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%.

Elettaria cardamomum (L.) Maton Essential Oil: An Interesting Source of Bioactive Specialized Metabolites as Inhibitors of Acetylcholinesterase and Butyrylcholinesterase.

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, IC50 24.9 µg/mL) and BChE (55.8% of inhibition, IC50 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.

Promising inhibition of diabetes-related enzymes and antioxidant properties of Ptilostemon casabonae leaves extract.

Type 2 diabetes (T2D) is a progressive metabolic disorder of glucose metabolism. One of the therapeutic approaches for the treatment of T2D is reducing postprandial hyperglycaemia through inhibition of the digestive enzymes α-glucosidase and α-amylase. In this context, aimed at identifying natural products endowed with anti-T2D potential, we focused on Ptilostemon casabonae (L.) Greuter, a species belonging to Asteraceae family. Enzymatic inhibition, antioxidant activity, phenolic composition and cellular assays were performed. This study revealed that the P. casabonae hydroalcoholic extract exerts a potent inhibitory activity against α-glucosidase. This activity is supported by an antioxidant effect, preventing ROS formation in a stressed cellular system. HPLC-PDA-MS/MS analysis, revealed a complex polyphenolic fraction. Among the tested pure compounds, 1,5-dicaffeoylquinic acid, apigenin and rutin displayed good α-glucosidase inhibitory activity. Our study suggested new potential of P. casabonae encouraging us to further testing the possible therapeutic potential of this extract.

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.

Free Amino Acids and Biogenic Amines Profiling and Variation in Wild and Sub-Endemic Cardueae Species from Sardinia and Corse.

The cardueae are a common species in the Mediterranean area where they grow spontaneously and are traditionally employed as food and for health purposes. In this work, five Cardueae, including two sub-endemic species (four Carduus and three Ptilostemon casabonae (L.) Greuter samples from different locations) were collected from Sardinia and the Corse islands. All the considered plants are characteristic of the area, in particular the sub-endemic species C. cephalanthus and P. casabonae. This work aims to obtain, for the first time, the amino compounds profile (primary metabolites) of these little-studied species to detect for any similarities and differences among the different samples using statistical analyses. A recently developed method was employed, where diethyl ethoxymethylenemalonate (DEEMM) derivatives are detected in a neutral loss scan mode using high performance liquid chromatography in tandem with a mass spectrometry technique. In total, 42 amino compounds were detected, of which 33 were fully identified and semi-quantified. Overall, the results show that DEEMM-derivatized amino compounds are qualitatively similar among the considered samples. Nonetheless, a discrimination at the genus level is possible. This work adds more information regarding the phytochemical composition regarding the primary metabolites of the considered samples, their discriminations and the search for compounds with potential health benefits.

Characterization and Biological Activity of Fiber-Type Cannabis sativa L. Aerial Parts at Different Growth Stages.

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.

Immobilization of phosphonium-based ionic liquid stationary phases extends their operative range to routine applications in the flavor, fragrance and natural product fields.

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 ([P66614+] [Cl-]), and trihexyl(tetradecyl)phosphonium bis[(trifluoromethyl)sulfonyl]imide ([P66614+][NTf2-]), 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 [P66614+][Cl-] IL and 180 °C for [P66614+][NTf2-]), 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 [P66614+][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 [P66614+][Cl-] and [P66614+][NTf2-] 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 [P66614+][Cl-] IL columns, and 200 °C for So-Im and 220° for Ha-Im in columns coated with the [P66614+] [NTf2-] 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.

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.

In Vitro Anti-HIV-1 Reverse Transcriptase and Integrase Properties of Punica granatum L. Leaves, Bark, and Peel Extracts and Their Main Compounds.

In a search for natural compounds with anti-HIV-1 activity, we studied the effect of the ethanolic extract obtained from leaves, bark, and peels of Punica granatum L. for the inhibition of the HIV-1 reverse transcriptase (RT)-associated ribonuclease H (RNase H) and integrase (IN) LEDGF-dependent activities. The chemical analyses led to the detection of compounds belonging mainly to the phenolic and flavonoid chemical classes. Ellagic acid, flavones, and triterpenoid molecules were identified in leaves. The bark and peels were characterized by the presence of hydrolyzable tannins, such as punicalins and punicalagins, together with ellagic acid. Among the isolated compounds, the hydrolyzable tannins and ellagic acid showed a very high inhibition (IC50 values ranging from 0.12 to 1.4 µM and 0.065 to 0.09 µM of the RNase H and IN activities, respectively). Of the flavonoids, luteolin and apigenin were found to be able to inhibit RNase H and IN functions (IC50 values in the 3.7-22 µM range), whereas luteolin 7-O-glucoside showed selective activity for HIV-1 IN. In contrast, betulinic acid, ursolic acid, and oleanolic acid were selective for the HIV-1 RNase H activity. Our results strongly support the potential of non-edible P. granatum organs as a valuable source of anti-HIV-1 compounds.

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.

Derivatization-targeted analysis of amino compounds in plant extracts in neutral loss acquisition mode by liquid chromatography-tandem mass spectrometry.

