Cannabis through the looking glass: chemo- and enantio-selective separation of phytocannabinoids by enantioselective ultra high performance supercritical fluid chromatography

By using the Inverted Chirality Columns Approach (ICCA) we have developed an enantioselective UHPSFC method to determine the enantiomeric excess (ee) of (-)-Δ(9)-THC in medicinal marijuana (Bedrocan®). The ee was high (99.73%), but the concentration of the (+)-enantiomer (0.135%) was not negligible, and it is worth a systematic evaluation of bioactivity

INVESTIGATION OF DIFFERENT EXTRACTION METHODS FOR HEMP SEEDS

Industrial hemp hearts were subjected to four different laboratory (research) scale solvent extraction techniques including soxhlet extraction, microwave pretreated soxhlet extraction, ultrasound extraction, and microwave extraction as well as extraction by non-solvent mechanical cold pressing. The maximum extraction yield of 54 % (w/w) was observed for both microwave as well as microwave pretreated soxhlet extracted seeds and the minimum run time of 30 minutes were noted for both ultrasound extraction and microwave extraction. The ratio of Linoleic acid to Linolenic acid was found to be close to 3:1 which is considered to be optimal for nutrition. Also, the ratio of unsaturated fatty acids to saturated fatty acids was in the range of 6.7 to 9.1. Several minor compounds like ɣ-Sitosterol, stigmasterol, rhodoxanthin, carotene and methyl cholate were identified in the oils which have not been previously reported. Antioxidants β-Carotene and ɣ-Tocopherol have also been identified in the oil sample. Moreover, the acid value, iodine value and oxidative stability of the oils indicated that the oil was of an improved quality and that the level of unsaturation was very high. The poly unsaturated fatty acids (PUFA) found in the oils have numerous health benefits, the most important being improvement of cardiovascular health. A perfect combination of unsaturated fatty acids and saturated fatty acids and an appropriate medley of different antioxidants not only qualifies hemp seed oil as an excellent source of nutrition, but also as an important plant based (vegetable) oil for the higher value nutraceutical and pharmaceutical industries

Comprehensive chromatographic profiling of cannabis from 23 USA States marketed for medical purposes

In this research, cannabis varieties represent 23 USA States were assayed by GC-FID to generate their complex chemical profiles informative for plants clustering. Results showed that 45 cannabinoids and terpenoids were quantified in all plant samples, where 8 cannabinoids and 18 terpenoids were identified. Among organics, Δ9-THC, CBN (cannabinoids) and Fenchol (terpenoid) not only showed the highest levels overall contents, but also were the most important compounds for cannabis clustering. Among States, Washington, Oregon, California and Hawaii have the highest cannabis content. GC-FID data were subjected to PCA and HCA to find (1) the variations among cannabis chemical profiles as a result of growing environment, (2) to reveal the compounds that were responsible for grouping cultivars between clusters and (3) finally, to facilitate the future profile prediction and States clustering of unknown cannabis based on the chemical profile. The 23 cannabis USA States were grouped into three clusters based on only Δ9-THC, CBN, C1 and Fenchol content. Cannabis classification based on GC-profile will meet the practical needs of cannabis applications in clinical research, industrial production, patients’ self-production, and contribute to the standardization of commercially-available cannabis cultivars in USA

Estudio teórico y aplicado del potencial de la espectrometría de movilidad iónica

