A New Blend of Litsea cubeba, Pinus mugo, and Cymbopogon winterianus Essential Oil Active as an Anti-tyrosinase Ingredient in Topical Formulations.

Tyrosinase is a target enzyme to be inhibited in order to reduce excessive melanin production and prevent typical age-related skin disorders. Essential oils are complex mixtures of volatile compounds, belonging mainly to monoterpenoids and sesquiterpenoids, which have been relatively little studied as tyrosinase inhibitors. Among the monoterpenoids, citral (a mixture of neral and geranial) is a fragrance compound in several essential oils that has shown interesting tyrosinase inhibitory activity. Although citral is listed as an allergen among the 26 fragrances in Annex III of the Cosmetics Directive 2003/15/EC, it can be safely used for the formulation of topical products in amounts that are not expected to cause skin sensitization, as shown by various commercially available products.The aim of this work was to evaluate two different formulations (oil/water emulsion, oily solution) containing a new combination of essential oils (Litsea cubeba, Pinus mugo, Cymbopogon winterianus) applied to the skin both in nonocclusive and partially occlusive modes. The blend is designed to reduce the concentration of citral to avoid potential skin reactions while taking advantage of the inhibitory activity of citral. Specifically, the amount of citral and other bioactive compounds (myrcene, citronellal) delivered through the skin was studied as a function of formulation and mode of application.The results show that an oil/water emulsion is preferable because it releases the bioactive compounds rapidly and minimizes their evaporative loss. In addition, semi-occluded conditions are required to prevent evaporation, resulting in higher availability of the bioactive compounds in viable skin.

Make the Quality Control of Essential Oils Greener: Fast Enantioselective GC-MS Analysis of Sweet and Bitter Orange as a Case Study.

Quality control of essential oils is fundamental for verifying their authenticity and conformity with quality standards, ensuring their safety and regulatory compliance, and monitoring their consistency. Companies that produce or market essential oils routinely evaluate the quality and authenticity of their products. However, they also must deal with increasing attention to environmental sustainability as well as practical considerations such as productivity, cost, and simplicity of methods. In this study, enantioselective gas chromatography (GC) was adopted to evaluate the quality of sweet and bitter orange essential oils, used as a case study. The analytical conditions were optimized and translated to fast GC to evaluate the impact of this approach on the environmental footprint of the analyses. The greenness of fast GC, compared with conventional GC, was quantitatively evaluated using a dedicated metric tool (AGREE), and important improvements have been calculated. The developed methods were applied to a set of commercial essential oils, and the data about the enantiomeric composition and relative percentage abundance were elaborated through multivariate statistics (principal component analysis). The results showed that fast chiral gas chromatography enables the classification of citrus essential oil samples and can be considered an environmentally friendly and sustainable approach for evaluating their quality.

Anharmonic Aspects in Vibrational Circular Dichroism Spectra from 900 to 9000 cm-1 for Methyloxirane and Methylthiirane.

Vibrational circular dichroism (VCD) spectra and the corresponding IR spectra of the chiral isomers of methyloxirane and of methylthiirane have been reinvestigated, both experimentally and theoretically, with particular attention to accounting for anharmonic corrections, as calculated by the GVPT2 approach. De novo recorded VCD spectra in the near IR (NIR) range regarding CH-stretching overtone transitions, together with the corresponding NIR absorption spectra, were also considered and accounted for, both with the GVPT2 and with the local mode approaches. Comparison of the two methods has permitted us to better describe the nature of active "anharmonic" modes in the two molecules and the role of mechanical and electrical anharmonicity in determining the intensities of VCD and IR/NIR data. Finally, two nonstandard IR/NIR regions have been investigated: the first one about ≈2000 cm-1, involving mostly two-quanta bending mode transitions, the second one between 7000 and 7500 cm-1 involving three-quanta transitions containing CH-stretching overtones and HCC/HCH bending modes.

Gas chromatography of essential oil: State-of-the-art, recent advances, and perspectives.

