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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., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

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 (Δ 9 -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%., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Elettaria cardamomum (L.) Maton (Zingiberaceae family) is a plant traditionally used in Ayurvedic and Chinese medicine. In this work, the essential oil of E. cardamomum was found to inhibit the enzymes AChE (62.6% of inhibition, IC 50 24.9 μg/mL) and BChE (55.8% of inhibition, IC 50 25.9 μg/mL) by performing an in vitro colorimetric assay using the Ellman method. A bio-guided fractionation approach was used to isolate fractions/pure compounds that were tested individually to evaluate their activity. The resulting oxygenated fraction was found to be active against both AChE (percentage inhibition 42.8%) and BChE (percentage inhibition 63.7%), while the hydrocarbon fraction was inactive. The activity was attributed to a pool of oxygenated terpenes (α-terpinyl acetate, 1,8-cineole, linalool, linalyl acetate, and α-terpineol) that synergistically contributed to the overall activity of the essential oil.

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.

Data on urban and rural diabetes prevalence ratios show a significantly lower presence of diabetes in rural areas. Several bioactive compounds of plant origin are known to exert anti-diabetic properties. Interestingly, most of them naturally occur in different plants present in mountainous areas and are linked to traditions of herbal use. This review will aim to evaluate the last 10 years of evidence-based data on the potential anti-diabetic properties of 9 plants used in the Piedmont Alps (North-Western Italy) and identified through an ethnobotanical approach, based on the Occitan language minority of the Cuneo province ( Sambucus nigra L., Achillea millefolium L., Cornus mas L., Vaccinium myrtillus L ., Fragaria vesca L., Rosa canina L., Rubus idaeus L., Rubus fruticosus/ulmifolius L., Urtica dioica L.), where there is a long history of herbal remedies. The mechanism underlying the anti-hyperglycemic effects and the clinical evidence available are discussed. Overall, this review points to the possible use of these plants as preventive or add-on therapy in treating diabetes. However, studies of a single variety grown in the geographical area, with strict standardization and titration of all the active ingredients, are warranted before applying the WHO strategy 2014-2023.

