Methods for the detection of adulteration of herbs and spices

The EU General Food Law not only aims at ensuring food safety but also to ‘prevent fraudulent or deceptive practices; the adulteration of food; and any other practices which may mislead the consumer’. Especially the partial or complete, deliberate and intentional substitution of valuable ingredients (e.g. saffron) for less valuable ones is of concern. Due to the variety of products on the market, an approach to detect food adulteration that works well for one species may not be easily applicable to another. Here we present a broadly applicable approach for the detection of substitution of biological materials based on digital PCR. By simultaneously measuring and forecasting the number of genome copies in a sample, fraud is detectable as a discrepancy between these two values. Apart from the choice of target gene, the procedure is identical across all species. It is scalable, rapid, and has a high dynamic range. We provide proof of concept by presenting the analysis of 141 samples of saffron (Crocus sativus) from across the Europeanmarket by DNA accounting and the verification of these results by NGS analysis.

Lievens, A., Paracchini, V., Pietretti, D., Garlant, L., Maquet, A. and Ulberth, F., DNA accounting: tallying genomes to detect adulterated saffron, FOODS, ISSN 2304-8158, 10 (11), 2021, p. 2670

DNA technology for food authentication is already well established, and with the advent of Next Generation Sequencing (NGS) and, more specifically, metabarcoding, compositional analysis of food at the molecular level has rapidly gained popularity. This has led to several reports in the media about the presence of foreign, non-declared species in several food commodities. As herbs and spices are attractive targets for fraudulent manipulation, a combination of digital PCR and metabarcoding by NGS was employed to check the purity of 285 oregano samples taken from the European market. By using novel primers and analytical approaches, it was possible to detect and quantify both adulterants and contaminants in these samples. The results highlight the high potential of NGS for compositional analysis, although its quantitative information (read count percentages) is unreliable, and other techniques are therefore needed to complement the sequencing information for assessing authenticity (‘true to the name’) of food ingredients.

Lievens, A., Paracchini, V., Garlant, L., Pietretti, D., Maquet, A. and Ulberth, F., Detection and Quantification of Botanical Impurities in Commercial Oregano (Origanum vulgare) Using Metabarcoding and Digital PCR, FOODS, ISSN 2304-8158, 12 (16), 2023, p. 2998

Consumers and companies associated with food or pharmaceuticals rely on spices and herbs in various forms. Their intricate supply chains, elevated prices, and low-volume production render them vulnerable to fraudulent practices. However, comprehensive methodologies to detect adulterants remain scarce, impeding national control laboratories from enforcing European and national legislation. In this study, we present quantitative real-time PCR (qPCR) methods designed to identify the top five adulterants of each of six commonly consumed spices and herbs: paprika/chili, turmeric, saffron, cumin, oregano and black pepper. The specificity of each method was confirmed by qPCR analysis of a large collection of relevant plant species. Each authentic sample was combined with its respective top five adulterants as identified in the European Union-wide coordinated control plan on herbs and spices in 2021 or in the existing literature. These binary mixtures were used to evaluate the method's performance with respect to sensitivity, linearity and trueness at four levels of adulterants concentration. Detection was also investigated in multi-adulterated samples. These SYBR™ Green-based qPCR methods enable the specific detection of adulterants, and their sensitivity allows for the distinction between inadvertent contamination and deliberate adulteration. Altogether, these methods contribute to safeguard the authenticity of these high-value commodities

Behr, M., Garlant, L., Pietretti, D., Pellegrin, C., Lievens, A., Boix Sanfeliu, A., Maquet, A. and Alvarellos Lopez, L., A robust set of qPCR methods to evaluate adulteration in major spices and herbs, FOOD CONTROL, ISSN 0956-7135, 165, 2024, p. 110623

In recent years, species identification in herbs has attracted considerable attention due to several cases of fraud; hence inexpensive high-throughput authentication methods are highly welcomed. Species authentication is often performed through DNA analysis and several specific regions (barcodes) are considered suitable. Each barcode (Bar) possesses different qualities in terms of universality and discrimination power. A multiplexed format where information can be extracted simultaneously from several barcode regions is seemingly appropriate to ensure the power of both universality and discrimination. In this approach, we amplified DNA from five different barcode regions in a multiplexed PCR format followed by high-resolution melting (HRM). This multiplexed Bar-HRM approach was first applied to plants spanning the plant kingdom and then gradually narrowing down the genetic variability within the Lamiaceae and the Solanaceae families to finally reach closely related cultivars. Universality was demonstrated through distinct melting profiles obtained for species originating from 29 different families spanning the angiosperms, gymnosperm, mosses, and liverwort (Marchantiophyta). Discrimination power was retained for species, sub-species, and a few cultivars through the application of multivariate statistics to the high-resolution melting profiles. This preliminary investigation has shown the potential to discriminate a vast amount of species within the whole plant kingdom. It requires no a priori knowledge of the species' DNA sequence and occurs in a closed system within 2.5 h at a reduced cost per sample compared to other DNA based approaches.