This review is an overview of the recent advances of gas chromatography in essential oil analysis; in particular, it focuses on both the new stationary phases and the advanced analytical methods and instrumentations. A paragraph is dedicated to ionic liquids as gas chromatography stationary phases, showing that, thanks to their peculiar selectivity, they can offer a complementary contribution to conventional stationary phases for the analysis of complex essential oils and the separation of critical pairs of components. Strategies to speed-up the analysis time, thus answering to the ever increasing request for routine essential oils quality control, are also discussed. Last but not least, a paragraph is dedicated to recent developments in column miniaturization in particular that based on microelectromechanical-system technology in a perspective of developing micro-gas chromatographic systems to optimize the energy consumption as well as the instrumentation dimensions. A number of applications in the essential oil field is also included.

Adulteration of Essential Oils: A Multitask Issue for Quality Control. Three Case Studies: Lavandula angustifolia Mill., Citrus limon (L.) Osbeck and Melaleuca alternifolia (Maiden & Betche) Cheel.

The quality control of essential oils (EO) principally aims at revealing the presence of adulterations and at quantifying compounds that are limited by law by evaluating EO chemical compositions, usually in terms of the normalised relative abundance of selected markers, for comparison to reference values reported in pharmacopoeias and/or international norms. Common adulterations of EO consist of the addition of cheaper EO or synthetic materials. This adulteration can be detected by calculating the percent normalised areas of selected markers or the enantiomeric composition of chiral components. The dilution of the EO with vegetable oils is another type of adulteration. This adulteration is quite devious, as it modifies neither the qualitative composition of the resulting EO nor the marker's normalised percentage abundance, which is no longer diagnostic, and an absolute quantitative analysis is required. This study aims at verifying the application of the two above approaches (i.e., normalised relative abundance and absolute quantitation) to detect EO adulterations, with examples involving selected commercial EO (lavender, bergamot and tea tree) adulterated with synthetic components, EO of different origin and lower economical values and heavy vegetable oils. The results show that absolute quantitation is necessary to highlight adulteration with heavy vegetable oils, providing that a reference quantitative profile is available.

Ionic liquids as gas chromatographic stationary phases: how can they change food and natural product analyses?

The volatile fraction of natural products often consists of complex mixtures of isomeric and/or homologous components with similar structural and physical characteristics (e.g. mono- and sesquiterpenoids) that are not easy to separate simultaneously with conventional GC stationary phases, even when used with multidimensional systems. The introduction of ionic liquids (ILs) as stationary phases has opened up new perspectives in this field as their unique solvation properties result in uncommon selectivity, which is completely different to that of classic polydimethylsiloxane (PDMS)- and polyethyleneglycol (PEG)-based columns. Because of their peculiar selectivity and high inertness, IL-based columns have already found several applications in the natural product field in mono- and multidimensional GC and preparative GC, leading to the exhaustive analysis of complex samples (including aqueous solutions), and the separation of challenging pair(s) of compounds. This article provides an overview of how IL-based columns can be exploited in the fields of food and natural products, explores the wide range of applications that have already been developed and highlights the main features of these promising stationary phases, which are expected to be further extended in the near future, in particular, for routine use. Graphical abstract.

Exploiting the versatility of vacuum-assisted headspace solid-phase microextraction in combination with the selectivity of ionic liquid-based GC stationary phases to discriminate Boswellia spp. resins through their volatile and semivolatile fractions.

The frankincense resins, secreted from Boswellia species, are an uncommon example of a natural raw material where every class of terpenoids is present in similar proportions. Diterpenoids (serratol, incensole, and incensole acetate) are used to discriminate samples from different species and origins. Headspace solid-phase microextraction has been used for frankincense analysis, although it requires long sampling time for medium- to low-volatility markers; headspace solid-phase microextraction under vacuum can overcome this limit. Gas chromatography is used for analysis but the separation of incensole and serratol needs polar stationary phases. In this study, we develop a method to discriminate frankincenses based on vacuum-assisted headspace solid-phase microextraction combined with fast gas chromatography-mass spectrometry with ionic liquid-based stationary phases. The optimized conditions for solid samples were: air evacuation below 0°C, 15 min of incubation time, and 15 min of extraction time. Losses of volatiles due to vial air-evacuation in the presence of the sample were minimized by sample amount above 100 mg and low sample temperature. Fast gas chromatography provides the baseline separation of all markers in 20 min. By applying vacuum sampling and fast gas chromatography, the total analysis was reduced to 50 min compared to 120 min (60 min sampling plus 60 min analysis) as previously reported. The method was successfully applied to commercial frankincense samples.