Amino compounds, such as amino acids and biogenic amines, are important metabolites that can be found in diverse natural matrices. The most common method for amino compound analysis nowadays is reversed-phase liquid chromatography tandem mass spectrometry (RPLC-MS/MS). However, due to the polar and the basic nature of amines, their RPLC retention is often insufficient or peaks are tailing. Derivatization is a way to overcome the issue and in the present work amino compounds are derivatized with diethyl ethoxymethylenemalonate (DEEMM) and analyzed by a RPLC triple quadrupole MS system in neutral loss scan (NLS) mode (loss of 46). This allows to target all compounds in the sample that undergo derivatization with DEEMM, so that the amino compound profile of the sample is obtained. To the best of our knowledge, the NLS acquisition mode has never been employed to target amino compounds after DEEMM derivatization. In the first part of the study, eight amino acids (arginine, aspartic acid, threonine, proline, tyrosine, tryptophan, phenylalanine and isoleucine) were employed as model compounds for method optimization, with good results in terms of DEEMM derivatives detection and repeatability. The developed method was successfully applied to a complex extract from the plant species Carduus nutans subsp. macrocephalus (Desf.) Nyman, with 18 amino acids and 3 other amines being identified. The proposed approach could be employed for straightforward identification of known and unknown amino compounds in different types of matrices.

Citral-Containing Essential Oils as Potential Tyrosinase Inhibitors: A Bio-Guided Fractionation Approach.

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 ß-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.

Wild Italian Hyssopus officinalis subsp. aristatus (Godr.) Nyman: From Morphological and Phytochemical Evidences to Biological Activities.

Three specimens of H. officinalis subsp. aristatus were collected in three areas of the Abruzzo region (Italy) and subjected to macroscopic and microscopic observation to support their botanical identification. The essential oils (EOs) obtained from the aerial parts of the samples were characterized with the object to define their phytochemical and pharmaceutical biology profile. They highlight three different chemotypes, including one never seen in previous literature (CIV17-EO, distilled from sample harvested in 2017 at Civitaretenga), that showed a fingerprinting with the predominance of (-)-limonen-10-yl-acetate (67.9%). In 2017 European Food Safety Authority (EFSA) reported the genotoxicity of similar compounds, therefore, to dismiss any safety concern for the CIV17-EO use as flavouring substance, the Ames test was performed with no evidence of mutagenic activity. Safety of use coupled with chemical characterization of this new chemotype set the stage for a better standardization of H. officinalis EOs. The ethanolic extracts, on the other hand, with qualitatively similar chemical profiles in which caftaric, chlorogenic and rosmarinic acid were the main molecules, showed interesting antioxidant activity and a slight cytotoxicity towards the A549 cell line that could indicate a starting point for the evaluation of an additional preventive tool for maintaining health status.

Analytical strategies for in-vivo evaluation of plant volatile emissions - A review.

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.

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.

Bio-Guided Fractionation Driven by In Vitro α-Amylase Inhibition Assays of Essential Oils Bearing Specialized Metabolites with Potential Hypoglycemic Activity.

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 Myristicafragrans 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 ß-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.

Evaluation of the Farming Potential of Echinacea Angustifolia DC. Accessions Grown in Italy by Root-Marker Compound Content and Morphological Trait Analyses.

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.

Identification of a new R3 MYB type repressor and functional characterization of the members of the MBW transcriptional complex involved in anthocyanin biosynthesis in eggplant (S. melongena L.).

Here we focus on the highly conserved MYB-bHLH-WD repeat (MBW) transcriptional complex model in eggplant, which is pivotal in the transcriptional regulation of the anthocyanin biosynthetic pathway. Through a genome-wide approach performed on the recently released Eggplant Genome (cv. 67/3) previously identified, and reconfirmed by us, members belonging to the MBW complex (SmelANT1, SmelAN2, SmelJAF13, SmelAN1) were functionally characterized. Furthermore, a regulatory R3 MYB type repressor (SmelMYBL1), never reported before, was identified and characterized as well. Through a qPCR approach, we revealed specific transcriptional patterns of candidate genes in different plant tissue/organs at two stages of fruit development. Two strategies were adopted for investigating the interactions of bHLH partners (SmelAN1, SmelJAF13) with MYB counterparts (SmelANT1, SmelAN2 and SmelMYBL1): Yeast Two Hybrid (Y2H) and Bimolecular Fluorescent Complementation (BiFC) in A. thaliana mesophylls protoplast. Agro-infiltration experiments highlighted that N. benthamiana leaves transiently expressing SmelANT1 and SmelAN2 showed an anthocyanin-pigmented phenotype, while their co-expression with SmelMYBL1 prevented anthocyanin accumulation. Our results suggest that SmelMYBL1 may inhibits the MBW complex via the competition with MYB activators for bHLH binding site, although this hypothesis requires further elucidation.

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.

Can the selectivity of phosphonium based ionic liquids be exploited as stationary phase for routine gas chromatography? A case study: The use of trihexyl(tetradecyl) phosphonium chloride in the flavor, fragrance and natural product fields.

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, [P66614+] [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 [P66614+][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 [P66614+][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 [P66614+][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.