Phosphonium-based ionic liquids (ILs) have proven to be successful stationary phases (SPs) for gas chromatography (GC) in several fields of application because of their unique selectivity and good chromatographic properties. This study focuses on the use of two ILs as GC SPs that are based on the phosphonium derivatives trihexyl(tetradecyl)phosphonium chloride ([P 66614 + ] [Cl - ]), and trihexyl(tetradecyl)phosphonium bis[(trifluoromethyl)sulfonyl]imide ([P 66614 + ][NTf 2 - ]), which have previously been shown to be complementary in terms of chromatographic selectivity and retention. Their application in routine analysis has been limited by their lower maximum allowable operating temperatures (MAOT) (200 °C for the [P 66614 + ][Cl - ] IL and 180 °C for [P 66614 + ][NTf 2 - ]), which restricts their use to samples that consist of analytes with relatively high volatility. A previous study carried out in the Authors' laboratory focused on extending the use of the [P 66614 + ][Cl - ] IL SP to the analysis of samples with analytes of medium-to-low volatility by optimizing column characteristics and operative conditions. This study addresses the immobilization of both the [P 66614 + ][Cl - ] and [P 66614 + ][NTf 2 - ] ILs to the inner wall of fused silica columns to increase their MAOT under soft and hard reaction conditions. The resulting MAOT depended on more or less drastic immobilization conditions, and reached 220 °C for soft immobilization (So-Im) and 240 °C for hard immobilization (Ha-Im) in the [P 66614 + ][Cl - ] IL columns, and 200 °C for So-Im and 220° for Ha-Im in columns coated with the [P 66614 + ] [NTf 2 - ] IL. The influence of immobilization on the separation power and performance of all the columns has been evaluated using i) the Grob test, ii) a model mixture of 41 compounds of different polarity, structure, and with different organic functional groups representative of the flavor and fragrance field, iii) a standard mixture of 37 fatty acid methyl esters, iv) the peppermint essential oil, v) two mixtures of sesquiterpenic alcohols (farnesols and santalols), and vi) a standard mixture of 16 pesticides. These test samples were also used to demonstrate the complementarity of the two phosphonium-based IL SPs in terms of selectivity and retention., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Currently, there is a renewed interest in cannabis-related products in different fields because of the rich phytocomplex of this plant, together with its fiber and agricultural features. In this context, the current study aims to chemically characterize different samples of fiber-type Cannabis sativa L. grown in Italy as a potential health promoting source. An ultrasound-assisted solid-liquid extraction (UA-SLE) method was first developed and optimized to obtain a fingerprinting of the investigated phytocomplex. Analyses were carried out through an ultra high performance liquid chromatography equipped with a photodiode array detector in series with triple quadrupole system with an electrospray ionization (ESI) interface (UHPLC-UV-ESI-MS/MS) and showed that the phytocomplex mainly includes flavonoids and non-psychotomimetic cannabinoids. The method was then applied to characterize and compare 24 samples of fiber-type Cannabis sativa L. aerial parts (mainly stems and leaves), which differed for the growth stages (from mid-vegetative to early flowering), growth land plots, and methods of drying (forced-draft oven or freeze-drying). The quali-quantitative analysis showed that a freeze-drying method seems to better preserve the chemical composition of the samples, while the location of the land plot and the growth stage of the plant (which did not comprise inflorescences) had minor influences on the chemical pattern. These results were also supported by spectrophotometric in-vitro assays (scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH • ) and 2,2'-azinobis-3-ethyl-benzthiazoline-6-sulphonate (ABTS +• ) radicals and inhibitory activity against tyrosinase and elastase enzymes) to investigate the potential biological activity of these samples and the contribution of non-psychotomimetic cannabinoids.

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., (© 2021 Wiley-VCH GmbH.)

As part of a project devoted to the phytochemical study of Ecuadorian biodiversity, new essential oils are systematically distilled and analysed. In the present work, Jungia rugosa Less (Asteraceae) has been selected and some wild specimens collected to investigate the volatile fraction. The essential oil, obtained from fresh leaves, was analysed for the first time in the present study. The chemical composition was determined by gas chromatography, coupled to mass spectrometry (GC-MS) for qualitative analysis, and to flame ionization detector (GC-FID) for quantitation. The calculation of relative response factors (RRF), based on combustion enthalpy, was carried out for each quantified component. Fifty-six compounds were identified and quantified in a 5% phenyl-polydimethylsiloxane non-polar column and 53 compounds in a polyethylene glycol polar column, including four undetermined compounds. The main feature of this essential oil was the exclusive sesquiterpenes content, both hydrocarbons (74.7% and 80.4%) and oxygenated (8.3% and 9.6%). Major constituents were: γ-curcumene (47.1% and 49.7%) and β-sesquiphellandrene (17.0% and 17.9%), together with two abundant undetermined oxygenated sesquiterpenes, whose abundance was 6.7-7.2% and 4.7-3.3%, respectively. In addition, the essential oil was submitted to enantioselective evaluation in two β-cyclodextrin-based enantioselective columns, determining the enantiomeric purity of a minor component (1 S ,2 R ,6 R ,7 R ,8 R )-(+)-α-copaene. Finally, the AChE inhibition activity of the EO was evaluated in vitro. In conclusion, this volatile fraction is suitable for further investigation, according to two main lines: (a) the purification and structure elucidation of the major undetermined compounds, (b) a bio-guided fractionation, intended to investigate the presence of new sesquiterpene AChE inhibitors among the minor components.

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.