Melaleuca alternifolia Essential Oil: Evaluation of Skin Permeation and Distribution from Topical Formulations with a Solvent-Free Analytical Method.

Melaleuca alternifolia essential oil (tea tree oil) is widely used as an ingredient in skin care products because of its recognized biological activities. The European Scientific Committee on Consumer Products constantly promotes research and collection of data on both skin distribution and systemic exposure to tea tree oil components after the application of topical formulations. This study quantitatively evaluates permeation, skin layer distribution (stratum corneum, epidermis, and dermis), and release into the surrounding environment of bioactive tea tree oil markers (i.e., α-pinene, ß-pinene, α-terpinene, 1,8-cineole, γ-terpinene, 4-terpineol, α-terpineol) when a 5% tea tree oil formulation is applied at a finite dosing regimen. Permeation kinetics were studied in vitro on pig ear skin using conventional static glass Franz diffusion cells and cells ad hoc modified to monitor the release of markers into the atmosphere. Formulation, receiving phases, and skin layers were analyzed using a fully automatic and solvent-free method based on headspace solid-phase microextraction/gas chromatography-mass spectrometry. This approach affords, for the first time, to quantify tea tree oil markers in the different skin layers while avoiding using solvents and overcoming the existing methods based on solvent extraction. The skin layers contained less than 1% of each tea tree oil marker in total. Only oxygenated terpenes significantly permeated across the skin, while hydrocarbons were only absorbed at trace level. Substantial amounts of markers were released into the atmosphere.

Antiviral Activity of a Arisaema Tortuosum Leaf Extract and Some of its Constituents against Herpes Simplex Virus Type 2.

Infections caused by HSV-2 are a public health concern worldwide, and there is still a great demand for the discovery of novel anti-herpes virus agents effective against strains resistant to current antiviral agents. In this context, medicinal plants represent an alternative source of active compounds for developing efficient antiviral therapies. The aim of this study was to evaluate the antiviral activity of Arisaema tortuosum, a plant used in the traditional medicine of India. A chloroform soluble fraction of the leaves exhibited anti-HSV-2 activity with a selectivity index of 758. The extract was also active against acyclovir-resistant HSV-2 and HSV-1. The mechanism of action of the extract was investigated evidencing inhibition of both early and late events of the HSV-2 replicative cycle. A HPLC-PDA-MS/MS analysis showed the presence of flavonoids including apigenin and luteolin in the chloroform extract (CE). Apigenin and luteolin showed a high inhibitory activity with EC50 values of 0.05 and 0.41 µg/mL, respectively. Both compounds exhibited antiviral activity when added up to 6 h post infection and were able to reduce the viral progeny production. In addition, apigenin interfered with cell-to-cell virus spread.

Punica granatum Leaf Ethanolic Extract and Ellagic Acid as Inhibitors of Zika Virus Infection.

