A strategy based on fingerprinting and chemometrics for the detection of regulated plants in plant food supplements from the Belgian market: Two case studies.

The sale and consumption of plant food supplements is increasing, especially in the western world. A lot of these supplements can be bought through internet, where a lot of illegal trade is going on. Every year seized dietary supplements are send to laboratories in order to screen for the presence of chemical adulterants or illegally added active pharmaceutical ingredients, though also herbal adulteration occurs and is given less attention. In this paper a two-step approach is presented based on fingerprints recorded by both infrared spectroscopy as liquid chromatography with UV-detection for the screening of five regulated plants used in respectively dietary supplements for slimming and potency enhancement. Both types of fingerprints are combined with chemometric techniques in order to obtain classification models. A first classification model is calculated based on the infrared data and gives a first idea about the plant suspected to be present. This suspicion is then confirmed based on binary classification models calculated with the chromatographic data obtained for the suspected plant. In general, good classification models were obtained for each of the targeted plants. The approach was applied in a small market study comprising 35 dietary supplements for slimming and 34 for male potency enhancement. In total 21 samples were found to contain one of the five targeted plants.

Combining attenuated total reflectance- infrared spectroscopy and chemometrics for the identification and the dosage estimation of MDMA tablets.

The principal objective of the Belgian Early Warning System on drugs (BEWSD) of the Reitox national focal point is to collect and disseminate information on the appearance of drugs circulating on the local drug market. To this end, one of the actions of BEWSD is to annually collect substances that are circulating on the Belgian summer festivals. Every year a high number of seized samples is collected and submitted to qualitative and quantitative analysis. The goal is twofold: next to the monitoring of the drug market, a surveillance on the quality of drugs is established with main focus on the evolution of MDMA dosages in ecstasy as well as the emergence of new psychoactive substances (NPS). The presented paper focusses on the characterization of ecstasy tablets, seized at summer festivals in the seasons 2016 and 2017. Samples were classically analysed with GC-MS and UV spectroscopy, representing a certain cost and time for analysis. In this paper, an approach is presented based on infrared spectroscopy to first differentiate ecstasy from other party drugs and further quantify MDMA in the tablets. To this end, a comparison was done between NIR and Mid-IR spectroscopy in combination with partial least squares-discriminant analysis (PLS-DA) and -regression (PLS). For the differentiation between MDMA positive and negative tablets, the best results were obtained by NIR and PLS-DA and resulted in a correct classification rate of 96% for an external test set. In addition, for the quantification of MDMA, the best results were also obtained with a PLS model based on NIR spectra. A RMSEP for the external test set of 3.86 was obtained and an R2 value between real and predicted values of 0.88. This means that for the dosage MDMA per tablet, when taking into account the range of masses of the tablets in our sample set, the error varies between 8 mg for low mass tablets and 27.8 mg for high mass tablets. These are acceptable values for a first estimation and give a first indication of the risk the tablet may represent. The presented approach will be of use for on-site analysis. Moreover, when applied in a laboratory environment, it will reduce the workload for the analysis of MDMA tablets and thus liberating resources.

Detection and identification of multiple adulterants in plant food supplements using attenuated total reflectance-Infrared spectroscopy.

Due to the rising popularity of dietary supplements, especially plant food supplements, and alternative herbal medicines, a whole market developed and these products became freely available through internet. Though several searches revealed that at least a part of these products, especially the ones obtained from websites disclosing their physical identity, are aldulterated with pharmaceutical compounds. This causes a threat for public health, since these compounds are not declared and therefore adverse effects will not immediately be related to the product. The more the adulterants can interfere with other medicinal treatments. Since the present active pharmaceutical ingredients are not declared on the package and the products are sold as 100% natural or herbal in nature, it is very difficult for custom personnel to discriminate between products to be confiscated or not. Therefore easy to apply analytical approaches to discriminate between adulterated and non-adulterated products are necessary. This paper presents an approach based on infrared spectroscopy combined with attenuated total reflectance (ATR) and partial least squares- discriminant analysis (PLS-DA) to easily differentiate between adulterated and non- adulterated plant food supplements and to get a first idea of the nature of the adulterant present. The performance of PLS-DA models based on Mid-IR and NIR data were compared as well as models based on the combined data. Further three preprocessing strategies were compared. The best performance was obtained for a PLS-DA model using Mid-IR data with the second derivative as preprocessing method. This model showed a correct classification rate of 98.3% for an external test set. Also eight real samples were screened using the model and for seven of these samples a correct classification was obtained. Generally it could be concluded that the obtained model and the presented approach could be used at customs to discriminate between adulterated and non-adulterated herbal food supplements and even get a first idea of the nature of the adulterant present. The more the presented approach hardly needs sample preparation.

Detection of regulated herbs and plants in plant food supplements and traditional medicines using infrared spectroscopy.

The identification of a specific toxic or regulated plant in herbal preparations or plant food supplements is a real challenge, since they are often powdered, mixed with other herbal or synthetic powders and compressed into tablets or capsules. The classical identification approaches based on micro- and macroscopy are therefore not possible anymore. In this paper infrared spectroscopy, combined with attenuated total reflectance was evaluated for the screening of plant based preparations for nine specific plants (five regulated and four common plants for herbal supplements). IR and NIR spectra were recorded for a series of self-made triturations of the targeted plants. After pretreatment of the spectral data chemometric classification techniques were applied to both data sets (IR and NIR) separately and the combination of both. The results show that the screening of herbal preparations or plant food supplements for specific plants, using infrared spectroscopy, is feasible. The best model was obtained with the Mid-IR data, using SIMCA as modelling technique. During validation of the model, using an external test set, 21 of 25 were correctly classified and six of the nine targeted plants showed no misclassifications for the selected test set. For the other three a success rate of 50% was obtained. Mid-IR combined with SIMCA can therefore be applied as a first step in the screening of unknown samples, before applying more sophisticated fingerprint approaches or identification tests described in several national and international pharmacopoeia. As a proof of concept five real suspicious samples were successfully screened for the targeted regulated plants.