Accurate, reliable, and informative mapping of untargeted and targeted components across many samples is here performed by combining off-line GC-Olfactometry (GC-O) and comprehensive two-dimensional gas chromatography (GC×GC) coupled to time-of-flight mass spectrometry with variable ionization energy (TOF MS featuring Tandem Ionization™). In particular, untargeted and targeted (UT) features patterns are processed by chromatographic fingerprinting, giving differential priority to potent odorants' retention-times regions. Distinguishing peppermint essential oil (EO) from Piedmont (Italy - Mentha × piperita L. var. Italo-Mitcham - Menta di Pancalieri EO), with its unique sensory fingerprint (i.e., freshness and long-lasting sweetness), from high-quality peppermint EOs produced in other areas poses a great challenge. Chromatographic UT fingerprinting provided a great chemical dimensionality by mapping more than 350 peak-regions at 70 eV and 135 at 12 eV. From them, 95 components were identified and responses compared to available literature. Then, potent odorants, detected by GC-O using the aroma extraction dilution analysis (AEDA), were tracked over the chromatographic space and tentatively identified. With the highest flavor dilution (FD), 1,8-cineole (eucalyptus, fresh, camphoraceous); menthone (minty, herbaceous); and menthofuran (minty, musty, petroleum-like) were highlighted. Responsible for creamy and coumarinic notes were the diasteroisomers of (3,6)-dimethyl-4,5,6,7-tetrahydrobenzo[b]-furan-2(3H)-one (i.e., menthofurolactones), detected in higher relative abundance in Pancalieri EOs. By prioritizing the investigation of volatiles on higher LogFD retention regions, including 131 untargeted/targeted features, Pancalieri EOs were separately clustered from United States samples. Besides pre-targeted analytes, additional untargeted features were post-processed for identification within marker chemicals. Myrtenyl methyl ether, ethyl 3-methyl butanoate, propyl-2-methylbutanoate, and (E)-2-hexenal were putatively identified. Of the "unknown - knowns" with diagnostic roles, all metadata were collected including low energy spectra at 12 eV, which were found to be highly complementary to 70 eV spectra., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)

Excessive melanin production causes serious dermatological conditions as well as minor aesthetic problems (i.e., freckles and solar lentigo). The downregulation of tyrosinase is a widespread approach for the treatment of such disorders, and plant extracts have often proven to be valuable sources of tyrosinase inhibitors. Citral (a mixture of neral and geranial) is an important fragrance ingredient that has shown anti-tyrosinase potential. It is highly concentrated in the essential oils (EOs) of Cymbopogon schoenanthus (L.) Spreng., Litsea cubeba (Lour.) Pers., Melissa officinalis L., and Verbena officinalis L. However, only L. cubeba EO has been investigated for use as a potential skin-whitening agent. This work evaluates the in vitro tyrosinase inhibitory activity of these EOs and studies, using bio-assay oriented fractionation, whether their differing chemical compositions influence the overall EO inhibitory activities via possible synergistic, additive, and/or competitive interactions between EOs components. The inhibitory activity of C. schoenanthus EO and that of M. officinalis EOs, with negligible (+)-citronellal amounts, were in-line with their citral content. On the other hand, L. cubeba and V. officinalis EOs inhibited tyrosinase to considerably greater extents as they contained β-myrcene, which contributed to the overall EO activities. Similar observations were made for M. officinalis EO, which bears high (+)-citronellal content which increased citral activity.