Zika virus, an arthropod-borne flavivirus, is an emerging healthcare threat worldwide. Zika virus is responsible for severe neurological effects, such as paralytic Guillain-Barrè syndrome, in adults, and also congenital malformations, especially microcephaly. No specific antiviral drugs and vaccines are currently available, and treatments are palliative, but medicinal plants show great potential as natural sources of anti-Zika phytochemicals. This study deals with the investigation of the composition, cytotoxicity, and anti-Zika activity of Punica granatum leaf ethanolic extract, fractions, and phytoconstituents. P. granatum leaves were collected from different areas in Italy and Greece in different seasons. Crude extracts were analyzed and fractionated, and the pure compounds were isolated. The phytochemical and biomolecular fingerprint of the pomegranate leaves was determined. The antiviral activities of the leaf extract, fractions, and compounds were investigated against the MR766 and HPF2013 Zika virus strains in vitro. Both the extract and its fractions were found to be active against Zika virus infection. Of the compounds isolated, ellagic acid showed particular anti-Zika activities, with EC50 values of 30.86 µM for MR766 and 46.23 µM for HPF2013. The mechanism of action was investigated using specific antiviral assays, and it was demonstrated that ellagic acid was primarily active as it prevented Zika virus infection and was able to significantly reduce Zika virus progeny production. Our data demonstrate the anti-Zika activity of pomegranate leaf extract and ellagic acid for the first time. These findings identify ellagic acid as a possible anti-Zika candidate compound that can be used for preventive and therapeutic interventions.

Sustainable Micro-Scale Extraction of Bioactive Phenolic Compounds from Vitis vinifera Leaves with Ionic Liquid-Based Surfactants.

This paper proposes a new sustainable and simple strategy for the micro-scale extraction of phenolic compounds from grapevine leaves with analytical purpose. The method is based on a microwave-assisted solid-liquid extraction approach (MA-SLE), using an aqueous solution of an ionic liquid (IL)-based surfactant as extraction phase. The method does not require organic solvents, nor any clean-up step, apart from filtration prior to the injection in the analytical system. Two IL-based surfactants were evaluated, and the method was optimized by using experimental designs, resulting in the use of small amounts of sample (100 mg) and extraction phase (2.25 mL), low concentrations of the selected 1-hexadecyl-3-butyl imidazolium bromide IL (0.1 mM), and 30 min of extraction time. The proposed methodology was applied for the determination of the polyphenolic pattern of six different varieties of Vitis vinifera leaves from the Canary Islands, using high-performance liquid chromatography and photodiode array detection for the quantification of the compounds. The proposed MA-SLE approach was greener, simpler, and more effective than other methods, while the results from the analysis of the leaves samples demonstrate that these by-products can be exploited as a source of natural compounds for many applications.

Grapevine Green Pruning Residues as a Promising and Sustainable Source of Bioactive Phenolic Compounds.

Green pruning residues (GPRs) and leaves from 16 red and white Vitis vinifera L. cultivars from Piedmont (Italy) were studied. The investigated samples were extracted by ultrasound-assisted extraction optimized by an experimental design, and quali- and quantitatively analyzed by HPLC-PDA-MS/MS. GPRs and leaves show a similar polyphenolic pattern, with quercetin 3-O-glucuronide, caftaric acid, and quercetin 3-O-glucoside as the main components, although in variable proportions. The HPLC results were related to the antioxidant activity, measured as total phenolic content and through DPPH and ABTS assays with similar results. Colorimetric in vitro assays, offline combined with HPLC-PDA analysis, determine which compounds contribute to the antioxidant activity in terms of radical scavenging abilities. Valorization of GPRs is a potential source of natural compounds that could be of interest in the health field, increasing their economic value together with a positive effect on the environment.

Arabidopsis thaliana ITS sequence-specific DNA extraction by ion-tagged oligonucleotides coupled with a magnetic ionic liquid.

This study reports a follow-up investigation on the capture of specific DNA sequences using ion-tagged oligonucleotides (ITOs) and magnetic ionic liquids (MIL). Five allylimidazolium salts bearing octyl substituents ([AOIM+]-ITOs) were used for the selective extraction of the internal transcribed spacer region (ITS) from Arabidopsis thaliana. In this work, the ability of the [AOIM+]-ITOs to enhance the extraction of longer target sequences (~ 700 bp) of plant origin was shown. Moreover, the independence of the probe binding position and the importance of complementarity to the target region for the extraction performance were demonstrated. To test the specificity of the ITOs, the same experiments were performed using the ITS region from another plant species, with a lower target capture for the probes which were specific for the A. thaliana sequence. Finally, extraction in the presence of interferences (heterogenous DNA, primary and secondary metabolites, proteins) provided interesting and insightful results. This work illustrates the feasibility and versatility of these probes when coupled to MILs for rapid, cost-effective, and environmentally sensitive sample preparation in the extraction of specific target sequences from different origins. Graphical abstract.