Discriminating nicotine and non-nicotine containing e-liquids using infrared spectroscopy.

In a few countries, including Belgium, nicotine-containing e-cigarettes and e-liquids are considered medicines, and therefore cannot freely be sold, but should be distributed in a pharmacy. The fact that in the neighbouring countries these products are freely available, poses a problem for custom personnel, the more the nicotine content of the products is not always labelled, especially when they are bought through internet. Therefore there is a need for easy-to-use equipment and methods to perform a first on site screening of intercepted samples, both for border control as to check label compliance of the sample. The use of attenuated total reflectance-infrared spectroscopy (ATR-IR) and near infrared spectroscopy (NIR), combined with chemometrics was evaluated for the discrimination between nicotine containing and non-nicotine containing samples. It could be concluded that both ATR-IR and NIR could be used for the discrimination when combined with the appropriate chemometric techniques. The presented techniques do not need sample preparation and result in models with a minimum of false negative samples. If a large enough training set can be established the interpretation can be fully automated, making the presented approach suitable for on-site screening of e-liquid samples.

ATR-FTIR spectroscopy and chemometrics: An interesting tool to discriminate and characterize counterfeit medicines.

Counterfeit medicines pose a huge threat to public health worldwide. High amounts of counterfeit pharmaceuticals enter the European market and therefore detection of these products is essential. Attenuated Total Reflection Fourier-Transform infrared spectroscopy (ATR-FTIR) might be useful for the screening of counterfeit medicines since it is easy to use and little sample preparation is required. Furthermore, this approach might be helpful to customs to obtain a first evaluation of suspected samples. This study proposes a combination of ATR-FTIR and chemometrics to discriminate and classify counterfeit medicines. A sample set, containing 209 samples in total, was analyzed using ATR-FTIR and the obtained spectra were used as fingerprints in the chemometric data-analysis which included Principal Component Analysis (PCA), k-Nearest Neighbours (k-NN), Classification and Regression Trees (CART) and Soft Independent Modelling of Class Analogy (SIMCA). First it was verified whether the mentioned techniques are capable to distinguish samples containing different active pharmaceutical ingredients (APIs). PCA showed a clear tendency of discrimination based on the API present; k-NN, CART and SIMCA were capable to create suitable prediction models based on the presence of different APIs. However k-NN performs the least while SIMCA performs the best. Secondly, it was tested whether these three models could be expanded to discriminate between genuine and counterfeit samples as well. k-NN was not able to make the desired discrimination and therefore it was not useful. CART performed better but also this model was less suited. SIMCA, on the other hand, resulted in a model with a 100% correct discrimination between genuine and counterfeit drugs. This study shows that chemometric analysis of ATR-FTIR fingerprints is a valuable tool to discriminate genuine from counterfeit samples and to classify counterfeit medicines.

Detection of sibutramine in adulterated dietary supplements using attenuated total reflectance-infrared spectroscopy.

Sibutramine is one of the most occurring adulterants encountered in dietary supplements with slimming as indication. These adulterated dietary supplements often contain a herbal matrix. When customs intercept these kind of supplements it is almost impossible to discriminate between the legal products and the adulterated ones, due to misleading packaging. Therefore in most cases these products are confiscated and send to laboratories for analysis. This results inherently in the confiscation of legal, non-adulterated products. Therefore there is a need for easy to use equipment and techniques to perform an initial screening of samples. Attenuated total reflectance-infrared (ATR-IR) spectroscopy was evaluated for the detection of sibutramine in adulterated dietary supplements. Data interpretation was performed using different basic chemometric techniques. It was found that the use of ATR-IR combined with the k-Nearest Neighbours (k-NN) was able to detect all adulterated dietary supplements in an external test set and this with a minimum of false positive results. This means that a small amount of legal products will still be confiscated and analyzed in a laboratory to be found negative, but no adulterated samples will pass the initial ATR-IR screening.

Detection of whitening agents in illegal cosmetics using attenuated total reflectance-infrared spectroscopy.

Cosmetic products containing illegal whitening agents are still found on the European market. They represent a considerable risk to public health, since they are often characterised by severe side effects when used chronically. The detection of such products at customs is not always simple, due to misleading packaging and the existence of products containing only legal components. Therefore there is a need for easy to use equipment and techniques to perform an initial screening of samples. The use of attenuated total reflectance-infrared (ATR-IR) spectroscopy, combined with chemometrics, was evaluated for that purpose. It was found that the combination of ATR-IR with the simple chemometric technique k-nearest neighbours gave good results. A model was obtained in which a minimum of illegal samples was categorised as legal. The correctly classified illegal samples could be attributed to the illegal components present.

Comparative dissolution study on counterfeit medicines of PDE-5 inhibitors.

Counterfeit medicines are a growing problem in both developing and industrialised countries. In general the evaluation of these medicines is limited to the identification and the dosage of the active ingredients. In this study in vitro dissolution tests were conducted on two sets of counterfeit medicines containing PDE-5 inhibitors (sildenafil citrate and tadalafil). The dissolution profiles were statistically compared to the ones of the genuine products using the f2-method and a comparison at each time point using the Cochran test. The results showed low equivalences between counterfeit and genuine products as well as higher variations around the mean dissolution value at the different time points for the counterfeit products.