Biogenic volatile organic compounds (BVOCs) are metabolites emitted by living plants that have a fundamental ecological role since they influence atmospheric chemistry, plant communication and pollinator/herbivore behaviour, and human activities. Over the years, several strategies have been developed to isolate and identify them, and to take advantage of their activity. The main techniques used for in-vivo analyses include dynamic headspace (D-HS), static headspace (S-HS) and, more recently, direct contact (DC) methods in association with gas chromatography (GC) and mass spectrometry (MS). The aim of this review is to provide insight into the in-vivo characterisation of plant volatile emissions with a focus on sampling, analysis and possible applications. This review first provides a critical discussion of the challenges associated with conventional approaches and their limitations and advantages. Then, it describes a series of applications of in-vivo volatilomic studies to enhance how the information they provide impact on our knowledge of plant behaviour, including the effects of abiotic (damage, flooding, climate) and biotic (insect feeding) stress factors in relation to the plants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A novel chemical profile essential oil, distilled from the aerial parts of Clinopodium taxifolium (Kunth) Govaerts (Lamiaceae), was analysed by Gas Chromatography-Mass Spectrometry (GC-MS, qualitative analysis) and Gas Chromatography with Flame Ionization Detector (GC-FID, quantitative analysis), with both polar and non-polar stationary phase columns. The chemical composition mostly consisted of sesquiterpenes and sesquiterpenoids (>70%), the main ones being ( E )- β -caryophyllene (17.8%), α -copaene (10.5%), β -bourbonene (9.9%), δ -cadinene (6.6%), cis -cadina-1(6),4-diene (6.4%) and germacrene D (4.9%), with the non-polar column. The essential oil was then submitted to enantioselective GC analysis, with a diethyl- tert -butyldimethylsilyl- β -cyclodextrin diluted in PS-086 chiral selector, resulting in the following enantiomeric excesses for the chiral components: (1 R ,5 S )-(-)- α -thujene (67.8%), (1 R ,5 R )-(+)- α -pinene (85.5%), (1 S ,5 S )-(-)- β -pinene (90.0%), (1 S ,5 S )-(-)-sabinene (12.3%), ( S )-(-)-limonene (88.1%), ( S )-(+)-linalool (32.7%), ( R )-(-)-terpinen-4-ol (9.3%), ( S )-(-)- α -terpineol (71.2%) and ( S )-(-)-germacrene D (89.0%). The inhibition activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) of C. taxifolium essential oil was then tested, resulting in selective activity against BChE with an IC 50 value of 31.3 ± 3.0 μg/mL (positive control: donepezil, IC 50 = 3.6 μg/mL).

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 EC 50 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., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by unpaired blood glycaemia maintenance. T2DM can be treated by inhibiting carbohydrate hydrolyzing enzymes (α-amylases and α-glucosidases) to decrease postprandial hyperglycemia. Acarbose and voglibose are inhibitors used in clinical practice. However, these drugs are associated with unpleasant gastrointestinal side effects. This study explores new α-amylase inhibitors deriving from plant volatile specialized metabolites. Sixty-two essential oils (EOs) from different plant species and botanical families were subjected to α-amylase in vitro enzymatic assay and chemically characterized using gas chromatography coupled to mass spectrometry. Several EOs were found to be potential α-amylase inhibitors, and Eucalyptus radiata , Laurus nobilis , and Myristica fragrans EOs displayed inhibitory capacities comparable to that of the positive control (i.e., acarbose). A bio-guided fractionation approach was adopted to isolate and identify the active fractions/compounds of Eucalyptus radiata and Myristica fragrans EOs. The bio-guided fractionation revealed that EOs α-amylase inhibitory activity is often the result of antagonist, additive, or synergistic interactions among their bioactive constituents and led to the identification of 1,8-cineole, 4-terpineol, α-terpineol, α-pinene, and β-pinene as bioactive compounds, also confirmed when they were tested singularly. These results demonstrate that EO oils are a promising source of potential α-amylase inhibitors.