Ionic liquids as water-compatible GC stationary phases for the analysis of fragrances and essential oils.

Fragrances and products deriving from essential oils are often formulated or diluted in aqueous media, usually ethanol/water. Gas chromatography (GC) is the technique of choice to analyze volatiles. However, when using columns coated with conventional stationary phases, its application to aqueous samples often requires time-consuming and/or discriminative sample preparation techniques to extract the target analytes from the aqueous medium, so as to avoid its direct injection. In GC with conventional columns, water produces peak asymmetry, poor sensitivity and efficiency, strong adsorption, stationary phase degradation, and, last but not least, it is not easy to detect reliably when present in high amounts. In 2012, Armstrong's group introduced new fully water-compatible ionic-liquid (IL)-based GC capillary columns based on phosphonium and imidazolium derivative cations combined with trifluoromethanesulphonate. These columns were recently made available commercially by Supelco, under the trade name Watercol™. These derivatives maintain IL's unique selectivity and chromatographic properties, and enable water to be used as injection solvent, thus avoiding the sample preparation procedures required by conventional columns. This study reports and critically discusses the results of commercially available water-compatible IL columns for direct analysis of aqueous samples in the fragrance and essential oil fields by GC with thermal conductivity (TCD) and/or flame ionization detectors (FID). The results showed that water-compatible IL-based stationary phases can successfully be adopted for qualitative and quantitative analysis of fragrances and essential oils directly diluted in aqueous solvents. On the other hand, the study also shows that their inertness needs to be further increased and (possibly) the range of operative temperature extended when water is the main solvent of the sample.

The (+)-cis- and (+)-trans-Olibanic Acids: Key Odorants of Frankincense.

Frankincense (olibanum) is one of the oldest aromatic materials used by humans, but the key molecular constituents contributing to its characteristic odor remained unknown. Reported herein is the discovery that (1S,2S)-(+)-trans- and (1S,2R)-(+)-cis-2-octylcyclopropyl-1-carboxylic acids are highly potent and substantive odorants occurring in ppm amounts in all of the frankincense samples analyzed, even those showing radically different volatile compositions. These cyclopropyl-derived acids provide the very characteristic old churchlike endnote of the frankincense odor.

Direct Contact - Sorptive Tape Extraction coupled with Gas Chromatography - Mass Spectrometry to reveal volatile topographical dynamics of lima bean (Phaseolus lunatus L.) upon herbivory by Spodoptera littoralis Boisd.

BACKGROUND: The dynamics of plant volatile (PV) emission, and the relationship between damaged area and biosynthesis of bioactive molecules in plant-insect interactions, remain open questions. Direct Contact-Sorptive Tape Extraction (DC-STE) is a sorption sampling technique employing non adhesive polydimethylsiloxane tapes, which are placed in direct contact with a biologically-active surface. DC-STE coupled to Gas Chromatography - Mass Spectrometry (GC-MS) is a non-destructive, high concentration-capacity sampling technique able to detect and allow identification of PVs involved in plant responses to biotic and abiotic stresses. Here we investigated the leaf topographical dynamics of herbivory-induced PV (HIPV) produced by Phaseolus lunatus L. (lima bean) in response to herbivory by larvae of the Mediterranean climbing cutworm (Spodoptera littoralis Boisd.) and mechanical wounding by DC-STE-GC-MS. RESULTS: Time-course experiments on herbivory wounding caused by larvae (HW), mechanical damage by a pattern wheel (MD), and MD combined with the larvae oral secretions (OS) showed that green leaf volatiles (GLVs) [(E)-2-hexenal, (Z)-3-hexen-1-ol, 1-octen-3-ol, (Z)-3-hexenyl acetate, (Z)-3-hexenyl butyrate] were associated with both MD and HW, whereas monoterpenoids [(E)-ß-ocimene], sesquiterpenoids [(E)-nerolidol] and homoterpenes (DMNT and TMTT) were specifically associated with HW. Up-regulation of genes coding for HIPV-related enzymes (Farnesyl Pyrophosphate Synthase, Lipoxygenase, Ocimene Synthase and Terpene Synthase 2) was consistent with HIPV results. GLVs and sesquiterpenoids were produced locally and found to influence their own gene expression in distant tissues, whereas (E)-ß-ocimene, TMTT, and DMNT gene expression was limited to wounded areas. CONCLUSIONS: DC-STE-GC-MS was found to be a reliable method for the topographical evaluation of plant responses to biotic and abiotic stresses, by revealing the differential distribution of different classes of HIPVs. The main advantages of this technique include: a) in vivo sampling; b) reproducible sampling; c) ease of execution; d) simultaneous assays of different leaf portions, and e) preservation of plant material for further "omic" studies. DC-STE-GC-MS is also a low-impact innovative method for in situ PV detection that finds potential applications in sustainable crop management.

