Development of Gas-Chromatographic Method for Simultaneous Determination of Cannabinoids and Terpenes in Hemp.

An original gas chromatographic method has been developed for simultaneous determination of major terpenes and cannabinoids in plant samples and their extracts. The main issues to be addressed were the large differences in polarity and volatility between both groups of analytes, but also the need for an exhaustive decarboxylation of cannabinoid acidic forms. Sample preparation was minimised, also by avoiding any analyte derivatisation. Acetone was found to be the most appropriate extraction solvent. Successful chromatographic separation was achieved by using a medium polarity column. Limits of detection ranged from 120 to 260 ng/mL for terpenes and from 660 to 860 ng/mL for cannabinoids. Parallel testing proved the results for cannabinoids are comparable to those obtained from established HPLC methods. Despite very large differences in concentrations between both analyte groups, a linear range between 1 and 100 µg/mL for terpenes and between 10 and 1500 µg/mL for cannabinoids was determined.

Simplified LC-MS Method for Analysis of Sterols in Biological Samples.

We developed a simple and robust liquid chromatographic/mass spectrometric method (LC-MS) for the quantitative analysis of 10 sterols from the late part of cholesterol synthesis (zymosterol, dehydrolathosterol, 7-dehydrodesmosterol, desmosterol, zymostenol, lathosterol, FFMAS, TMAS, lanosterol, and dihydrolanosterol) from cultured human hepatocytes in a single chromatographic run using a pentafluorophenyl (PFP) stationary phase. The method also avails on a minimized sample preparation procedure in order to obtain a relatively high sample throughput. The method was validated on 10 sterol standards that were detected in a single chromatographic LC-MS run without derivatization. Our developed method can be used in research or clinical applications for disease-related detection of accumulated cholesterol intermediates. Disorders in the late part of cholesterol synthesis lead to severe malformation in human patients. The developed method enables a simple, sensitive, and fast quantification of sterols, without the need of extended knowledge of the LC-MS technique, and represents a new analytical tool in the rising field of cholesterolomics.

Synergistic complex from plants Solanaceae exhibits cytotoxicity for the human hepatocellular carcinoma cell line HepG2.

BACKGROUND: It had been demonstrated that sugars from various plants can act as potent agents, which induce apoptosis of cancer cells. METHODS: Using HPLC, we fractionated a mixture of two plant extracts from the plant family Solanaceae, namely Capsicum chinense and the plant family Amaryllidaceae namely Allium sativum. We evaluated the effect of different fractions on apoptosis of HepG2 cell line. The most effective fraction was further studied to determine its molecular composition using mass spectrometry (MS) and NMR. We further evaluated the effect of determined molecular composition found in the selected fraction by using a mixture of commercially available substances, which were found in the fraction and tested its pro-apoptotic effect on HepG2 cells. To get some insight into potential apoptotic mechanisms we studied caspase-3 activity and mitochondrial integrity in treated cells. RESULTS: Out of 93 fractions obtained by HPLC from the plant extract we found HPLC fraction 10 (10 min elution) was the most effective. MS and NMR studies revealed high presence of cellobiose together with vitamin C, sulphur (S) and trace amounts of selenium (Se). HPLC fraction 10 triggered apoptosis of HepG2 within 3 h in the 0.01-1.0 mg/mL concentration range. Furthermore, a mixture of pure cellobiose, vitamin C, S and Se (complex cellobiose/C/S/Se) had a very similar capacity in inducing apoptosis of HepG2 cells compared to HPLC fraction 10. Complex cellobiose/C/S/Se was capable of inducing caspase-3 activity and led to loss of mitochondrial integrity. The capacity of cellobiose alone to induce apoptosis of HepG2 was approximately 1000-fold lower compared to complex cellobiose/C/S/Se. CONCLUSION: In this study we present the highly synergistic effect of a unique complex consisting of cellobiose, vitamin C, sulphur and selenium on triggering the apoptosis of human hepatocellular carcinoma (HepG2) cell line.

Navigational Signals for Insect and Slug Parasitic Nematodes: The Role of Ascorbate-Glutathione System and Volatiles Released by Insect-Damaged Sweet Pepper Roots.

This study of underground multitrophic communication, involving plant roots, insects, and parasitic nematodes, is an emerging field with significant implications for understanding plant-insect-nematode interactions. Our research investigated the impact of wireworm (Agriotes lineatus L. [Coleoptera: Elateridae]) infestations on the ascorbate-glutathione system in sweet pepper (Capsicum annuum L.) plants in order to study the potential role in root-exudate-mediated nematode chemotaxis. We observed that an A. lineatus infestation led to a decrease in leaf ascorbate levels and an increase in root ascorbate, with corresponding increases in the glutathione content in both roots and leaves. Additionally, a pigment analysis revealed increased carotenoid and chlorophyll levels and a shift towards a de-epoxidized state in the xanthophyll cycle. These changes suggest an individual and integrated regulatory function of photosynthetic pigments accompanied with redox modifications of the ascorbate-glutathione system that enhance plant defense. We also noted changes in the root volatile organic compound (VOC). Limonene, methyl salicylate, and benzyl salicylate decreased, whereas hexanal, neoisopulegol, nonanal, phenylethyl alcohol, m-di-tert-butylbenzene, and trans-ß-ionone increased in the roots of attacked plants compared to the control group. Most notably, the VOC hexanal and amino acid exudate cysteine were tested for the chemotaxis assay. Nematode responses to chemoattractants were found to be species-specific, influenced by environmental conditions such as temperature. This study highlights the complexity of nematode chemotaxis and suggests that VOC-based biological control strategies must consider nematode foraging strategies and environmental factors. Future research should further explore these dynamics to optimize nematode management in agricultural systems.