Ion mobility spectrometry (IMS) is an analytical technique based on the separation of gaseous ions under the influence of an electric field through an inert gas atmosphere. Some of the main limitations of IMS, depending on the context, may be the limited quantification capacity of compounds in real samples since narrow linear quantification ranges are normally obtained; the low selectivity due to the low resolution power of this type of equipment; and the difficulty of unequivocally identifying compounds in real samples since the existing databases are not as up-to-date as for other technologies such as mass spectrometry (MS). Therefore, it is evident that there is a demand for more selective methodologies and that provide greater analyte detection and quantification capacity. With these premises, it can be said that the greatest current challenge of the IMS is to maximize the detection capacity of the technique in order to achieve the unambiguous identification of a high number of analytes. This challenge is currently utopian when working with complex samples. For this reason, the main motivation of this Doctoral Thesis was to seek solutions for the different challenges that the IMS currently faces in a theoretical and applied context. The basic objective of the research was to explore the potential of IMS by using theoretical and applied strategies to improve the detection and identification coverage of the analysis carried out with this technology. These new strategies were applied throughout the main steps of the analytical process and allowed improving basic analytical features such as the selectivity and sensitivity of optimized analysis methods and their detection capacity. The achievement of this basic objective leaded to analysis methods of standards and real samples, such as explosives, drugs, soil, rosemary plant, olives and mainly different types of olive oils. This basic objective was divided into three general objectives according to the different research topics to address in this Doctoral Thesis: a) To take benefits derived from the study of theoretical aspects of IMS for improving the interpretation of IMS spectra and from the use of additional features such as structural information to enhance qualitative analysis; b) To develop approaches to improve the detection and identification capacity in IMS analysis; and c) To exploit the opportunities of gas chromatography (GC)-IMS and IMS devices for food analysis as an expanding application area in IMS based on untargeted analysis methods. In this context, the Thesis has included the following studies: (i) To study about the fundamentals of the formation of product ions through the modeling of ions stability using ab initio computations to math these results with the spectral patterns and structure of ions [1]. (ii) To explore the fragmentation of ions using an external electric field and the potential of the extra information of these fragments to enhance the rates of categorization by chemical class using neural networks [2]. (iii) To explore a thermal desorption (TD)-IMS device to obtain spectral fingerprints of Cannabis herbal samples, with and without pretreatment for rapid assignment to their different chemotypes by using principal component análisis (PCA) and linear discriminant analysis (LDA) [3]. (iv) To achieve the selectivity in response to trinitrotoluene (TNT) through reactive removal of interfering ions following mobility isolation using a tandem IMS with reactive stage as detection system [4]. (v) To develop a pioneer online coupling of supercritical fluid extraction (SFE) as sample introduction system (SIS) prior IMS using a column filled with Tenax TA material as sorbent trap to coupled both devices to improve analytical properties such as sensitivity and selectivity of future IMS methods [5]. (vi) To carry out a bibliographical study which gather and critically discuss recent publications related to analytical techniques to distinguish olive oils according to their quality as extra virgin (EVOO), virgin (VOO) or lampante (LOO) [6]. (vii) To investigate and compare different chemometric approaches for olive oil classification as EVOO, VOO or LOO using GC-IMS to get the most robust model over time [7]. (viii) To evaluate the combination of the results of orthogonal instrumental techniques to differentiate EVOO, VOO or LOO to imitate the expert panels [8]. (ix) To analyze olive and olive oil samples according with their production system to classify them as organic or conventional using ultraviolet (UV)-IMS, GC-IMS, GC-MS and/or capillary electrophoresis (CE)-UV [9].La espectrometría de movilidad iónica (IMS en inglés) es una técnica analítica que se basa en la separación de iones gaseosos bajo la influencia de un campo eléctrico a través de una atmósfera de gas inerte. Algunas de las principales limitaciones de la IMS, dependiendo