Dual catalytic activity of hydroxycinnamoyl-coenzyme A quinate transferase from tomato allows it to moonlight in the synthesis of both mono- and dicaffeoylquinic acids.

Tomato (Solanum lycopersicum), like other Solanaceous species, accumulates high levels of antioxidant caffeoylquinic acids, which are strong bioactive molecules and protect plants against biotic and abiotic stresses. Among these compounds, the monocaffeoylquinic acids (e.g. chlorogenic acid [CGA]) and the dicaffeoylquinic acids (diCQAs) have been found to possess marked antioxidative properties. Thus, they are of therapeutic interest both as phytonutrients in foods and as pharmaceuticals. Strategies to increase diCQA content in plants have been hampered by the modest understanding of their biosynthesis and whether the same pathway exists in different plant species. Incubation of CGA with crude extracts of tomato fruits led to the formation of two new products, which were identified by liquid chromatography-mass spectrometry as diCQAs. This chlorogenate:chlorogenate transferase activity was partially purified from ripe fruit. The final protein fraction resulted in 388-fold enrichment of activity and was subjected to trypsin digestion and mass spectrometric sequencing: a hydroxycinnamoyl-Coenzyme A:quinate hydroxycinnamoyl transferase (HQT) was selected as a candidate protein. Assay of recombinant HQT protein expressed in Escherichia coli confirmed its ability to synthesize diCQAs in vitro. This second activity (chlorogenate:chlorogenate transferase) of HQT had a low pH optimum and a high Km for its substrate, CGA. High concentrations of CGA and relatively low pH occur in the vacuoles of plant cells. Transient assays demonstrated that tomato HQT localizes to the vacuole as well as to the cytoplasm of plant cells, supporting the idea that in this species, the enzyme catalyzes different reactions in two subcellular compartments.

Versatile DNA extraction from diverse plant taxa using ionic liquids and magnetic ionic liquids: a methodological breakthrough for enhanced sample utility.

BACKGROUND: There is a growing demand for fast and reliable plant biomolecular analyses. DNA extraction is the major bottleneck in plant nucleic acid-based applications especially due to the complexity of tissues in different plant species. Conventional methods for plant cell lysis and DNA extraction typically require extensive sample preparation processes and large quantities of sample and chemicals, elevated temperatures, and multiple sample transfer steps which pose challenges for high throughput applications. RESULTS: In a prior investigation, an ionic liquid (IL)-based modified vortex-assisted matrix solid phase dispersion approach was developed using the model plant, Arabidopsis thaliana (L.) Heynh. Building upon this foundational study, the present study established a simple, rapid and efficient protocol for DNA extraction from milligram fragments of plant tissue representing a diverse range of taxa from the plant Tree of Life including 13 dicots and 4 monocots. Notably, the approach was successful in extracting DNA from a century old herbarium sample. The isolated DNA was of sufficient quality and quantity for sensitive molecular analyses such as qPCR. Two plant DNA barcoding markers, the plastid rbcL and nuclear ribosomal internal transcribed spacer (nrITS) regions were selected for DNA amplification and Sanger sequencing was conducted on PCR products of a representative dicot and monocot species. Successful qPCR amplification of the extracted DNA up to 3 weeks demonstrated that the DNA extracted using this approach remains stable at room temperature for an extended time period prior to downstream analysis. CONCLUSIONS: The method presented here is a rapid and simple approach enabling cell lysis and DNA extraction from 1.5 mg of plant tissue across a broad range of plant taxa. Additional purification prior to DNA amplification is not required due to the compatibility of the extraction solvents with qPCR. The method has tremendous potential for applications in plant biology that require DNA, including barcoding methods for agriculture, conservation, ecology, evolution, and forensics.