The Echinacea genus includes a number of species that are commercially employed for the preparation of herbal products. Echinacea angustifolia DC. is one of these and is widely used, mainly for its immunomodulating properties, as it contains a wide range of compounds that belong to different chemical classes. In particular, echinacoside, cynarin and lipophylic alkylamides are the main specialized metabolites of the roots and can be considered to be marker compounds. In this work, 65 E. angustifolia accessions have been compared in a field trial in Italy, with the aim of investigating the variability/stability of the weight and chemical composition of their roots in order to identify the accessions that are most promising for future genetic-improvement programs. The morphological characteristics of the aerial parts have also been investigated. Seventeen samples were discarded due to germination or plantlet-development issues. Seven of the remaining accessions were identified as being different Echinacea species after a combined phytochemical and morphological evaluation. The morphological traits of the epigeal part, the root weight and the chemical composition data of the 41 confirmed E. angustifolia accessions were submitted to multivariate statistical analysis and a moderately homogenous sample distribution, with low selected-marker variability, was observed. Good echinacoside content was detected in almost all roots (>0.5%). However, two groups of accessions stood out because of their interesting features: One group possessed small roots, but had a high concentration of marker compounds, while another had highly developed roots and a good amount of marker compounds. These accessions can therefore be exploited for future selection work.

Hemp (Cannabis sativa L.) has become widely used in several sectors due to the presence of various bioactive compounds such as terpenes and cannabidiol. In general, terpenes and cannabidiol content is determined separately, which is time consuming. Thus, a fast gas chromatography with flame ionization detection method was validated for simultaneous determination of both terpenes and cannabidiol in hemp. The method enabled a rapid detection of 29 different terpenes and cannabidiol within a total analysis time of 16 min, with satisfactory sensitivity (limit of detection = 0.03-0.27 µg/mL, limit of quantitation = 0.10-0.89 µg/mL). The inter- and intraday precision (RSD) was <7.82 and <3.59%, respectively. Recoveries at two spiked concentration levels (low, 3.15 µg/mL; high, 20.0 µg/mL) were determined on both apical leaves (78.55-101.52%) and inflorescences (77.52-107.10%). The reproducibility (RSD) was <5.94 and <5.51% in apical leaves and inflorescences, respectively. The proposed and validated method is highly sensitive, robust, fast, and accurate for determination of the main terpenes and cannabidiol in hemp and could be routinely used for quality control., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Room temperature ionic liquids (ILs) are well established stationary phases (SPs) for gas chromatography (GC) in several fields of applications because of their unique and tunable selectivity, low vapor pressure and volatility, high thermal stability (over 300 °C), and good chromatographic properties. This study is focused on an IL based on a phosphonium derivative (trihexyl(tetradecyl)phosphonium chloride, [P 66614 + ] [Cl - ]), previously shown to be suitable as a gas chromatographic SP because of its unique selectivity. In particular, it aims to establish the operative conditions to apply [P 66614 + ][Cl - ] to routine analysis of samples containing medium to high volatility analytes with different polarity, organic functional groups and chemical structure. In the first part, the study critically evaluates long term [P 66614 + ][Cl - ] column stability and maximum allowable operating temperatures (MAOT). The relatively low MAOT (210 °C) requires the adoption of a dedicated approach for analytes eluting above this temperature based on a suitable combination of efficiency and selectivity, and column characteristics (length, inner diameter and film thickness) and operative conditions. The performance of [P 66614 + ][Cl - ] as a GC SP have been validated through the Grob test, a model mixture of 41 compounds of different polarity, structure, and with different organic functional groups in the flavor and fragrance field, a standard mixture of 37 fatty acid methyl esters, some essential oils containing pairs or groups of compounds of different volatility critical to separate in particular peppermint, thyme, oregano, sandalwood and frankincense. The above approach has produced highly satisfactory separations with all of the samples investigated., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests, (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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, β -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., Competing Interests: The authors declare that they have no conflict of interest., (Georg Thieme Verlag KG Stuttgart · New York.)