del contexto, pueden ser la limitada capacidad de cuantificación de compuestos en muestras reales ya que se obtienen normalmente rangos lineales de cuantificación muy estrechos; la escasa selectividad debido al bajo poder de resolución de este tipo de equipos; y la dificultad de identificación de forma inequívoca de compuestos en muestras reales ya que las bases de datos existentes no están tan actualizadas como para otras tecnologías como la espectrometría de masas (MS en inglés). Por tanto, resulta evidente que existe una demanda de metodologías más selectivas y que proporcionen mayor capacidad de detección y cuantificación de analitos. Con estas premisas, se puede decir que el mayor reto actual de la IMS es maximizar la capacidad de detección de la técnica con el fin de conseguir la identificación inequívoca de un alto número de analitos. Este reto es actualmente utópico cuando se trabaja con muestras complejas. Por ello, la principal motivación de esta Tesis Doctoral fue buscar soluciones para los distintos retos a los que se enfrenta actualmente la IMS en un contexto teórico y aplicado. El objetivo básico de la investigación fue explorar el potencial de la IMS mediante el uso de estrategias teóricas y aplicadas para mejorar la capacidad de detección e identificación de los análisis realizados con esta tecnología. Estas nuevas estrategias se aplicaron a lo largo de las etapas principales del proceso analítico y permitieron mejorar características analíticas básicas, como la selectividad y la sensibilidad, de los métodos de análisis optimizados y su capacidad de detección. El logro de este objetivo básico condujo a métodos de análisis de estándares y muestras reales, como explosivos, drogas, suelo, plantas de romero, aceitunas y principalmente diferentes tipos de aceites de oliva. Este objetivo básico se dividió en tres objetivos generales de acuerdo con los diferentes temas de investigación para abordar en esta Tesis Doctoral: a) aprovechar los beneficios derivados del estudio de los aspectos teóricos de la IMS para mejorar la interpretación de los espectros de IMS y del uso de características adicionales como información estructural para mejorar el análisis cualitativo; b) desarrollar herramientas para mejorar la capacidad de detección e identificación en los análisis de IMS; y c) aprovechar las oportunidades de los instrumentos de cromatografía de gases (GC en inglés)-IMS e IMS para el análisis de alimentos como un área de aplicación en expansión en IMS basado en métodos de análisis no dirigidos. En este contexto, la Tesis ha incluido los siguientes estudios: (i) Estudiar los fundamentos de la formación de iones producto a través del modelado computacional de la estabilidad de los iones utilizando cálculos ab initio para combinarlos con los patrones espectrales y la estructura de los iones [1]. (ii) Explorar la fragmentación de iones utilizando un campo eléctrico externo y el potencial de la información adicional de estos fragmentos para mejorar las tasas de categorización por clase química utilizando redes neuronales [2]. (iii) Explorar un equipo de desorción térmica (TD en inglés)-IMS para obtener huellas espectrales de muestras de plantas de cannabis, con y sin pretratamiento, para la rápida asignación de los diferentes quimiotipos mediante análisis de componentes principales (PCA en inglés) y análisis discriminante lineal (LDA en inglés) [3]. (iv) Lograr la respuesta selectiva del trinitrotolueno (TNT en inglés) a través de la eliminación con etapa reactiva de iones interferentes usando el aislamiento de iones con un IMS en tándem con etapa reactiva como sistema de detección [4]. (v) Desarrollar un acoplamiento on-line pionero de la extracción con fluidos supercríticos (SFE en inglés) como sistema de introducción de muestra previo a la IMS utilizando una columna rellena con el material Tenax TA como trampa sorbente para acoplar ambos dispositivos para mejorar propiedades analíticas como la sensibilidad y la selectividad de futuros métodos IMS [5]. (vi) Realizar un estudio bibliográfico que reúna y discuta críticamente las publicaciones recientes relacionadas con técnicas analíticas para distinguir los aceites de oliva según su calidad como virgen extra (AOVE), virgen (AOV) o lampante (AOL) [6]. (vii) Investigar y comparar diferentes estrategias quimiométricas para la clasificación del aceite de oliva como AOVE, AOV o AOL utilizando la GC-IMS para obtener el modelo más robusto con el tiempo [7]. (viii) Evaluar la combinación de los resultados de técnicas instrumentales ortogonales para diferenciar AOVE, AOV o AOL para imitar los paneles de expertos [8]. (ix) Analizar muestras de aceitunas y aceite de oliva de acuerdo con su sistema de producción para clasificarlas como ecológicas o convencionales usando ultravioleta (UV)-IMS, GC-IMS, GC-MS y/o electroforesis capilar (CE en inglés)- UV [9]