Ballin, N., Omar Onaindia, J., Jawad, H., Fernandez Carazo, R. and Maquet, A., High-resolution melting of multiple barcode amplicons for plant species authentication, FOOD CONTROL, ISSN 0956-7135, 105, 2019, p. 141-150, JRC115129

Products with a Protected Denomination of Origin (PDO) are vulnerable to misdescription of their true geographical origin. In this work a method has been developed that allows the authentication of La Vera paprika powder (Pimentón de la Vera), a PDO product from the central-west Spanish region, Extremadura. The mass fractions of Br, Ca, Cr, Cl, Cu, Fe, K, Mn, Ni, P, Rb, S, Sr and Zn determined by energy dispersive X-ray fluorescence (ED-XRF) are used for classification purposes by multivariate analysis using Soft Independent Modelling of Class Analogy (SIMCA) (PCA-Class) and Partial Least Square-Discriminant Analysis (PLS-DA). Sixty-seven paprika samples purchased in supermarkets around Europe and on-line via the official web-site of Pimentón de La Vera, were used to build up the models for prediction purposes. The PCA-class model of La Vera paprika powder had a sensitivity of 82%, a specificity of 100% and an accuracy of 91%, whereas the PLS-DA model had a sensitivity of 100%, a specificity of 91% and an accuracy of 96%.

Fiamegkos, I., Dumitrascu, C., Papoci, S. and De La Calle Guntinas, M.B., Authentication of PDO paprika powder (Pimenton ´ de la Vera) by multivariate analysis of the elemental fingerprint determined by ED-XRF. Afeasibility study, FOOD CONTROL, ISSN 0956-7135, 120 (107496), 2021, p. 1-10

Oregano is frequently adulterated as demonstrated in a recent control plan organised by the European Commission. In this work, the elemental profiles of 282 oregano samples analysed by energy dispersive X-ray fluorescence were used in combination with multivariate analyses to detect adulteration, in particular with olive leaves. The analyses were carried out in the frame of the coordinated control plan on the authenticity of herbs and spices organised by the European Commission. Partial Least Square Discriminant Analyses, allowed the detection of adulterated samples with a sensitivity of 81 %, and a specificity of 92 %; among the adulterated samples PLS-DA allowed the detection of samples that contained olive leaves with a sensitivity of 94 % and a specificity of 92 %. Copper mass fraction is of relevance because it is significantly higher in samples adulterated with olive leaves. The ratio Cu/Zn allowed the identification of adulterated samples with a sensitivity and specificity of 85 % without the need to use modelling techniques. The elemental profile of oregano obtained by EDXRF was also used to authenticate the geographical origin declared in the labels.

PAPOCI, S. and DE LA CALLE GUNTINAS, M.B., Use of energy dispersive X-ray fluorescence as screening method to detect oregano adulteration, CURRENT RESEARCH IN FOOD SCIENCE, 10, 2025, p. 101099,

Multi-elemental analyses using ED-XRF have proven their efficacy in food authentication, but sample intake can be a drawback in the analysis of expensive matrices such as saffron. In this work, small sample holders and the double pellet method (a thin sample layer on a wax pellet) were combined to determine 19 elements of interest in food analysis, using only 1 gram of sample. The trueness of these two approaches was evaluated with 17 different organic matrix reference materials of (lichen, wheat flour, rice flour, bran, brown bread, cabbage, vegetable feed, pine needles, and spinach, tomato and tobacco leaves). Double pellets provided accurate results (recovery 100%) for Mg, Al, Si, P, Cl, S, K, Ca, Cr, Mn and Fe, but the penetration of the radiation into the wax layer of the double pellet hindered quantitative recovery for heavier elements. Contrarily, small sample holders were less accurate for the very light elements (Mg, Al and Si) but the recovery was satisfactory (> 80%) for the remaining ones, including the heavier elements (Cu, Zn, Rb, Sr, Cd and Ba). Ni and Br determinations were affected by a possible matrix dependent interference. The method was validated to analyse 21 Iranian saffron samples collected on the EU market.

Ghidotti, M., Papoci, S. and De La Calle Guntinas, M.B., Validation of an Energy Dispersive X-Ray Fluorescence method for small sample intake in a wide range of matrices. Application to the analysis of saffron, SPECTROCHIMICA ACTA PART B-ATOMIC SPECTROSCOPY, ISSN 0584-8547, 189, 2022, p. 106369

The price of Cinnamomum verum (Ceylon cinnamon) is around twice as high as that of the other cinnamon varieties commonly grouped under the name cassia cinnamon, making of the former spice an attractive target for fraudsters. This work demonstrates that elemental profiles obtained by Energy Dispersive-X Ray Fluorescence in combination with multivariate analyses, can be used as screening method to detect Ceylon cinnamon adulteration. Thirty-six elements were analysed in 52 commercially available cinnamon samples, 29 Ceylon, 8 cassia and 15 for which no indication about variety was provided. Fifty-eight per cent of the samples was either adulterated or did not met international quality criteria. Four of the ground cinnamon samples labelled as Ceylon cinnamon were found to be pure cassia or a mixture with a high cassia content, and 26 samples were suspect of other types of adulteration including replacement of bark with other parts of the cinnamon tree. Head Space-Gas Chromatography-Mass Spectrometry, and ash determination by thermogravimetric analysis confirmed the conclusions reached by elemental analysis. Only one sample labelled as Ceylon cinnamon and that according to its volatile composition was cassia cinnamon, was not flagged as suspicious by elemental analysis.

Ghidotti, M., Papoci, S., Pietretti, D., Ždiniaková, T. and De La Calle Guntinas, M.B., Use of elemental profiles determined by Energy Dispersive X-ray Fluorescence and multivariate analyses to detect adulteration in Ceylon cinnamon., ANALYTICAL AND BIOANALYTICAL CHEMISTRY, ISSN 1618-2642, 415 (22), 2023, p. 5437-5449