The use of a thick sorbent coating in headspace sorptive extraction (HSSE) increases the amount of analytes extracted at equilibrium as well as the time needed to reach it. In this work we propose HSSE sampling under vacuum conditions to reduce equilibration times. A theoretical model is presented that describes the pressure dependence of the so-called vacuum-assisted HSSE (Vac-HSSE) method, and predicts the reduction in equilibration times when lowering the sampling pressure. We take advantage of the theoretical formulation to reach some general conclusions for HSSE on the relationship between the physical characteristics of the stir bar, uptake rates and equilibration times. The theoretical predictions were experimentally verified using water solutions spiked with naphthalene, acenaphthene and fluoranthene as model compounds. The effects of sampling temperature and extraction time under vacuum and regular pressure conditions were thoroughly investigated. The positive combined effect of heating the sample under low sampling pressure pointed that high humidity did not affect the performance of the extraction phase; an effect commonly recorded in headspace solid-phase microextraction. The extraction time profiles built at 25 and 55 °C visualized the substantial improvement in extraction kinetics with Vac-HSSE compared to the regular HSSE method. The results on naphthalene (assumed to evaporate relatively fast from the water sample) provided evidence that at 1 atm gas-sided resistance limited analyte uptake by the stir-bar and that this limitation could be effectively reduced by adopting the vacuum sampling approach. The accelerations of acenaphthene and fluoranthene suggested that gas-phase constraints limited both the evaporation and analyte uptake processes. Independent method optimization of HSSE under each pressure condition yielded a shorter sampling time for Vac-HSSE compared to the regular HSSE procedure (30 min vs. 60 min respectively). The analytical performances of the two optimized methods were evaluated and it was concluded that Vac-HSSE was performing similar (naphthalene and acenaphthene) or better (fluoranthene) than regular HSSE in half the sampling time needed., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors declare no conflict of interest.

Qualitative and quantitative determination of volatile markers in aqueous based fragrances assumes ever-increasing importance, because of both the need for quality control and the safety-regulatory limitations introduced for several compounds. This study reports and critically discusses the results of applying new water-compatible ionic-liquid (IL) GC stationary phases, based on phosphonium and imidazolium derivative cations combined with trifluoromethanesulphonate (Watercol™) to the direct quantitative analysis of aqueous samples in the perfume field with GC-MS. Narrow-bore columns of different lengths, especially prepared for this study, were adopted to minimize the amount of water reaching the MS detector after GC separation. All GC-MS analysis steps were investigated, to achieve results compatible with quality control requirements for the volatiles of interest in this field, in terms of LODs, LOQs, and repeatability. Reliability of the GC-MS results was demonstrated by determining volatile allergens in two commercial perfumes, as per EU regulations concerning no-declaration limits for leave-on (0.001%) and rinse-off (0.01%) cosmetic products., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The quality assessment of the green coffee that you will go to buy cannot be disregarded from a sensory evaluation, although this practice is time consuming and requires a trained professional panel. This study aims to investigate both the potential and the limits of the direct headspace solid phase microextraction, mass spectrometry electronic nose technique (HS-SPME-MS or MS-EN) combined with chemometrics for use as an objective, diagnostic and high-throughput technique to be used as an analytical decision maker to predict the in-cup coffee sensory quality of incoming raw beans. The challenge of this study lies in the ability of the analytical approach to predict the sensory qualities of very different coffee types, as is usual in industry for the qualification and selection of incoming coffees. Coffees have been analysed using HS-SPME-MS and sensory analyses. The mass spectral fingerprints (MS-EN data) obtained were elaborated using: (i) unsupervised principal component analysis (PCA); (ii) supervised partial least square discriminant analysis (PLS-DA) to select the ions that are most related to the sensory notes investigated; and (iii) cross-validated partial least square regression (PLS), to predict the sensory attribute in new samples. The regression models were built with a training set of 150 coffee samples and an external test set of 34. The most reliable results were obtained with acid, bitter, spicy and aromatic intensity attributes. The mean error in the sensory-score predictions on the test set with the available data always fell within a limit of ±2. The results show that the combination of HS-SPME-MS fingerprints and chemometrics is an effective approach that can be used as a Total Analysis System (TAS) for the high-throughput definition of in-cup coffee sensory quality. Limitations in the method are found in the compromises that are accepted when applying a screening method, as opposed to human evaluation, in the sensory assessment of incoming raw material. The cost-benefit relationship of this and other screening instrumental approaches must be considered and weighed against the advantages of the potency of human response which could thus be better exploited in modulating blends for sensory experiences outside routine.

