Natural materials, such as essential oils (EO), hydrosols, concretes, absolutes, resinoids and other specialties, are numerous and of great importance in the flavor and fragrance industry. For many years, the historic use of perfume raw materials has consistently increased because of the strong demand for natural ingredients in many fields (flavor, fragrance, cosmetics, aroma, phytotherapy or nutraceuticals).
The production varies from large scale (e.g., orange oil, >65,000 metric tonnes) with low to moderate prices, to a much smaller scale due to the rarity often associated with high prices (agarwood or iris EO, rose absolute with production levels of 2,000-10,000 kg).
Their use constitutes a strong marketing and technical advantage, but the high cost of these natural materials often leads to many different ways of adulteration. Thus, authentication is an important issue for industrial users, authorities and consumers.
From a regulatory point of view, quality standards have been more and more established (ISO and AFNOR standards, for example). However, authentication is also important for consumer safety and economic reasons (to avoid unfair competition).
Adulteration of essential oils and extracts can be made by several means, such as addition of cheaper synthetic materials, other natural isolates, and/or vegetable oils, etc. The most important known cases of adulteration are reported in T-1. In this article we will not only discuss the conventional methods used to determine adulteration, but also new innovative techniques that may be added to the analyst’s toolbox for authentication.
Classical Quality Control Techniques
Authentication of natural materials constitutes a challenge for analytical chemists. Their chemical compositions are complex to very complex and may vary depending on harvest season and practices, habitat, conservation, isolation techniques and many other factors. Furthermore, compliance with a standard of quality does not necessarily reveal adulteration. In some cases, aging, processing or storage can induce terpenoid rearrangement, polymerization, oxidation, hydrolysis, isotopic fractionation, racemization of asymmetric centers and other chemical modifications that can lead to out-of-specification optical activity value or chemical composition even without adulteration.
Many analytical techniques can be used for authentication purposes: organoleptic, physical, chemical, chromatographic, spectroscopic and thermal techniques. Olfactory analysis is generally performed by trained evaluators comparing with carefully stored standards. In the case of physical measurement (density, refractive index, optical activity), the need for comparison with standard values is crucial. These two types of analyses are simple, cheap and fast for the identification of important falsification, but not for identifying subtle adulterations.
Quality control is more often realized using chromatographic (GC, HPLC, HPTLC) or spectroscopic techniques. Enantiomeric chromatography, NMR and IRMS are more specific methods used for authentication issues. Chiral chromatography is useful when optically active compounds are of interest in the natural extracts to confirm naturality or origins.14C radio counting helps distinguishing vegetable from fossil origin, whereas SIRA (Stable Isotopic Ratio Analysis) by IRMS of13C,2H or18O enables discrimination of synthetic or geographic origin. For more challenging issues SNIF-NMR of2H/1H or13C/12C isotopic ratio measurement offers the in-depth analyses of molecules.
Metabolomics: New Global Analytical Approach
Metabolomics is known and used in many areas, such as medical, food, nutrition, plant study, etc. However, so far, studies on natural ingredients dedicated to fragrances are very limited to EO and solvent extracts like absolutes [3- 6]. Nevertheless, this technique has been found to be efficient for authentication issues on botanical species, geographical origins or adulterations.
But what is precisely? Metabolomics consists in the chemical study of the metabolome of a species, e.g., the small molecules (metabolites) present in a living organism (animal or plant). Thus, an essential oil or extract originating from a part of an organism, is constituted of metabolites.
As shown in F-1 an “omics” discipline can be used to describe each step from the genetic information to the phenotypic data given by the metabolome.
Why is metabolomics relevant to authentication issues? Metabolomics can be used in two different approaches: untargeted or targeted. The first one is a screening of metabolites, and the second one is focused on a defined family of metabolites.
The first one, global approach, also called fingerprinting is discussed in this article: It appears as a new tool available for authentication issues. This technique combines experimental data of volatile and/or non-volatile molecules obtained by GC/MS, UHPLC-HRMS, NMR, IR etc., and multivariate statistical data analyses (Principal Component Analysis -PCA-, Partial Least Square Data Analysis -PLS-DA). Robust models are created with this broad information.
Viola odorata leaf absolute from French and Egyptian geographical origins were compared and differentiated through a metabolomics approach (F-2). A first part of volatile and non-volatile compounds from absolutes were analyzed by UHPLC-ToF/MS and data were treated by PLS-DA. Both sample groups were clearly discriminated. In the same work, a case of adulteration with a non-volatile compound (methyl linoleate) was specifically evident (F-2). Other interesting cases of botanical species, geographical origins and adulterations were discussed in a study on Rosa centifolia and Rosa damascena absolutes. 
Metabolomics Benefits and Drawbacks
Metabolomics is a new technique of authentication used in flavor and fragrance industries. It completes the panel of existing methods with additional information. This technique is of particular interest to study the non-volatile metabolites present in an organic extract when working with UHPLC-HRMS. Usual natural product chemistry can be conducted to isolate and identify some metabolite markers responsible for a discrimination (preparative chromatography, MS and NMR). Nevertheless, it will not always be possible to understand why one specific sample is an outlier of its theoretical sample group. Then metabolomics cannot be considered as a technique of replacement but rather a complementary one.
Model robustness was tested in the work on Rosa sp.  between analyses obtained at time t and at t + 4 months. Models addressing issues with marked differences among groups were shown to be reusable while this was not the case for models with slighter differences. Besides, this is absolutely not a “push-button” technique. Experimented analysts are needed: for rigorous work, high innovative instrument use (especially with UHPLC-ToF/MS for example) and data interpretation.