A Comparison of Antioxidant Potential, Total Phenolic Content, and Cannabidiol (CBD) Content of Cannabis Infused Hemp, MCT, and Olive Oils

The Cannabis industry has seen immense growth in recent years and research on this plant and its constituents has been growing to keep up with industry demand. The majority of research has focused on commercial-scale products and industrial processing, but there is a lack of research on the smaller scale manufacturing side of the Cannabis industry that includes homemade Cannabis products. Popular Cannabis products are oil-based tinctures that are made by infusing Cannabis plant material in a heated source of edible oil. The types of oils used for this process vary, and there is not an established standardized oil type that has been shown to be the optimal choice for reaping the most benefits from Cannabis infusion. The goal of infusing Cannabis in oil is to extract the desirable potentially neurologically active cannabinoid plant molecules that also serve as antioxidants, specifically cannabidiol (CBD). To determine the effect of oil type on extraction ability of Cannabis, different oil types were used to infuse a high-CBD strain of Cannabis and measure antioxidant potential, total phenolic content, and CBD content of the resulting oils. Hemp oil, MCT oil, and olive oil were used as infusion solvents for the ground decarboxylated Cannabis flowers. Consistency in the protocol was followed for the strain of Cannabis, decarboxylation process, grinding process, heated infusion process, and storage conditions. Additionally, control standards were established by implementing the heating process for the oils without Cannabis infusion. Antioxidant potential was assessed using Trolox Equivalent Antioxidant Capacity (TEAC) assay, and total phenolic content was assessed using Gallic Acid Equivalence (GAE) assay. CBD content of the CBD oils was assessed using high-performance liquid chromatography with ultraviolet detection (HPLC-UV). For antioxidant potential, hemp CBD oil had the greatest antioxidant potential, but the other CBD oils had a significant increase in antioxidant potential compared to their control oils whereas hemp CBD oil did not. For total phenolic content, olive CBD oil had the highest total phenolic content. For CBD content, hemp CBD oil and olive CBD oil had the highest CBD content

Supercritical CO2 extraction of vegetable oils: determination of fatty acids concentration in sea buckthorn oil

[EN] The main focus of this project is the use supercritical CO2 extraction. The project is entirely focused on it in order to determine if scCO2 can be a proper replacement for soxhlet extraction . This extraction method has actually been used at industrial level but its scale can be much wider in the future, so it is the spotlight of further researches. As CO2 is not toxic and its use in supercritical fluid extraction leaves almost no waste, its application in a large range is a total environmental and economic revolution, so it could turn to be the most widespread process in food industry. For achieving the objective set, a comparison between the compositions of soxhlet extracts and scCO2 extracts has been done. For the determination of free fatty acid composition in each extract FAME(Fatty Acid Methyl Esters) method has been carried out, and the total lipid composition has been analysed by gas chromatography using a flame ionization detector (GC-FID)

Cannabinoids Enriched Extracts from Industrial Hemp Residues

Obtaining phytocannabinoids, associated with various medicinal and therapeutic properties with no reported side effects, is one of the hot topics. The phychotropic Δ9-tetrahydrocannabinol (THC) is less than 0.2 % in industrial cultivars therefore can be grown legally in many EU countries. Harvesting and processing of hemp for fiber or seeds generates large amount of wastes containing substantial amounts of bioactives such as cannabidiol (CBD) which are the primary cannabinoids along with cannabidiolic acid (CBDA), cannabinol (CBN), cannabigerol (CBG), and cannabichromene (CBC). The aim of this work was to optimize the extraction of cannabinoids from industrial hemp threshing residue using supercritical carbon dioxide extraction in pilot scales. The effects of extraction pressure and temperature on the extraction yield were evaluated. Three ground and pelleted samples of the same type but with different harvesting time were also compared. After derivatization of the samples the cannabinoids and the minor THCs were quantified by GC-MS. The extraction yields were between 0.2 – 6.59 g/100 g dry mass depending on the source of hemp residue and on the process parameters of the extraction process. By increasing the pressure of extraction (in the range of 25-45 MPa at 45 °C) the extraction yields increased, meanwhile the yields of cannabinoids showed no significant increase. The volatile compounds were successfully separated from the cannabinoids with fractionated separation. From hemp threshing residues essential oil free extracts with high content of cannabinoids were obtained at 35 MPa extraction pressure and 45 °C temperature setting the first separator at 8 MPa and 40 °C.&nbsp