The fresh leaves of Coreopsis triloba S.F. Blake, collected at Cerro Villonaco in Loja, Ecuador, were investigated with respect to their essential oil (EO). The chemical composition was determined qualitatively through gas chromatography coupled with mass spectrometry (GC-MS) and quantitatively by gas chromatography coupled with flame ionization (GC-FID), using relative response factors (RRF) based on the enthalpy of combustion. The essential oil contained between 92.5% and 93.4% of monoterpene hydrocarbons, with ( E )- β -ocimene being the main component (35.2-35.9%), followed by β -phellandrene (24.6-25.0%), α-pinene (15.3-15.9%), myrcene (10.9-11.0%), sabinene (2.2-2.4%), ( Z )- β -ocimene (1.5%), and germacrene D (1.2-1.3%). The enantiomeric distribution of α-pinene, β -pinene, limonene, and germacrene D was also determined. The main components responsible for the aroma were identified through aroma extract dilution analysis (AEDA), a gas chromatography-olfactometry (GC-O) based technique, being α -pinene, β -pinene (0.6%), terpinolene (0.1%), α- copaene (0.1-0.3%), β- phellandrene, and ( E )-4,8-dimethyl-1,3,7-nonatriene (0.1-0.2%) the main olfactory constituents according to the decreasing factor of dilution (FD) order. The biological tests showed IC 50 inhibition values of 42.2 and 6.8 µg/mL for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), respectively.

A correct botanical identification and analytical quality control of volatile key-markers responsible for aroma and biological activities is necessary to monitor volatile compounds transferred from a plant to the related herbal tea and human intake to guarantee their safe use. This is mainly true for markers limited by regulations or by a recommended maximum amount of consumption per day. GC-MS is the elective technique to analyze volatiles, provided that for aqueous samples (herbal teas) an appropriate sample preparation procedure, and/or a water-compatible GC stationary phases are applied. Solid Phase Micro Extraction (SPME) on-line coupled to GC-MS in a fully automatic approach is here applied to sample and quantify key markers in plant material (headspace) and in the corresponding herbal tea (direct immersion). In parallel, a new generation of GC columns coated with ionic liquid based stationary phases compatible with aqueous samples (Watercol ™ ) was applied to test direct injection of aqueous samples (DAI-GC-FID). The latter approach fully bypasses sample preparation thus speeding up quality control. This study deals with the quantitation of menthol, α- and β-thujone, estragole, and anethole contained in several plant species commonly used for herbal teas (i.e. peppermint, sage, wormwood, fennel, aniseed) and regulated by International Organizations. The two methods gave comparable results and are characterized by high repeatability, linearity and accuracy, although, as expected, their sensitivity was different because DAI-GC-FID implies injection of the sample as such without analyte concentration as for DI-SPME-GC-MS. For instance, LOD and LOQ of estragole were 0.03 and 0.1 mg L -1 with DI-SPME-GC-MS and 0.1 and 0.8 mg L -1 with DAI-GC-FID. The two methods are fully complementary and their adoption depends on the amount of marker(s) to be quantified., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Ptilostemon casabonae (L.) Greuter is a Mediterranean endemism traditionally used for its health-giving properties. Little is known about this species, therefore this study provides additional information about the phytochemical and biomolecular patterns of this plant, to have a combined fingerprint as a taxonomic tool. Several P. casabonae specimens were therefore collected from three different sites, two from Sardinia (Italy) and one from Corsica and the hydroalcoholic extracts of their aerial parts were investigated through HPLC-PDA-MS/MS analysis to study the phenolic composition. Quercetin, luteolin, kaempferol, apigenin and diosmetin O-glycosides, and caffeoylquinic acid derivatives were found as main components. Samples from the three sites showed similar phenolic profiles, although statistical analyses highlighted some quantitative differences for several compounds. The biomolecular analysis included amplification and sequencing of ITS, 5S-rRNA-NTS and psbA regions. No difference was found in the nucleotides among the P. casabonae samples from different geographical origins; however, a comparison with other Ptilostemon species sequences from Genbank, revealed an interspecific variability of ITS and psbA regions. The combination of the results of the phytochemical and biomolecular studies provide information on P. casabonae useful to depict this little-known plant, which can also be applied for future investigations and to obtain a fingerprint of it. Moreover, the stability of the phenolic profile within the species affords to identify a set of specialised metabolites useful for its chemotaxonomic characterization. At the same time, the stability of the biomolecular profile of P. casabonae, and the identification of sequences specific for this species, enables to identify useful biomolecular markers to distinguish it unequivocally., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