Barcoding and Metabarcoding for Botanical Identification
Each plant is characterized by its unique genetic signature. However, two close species, having only recently evolved separately from each other, have nearly identical genetic signatures. Those DNA mutations, accumulated in the course of geologic times, reflect the natural evolution of plant and animal species and allow to trace back the evolutionary history. Conversely, knowing the proper genetic signature to each species, it is therefore possible to locate whichever organism in the tree of life and thus to determine its identity. The DNA barcoding is based on this principle. This genetic tool is able to identify an organism at a species level. No matter the form (entire or ground plant, fresh, dry or frozen) or the tissue (leaf, seed, root) that constitutes the raw material, DNA can be recovered, amplified and sequenced, and then the botanical identity can be deduced by comparison to a database of reference sequences from an extensive list of plant species.
DNA Metabarcoding as a New Genetic Tool for Perfumery Product Analysis
The DNA metabarcoding is an evolution of the DNA barcoding, following recent innovations of sequencing technologies. It is able to detect even traces of DNA in a large set of products. Next generation sequencers now allow to analyze small DNA fragments, and in a particular mix of sequences.
The genetic marker used for DNA metabarcoding is small enough to allow the application of this method to highly processed materials (enduring, for example, high temperature steps that will degrade DNA). The DNA metabarcoding is based on the same principle as the DNA barcoding: by isolating, amplifying and sequencing DNA fragments present in a product. It is still possible to propose a name for the plant species that left DNA traces in this product: the plant(s) that was(were) isolated for the need of this product, but also the contaminating plants that came along the production process (as weeds in the culture, pollens deposited during the transformation process and on the containers).
This method has shown its efficiency in ecology (for example, soil , water , diet  analyses) and paleontology . Interestingly, this technology has also been applied successfully to honey  for biodiversity purpose, but that is also used as an ingredient for cosmetics. However, although DNA barcoding from plant raw material tends to become more popular , such analyses on ingredients and final products are still very scarce .
The challenge of this study was to retrieve violet DNA from its concrete and its absolute to perform a DNA metabarcoding analysis on those perfumery products.
How to Proceed?
The first step is to isolate the DNA contained in the extract (EO, concrete, absolute) from the other components. Various solvents are applied to separate DNA molecules from co-extracted biological components (lipids, sugars, proteins) or from chemical compounds (terpenes, alkaloids).
A genetic marker is amplified from the extracted DNA. This marker is a particular DNA fragment that is characterized by its universality (present in every plant) and its sequence variability (enough variability to allow an analysis with a resolution at the species level). Our genetic marker meets those conditions, and moreover is small enough to allow metabarcoding on degraded DNA in highly processed products. This marker has been amplified using patented universal primers , creating multiple copies.
Once amplified, the multiple copies have to be sequenced to know their respective sequences. The small size of the marker and the fact that we obtained a mix of different sequences imply the use of next generation sequencers. Those are able to read each DNA molecule individually. We then know which sequences are present and their respective number.
The bio-informatic analysis compares the resulting sequences with reference sequences in a public database, as well as our own database. We can then deduce which plant species have left DNA in the isolate (F-3).
Identifying Violet DNA in Violet Concrete
DNA barcoding was performed on Viola odorata of French and Egyptian origin. The comparison of their respective sequences for four genetic markers showed some discrete differences that make a genetic differentiation between both origins possible. We could also deduce from this analysis the sequences of both origins for our small genetic marker. This is a necessary step for the DNA metabarcoding.
DNA metabarcoding has been achieved on concrete and absolute extracted from the French violet. Approximately 42% of the plant DNA present in the concrete originated from a violet (F-4). This sequence is common to a number of Viola species and to both French and Egyptian violet strains, so that the analysis resolution is at the genus level. Other plant species, representing 58% of the total plant DNA, were encountered in this concrete: Trifolium, Oxalis... Those may represent plants that have been collected in the field along with Viola odorata. No Viola DNA has been retrieved in the absolute: this may have been lost during the washing steps that lead from the concrete to the absolute. However, results on another absolute show DNA from the extracted plant, so that the DNA metabarcoding is successful on other absolutes (unpublished results).
Natural isolates are rich and complex matrices. They are key products used in different industries ranging from food to pharmaceutics and cosmetics. In this context, authentication is a real concern for the consumer safety, as well as for the high level of quality of these natural materials coming from an ancestral savoir-faire.
Several techniques have proved their efficiency for specific cases of authentication (chiral chromatography, IRMS, SNIF-NMR etc.). More global techniques, including organoleptic properties and physical-chemical techniques, are also developed but they do not give information on the fraudulent origin.
Finally, hyphenated techniques like chromatography associated to MS and NMR are certainly the most informative techniques. These global analyses of a mixture combined to statistical treatments correspond to metabolomics approaches and they are becoming very powerful alternatives to investigate natural extracts. The metabolome appears as the final product of a range of closely related different “omics”. All different “omics” approaches addressed in this report are useful and necessary.
New tools like metabolomics can allow us to detect quality defects that are difficult to identify from different techniques. This is nevertheless not universal and cannot solve all issues. Genomics as an upstream level, gives additional information for authentication issues. Botanical species can be determined by these approaches. Results given by metabarcoding are quantitative, based on the number of DNA sequences of each plant present in the natural material or in the product. The relationship between the amount of DNA and the plant tissue mass has still to be calibrated. But metabarcoding shows potentials in botanical identification of the plants used for production of foods, cosmetics, pharmaceuticals, and in quantifying plants that have no signature in physico-chemical methods. These very relevant technologies for authentication issues, are destined to a bright future even if progress is expected for the coming years.