard and falsified antituberculosis drugs: a preliminary field analysis, Int. J. Tuberc. Lung Dis. 17 (2013) 308–311. [53] C. O'Sullivan, J. Sherma, A model procedure for the transfer of TLC pharmaceutical product screening methods designed for use in developing countries to quantitative HPTLC-densitometry methods, Acta Chromatogr. 24 (2012) 241–252. [54] K. Lianza, J. Sherma, Application of an expanded model procedure for transfer of TLC screening methods for substandard and fake drugs designed for use in developing countries to quantitative HPTLC-densitometry methods, J. Liq. Chromatogr. Relat. Technol. 36 (2013) 2446–2462. [55] N. Popovic, J. Sherma, Transfer of TLC screening methods designed for use in developing countries to quantitative HPTLC-densitometry methods for diazepam and amodiaquine tablets, Acta Chromatogr. 26 (2014) 615–623. [56] B.A. Liang, Fade to black: importation and counterfeit drugs, Am. J. Law Med. 32 (2006) 279–323. [57] K. Saini, N. Saini, A. Baldi, Impact of anti-counterfeiting trade agreement on pharma sector: a global perspective, J. Curr. Pharm. Res. 6 (2011) 4–10. [58] T.K. Mackey, B.A. Liang, The global counterfeit drug trade: patient safety and public health risks, J. Pharm. Sci. Exp. Pharmacol. 100 (2011) 4571–4579. [59] Directive 2011/62/EU of the European Parliament and of the Council of 8 June 2011 amending Directive 2001/83/EC on the Community code relating to medicinal products for human use, as regards the prevention of the entry into the legal supply chain of falsified medicinal products, O. J. of the European Union L 174 (2011) 74–87 https://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri= OJ:L:2011:174:0074:0087:EN:PDF. [60] Food and Drug Administration Safety and Innovation Act (FDASIA), https://www. fda.gov/RegulatoryInformation/LawsEnforcedbyFDA/ SignificantAmendmentstotheFDCAct/FDASIA/ucm20027187.htm. [61] Committee on Understanding the Global Public Health Implications of Substandard, Falsified, and Counterfeit Medical Products, Board on Global Health, Institute of Medicine, G.J. Buckley, L.O. Gostin (Eds.), Countering the Problem of Falsified and Substandard Drugs, National Academies Press (US), Washington (DC), 2013, https://www.ncbi.nlm.nih.gov/pubmed/24872973.

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