Ultrasound-assisted dispersive solid-liquid microextraction with eutectic solvents for the determination of cannabinoids in different hemp products.

The wide range of applications of hemp products, together with the environmental benefits that come from hemp cultivation are driving up the market demand for Cannabis sativa L. plant. One of the main restrictions for hemp cultivation and marketing concerns the content of delta-9-tetrahydrocannabidiol (Δ9-THC), which is known to have psychotomimetic effect. If the recent growing of hemp market is beneficial by an economic and environmental point of view, it is necessary to develop reliable analytical methods for the chemical characterization of hemp products, to guarantee the safety of use for the customers. This study aimed to develop a simple ultrasound-assisted dispersive solid-liquid microextraction (UA-DSLME) method for the extraction of cannabinoids in hemp products, using eutectic solvents (ESs) as extraction material. Two types of ESs were compared: one prepared with a [Ch+][Br-]-modified salts as hydrogen bond acceptor and one based on natural terpenoids. The ultrasound-assisted dispersive solid-liquid microextraction method was optimized to be applied for the analysis of aerial parts of hemp collected before flowering, hemp inflorescences and a commercial sample called CBD oil, and proved to be robust and versatile. Under optimal conditions, only 100 µL of ES and 2 mL of water as co-solvent were used in the US-assisted extraction, before the analysis in the UHPLC-PDA system. The developed approach allowed to obtain the same chemical profile of conventional methods, while improving the greenness of the method and the enrichment of the marker analytes. To overcome the strong matrix effect for cannabinoids, a matrix-matched calibration was used. Blank matrices of the samples under study were easily obtained by performing an exhaustive extraction of the marker analytes in the hemp samples. These matrices were successfully used for validation, achieving accuracy values between 82% and 118%.

Populus nigra L. bud absolute: a case study for a strategy of analysis of natural complex substances.

The new European regulations (e.g., REACH) require that Natural Complex Substances such as essential oils, absolutes, concretes, and resinoids are registered. This need implies that the chemical composition of these complex mixtures is characterized as exhaustively as possible in view of defining their toxicological risk. This study proposes an analysis strategy to be applied to the chemical characterization of poplar absolute as an example of Natural Complex Substances of vegetable origin. In the first part, the proposed strategy is described, and the advantages and the limitations related to the combination of conventional analytical techniques such as gas chromatography (GC) without and with sample derivatization and high-performance liquid chromatography (HPLC) are critically discussed. In the second part, the qualitative data obtained with GC and HPLC analysis of poplar bud absolute confirm the sample complexity which mainly consists of phenolic components. Fourteen compounds (i.e., phenolic acids, phenylpropanoids, and flavonoids) were then chosen as markers representative of the main classes of components characterizing poplar bud absolute. The marker quantitation carried out by GC-SIM-MS and HPLC-PDA analyses gives similar results confirming the reliability of both techniques. These results demonstrate that conventional analytical techniques can positively and effectively contribute to the study of the the composition of Natural Complex Substances, i.e., matrices for which highly effective separation is necessary, consisting mainly of isomers or homologous components. The combination of GC and HPLC techniques is ever more necessary for routine quality control when conventional instrumentations are used.