This paper focuses on several methodological aspects in the quantitation of volatiles in solid samples by headspace solid phase micro-extraction (HS-SPME) combined with gas chromatography and parallel detection by flame ionization detector and mass spectrometry (GC-FID/MS). Informative volatiles, including key odorants and process markers, from single-origin cocoa samples (Colombia, Ecuador, Mexico, Sao Tomè, and Venezuela) were captured at two processing stages along the chocolate production chain (nibs and cocoa mass). Accurate quantitation was achieved by multiple headspace extraction (MHE) in headspace linearity conditions and by external calibration. Quantitative results on selected analytes (3-hydroxy-2-butanone, 2-heptanol, 2,3,5-trimethylpyrazine, 2-ethyl-3,6-dimethylpyrazine, ethyl octanoate, benzaldehyde, 2-methylpropionic acid, 3-methylbutyric acid, ethyl phenylacetate, 2-phenylethyl acetate, guaiacol, 2-phenylethanol, and (E)-2-phenyl-2-butenal) provided reliable information about the key sensory notes of cocoa intermediates (odor activity values) and their origin specificities. Additional information about analytes release by the solid environment (cocoa nibs, mass, and powders) was achieved by modeling decay curves. Parallel detection by MS and FID enabled quantitative cross-validation, and FID-predicted relative response factors (RRFs) extended method quantitation capabilities to additional compounds that were not subjected to an external calibration procedure: 3-methylbutyl acetate (isoamyl acetate), 2-heptanone, heptanal, 2-nonanone, γ-butyrolactone, octanoic acid, 2-ethyl-5(6)-methylpyrazine, phenylacetic acid, phenol, 2-acetyl pyrrole, and 2,3-dihydro-3,5-dihydroxy-6-methyl(4H)-pyran-4-one. This procedure extends method capabilities and information potential with great consistency., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Nowadays, there is an increasing demand for fast and reliable plant biomolecular analyses. Conventional methods for the isolation of nucleic acids are time-consuming and require multiple and often non-automatable steps to remove cellular interferences, with consequence that sample preparation is the major bottleneck in the bioanalytical workflow. New opportunities have been created by the use of magnetic ionic liquids (MILs) thanks to their affinity for nucleic acids., Results: In the present study, a MIL-based magnet-assisted dispersive liquid-liquid microextraction (maDLLME) method was optimized for the extraction of genomic DNA from Arabidopsis thaliana (L.) Heynh leaves. MILs containing different metal centers were tested and the extraction method was optimized in terms of MIL volume and extraction time for purified DNA and crude lysates. The proposed approach yielded good extraction efficiency and is compatible with both quantitative analysis through fluorimetric-based detection and qualitative analysis as PCR amplification of multi and single locus genes. The protocol was successfully applied to a set of plant species and tissues., Conclusions: The developed MIL-based maDLLME approach exhibits good enrichment of nucleic acids for extraction of template suitable for targeted PCR; it is very fast, sustainable and potentially automatable thereby representing a powerful tool for screening plants rapidly using DNA-based methods.