Quantitative structure-mobility relationship analysis of imidazoline receptor ligands in CDs-mediated CE.

The performed quantitative structure-mobility relationship (QSMR) study has investigated relative migration times of 11 guanidine/imidazoline derivatives, imidazoline receptor ligands, in CE system containing one of CDs, α-, ß-, or γ-CD, using linear and nonlinear modeling methods. The analyzed ligands and their inclusion complexes with CDs were fully examined and optimized at semiempirical parametrized model 3 level. The density functional theory, such as B3LYP/6-31G+(d,p)/3-21G(d)/STO-3G(d,p)/STO-3G(d), and ab initio theory, such as HF/3-21G(d)/STO-3G(d), were applied for molecular descriptors computation of the optimized ligands and their complexes. Predictive performances of the developed QSMR models were tested by use of the cross-validation and external test set prediction. Obtained results for Q(2) values (0.869, 0.911, and 0.966 for CE system containing α-, ß-, and γ-CD, respectively) and root mean squared error of prediction (0.239, 0.242, and 0.288 for α-, ß-, and γ-CD, respectively) were proved high predictive power of the proposed models. Finally, multitarget QSMR model, using the ligands descriptors (X) and the relative migration time in presence of α-CD (Y1), ß-CD (Y2), and γ-CD (Y3), has been created. The multitarget QSMR model can be used as initial screening predictive tool for CE migration behavior of other related guanidine/imidazoline derivatives in presence of α-, ß-, and γ-CD.

The Role of Ascorbate-Glutathione System and Volatiles Emitted by Insect-Damaged Lettuce Roots as Navigation Signals for Insect and Slug Parasitic Nematodes.

The effect of wireworm-damaged lettuce roots on the antioxidative defense system (ascorbate-glutathione cycle, photosynthetic pigments) and movement of insect/slug parasitic nematodes towards determined root exudates was studied in a glasshouse experiment. Lettuce seedlings were grown in a substrate soil in the absence/presence of wireworms (Elateridae). The ascorbate-glutathione system and photosynthetic pigments were analyzed by HPLC, while volatile organic compounds (VOC) emitted by lettuce roots were investigated by GC-MS. Herbivore-induced root compounds, namely 2,4-nonadienal, glutathione, and ascorbic acid, were selected for a chemotaxis assay with nematodes Steinernema feltiae, S. carpocapsae, Heterorhabditis bacteriophora, Phasmarhabditis papillosa, and Oscheius myriophilus. Root pests had a negative effect on the content of photosynthetic pigments in the leaves of infested plants, indicating that they reacted to the presence of reactive oxygen species (ROS). Using lettuce as a model plant, we recognized the ascorbate-glutathione system as a redox hub in defense response against wireworms and analyzed its role in root-exudate-mediated chemotaxis of nematodes. Infected plants also demonstrated increased levels of volatile 2,4-nonadienal. Entomopathogenic nematodes (EPNs, S. feltiae, S. carpocapsae, and H. bacteriophora) proved to be more mobile than parasitic nematodes O. myriophilus and P. papillosa towards chemotaxis compounds. Among them, 2,4-nonadienal repelled all tested nematodes. Most exudates that are involved in belowground tritrophic interactions remain unknown, but an increasing effort is being made in this field of research. Understanding more of these complex interactions would not only allow a better understanding of the rhizosphere but could also offer ecologically sound alternatives in the pest management of agricultural systems.

Chemical and Genetic Variability of Istrian Foeniculum vulgare Wild Populations.

Wild Foeniculum vulgare populations from the region of Istria have been subjected to a genetic and chemical study. Headspace-gas chromatography analysis of volatile secondary metabolites and PCR-RFLP analysis of the ribosomal DNA internal transcribed spacer region has been chosen to analyze the chemical and genetic traits of single plants, respectively. Large intrapopulation and interpopulation differences have been observed in both chemical profiles and restriction patterns of PCR products. The data from chemical and genetic analyses were pooled and used to assess allele frequencies of three putative genetic loci on individual populations. The pooled allele frequencies were used to determine interpopulation distances for phenogram reconstruction. The combined use of chemical and genetic datasets for genetic variation analysis proved to be a more comprehensive approach for such a study, compared to the use of single datasets, even using such relatively simple analytical tools.

Cytosine Methylation in Genomic DNA and Characterization of DNA Methylases and Demethylases and Their Expression Profiles in Viroid-Infected Hop Plants (Humulus lupulus Var. 'Celeia').

Abiotic and biotic stresses can lead to changes in host DNA methylation, which in plants is also mediated by an RNA-directed DNA methylation mechanism. Infections with viroids have been shown to affect DNA methylation dynamics in different plant hosts. The aim of our research was to determine the content of 5-methylcytosine (5-mC) in genomic DNA at the whole genome level of hop plants (Humulus lupulus Var. 'Celeia') infected with different viroids and their combinations and to analyse the expression of the selected genes to improve our understanding of DNA methylation dynamics in plant-viroid systems. The adapted HPLC-UV method used proved to be suitable for this purpose, and thus we were able to estimate for the first time that the cytosine methylation level in viroid-free hop plants was 26.7%. Interestingly, the observed 5-mC level was the lowest in hop plants infected simultaneously with CBCVd, HLVd and HSVd (23.7%), whereas the highest level was observed in plants infected with HLVd (31.4%). In addition, we identified three DNA methylases and one DNA demethylase gene in the hop's draft genome. The RT-qPCR revealed upregulation of all newly identified genes in hop plants infected with all three viroids, while no altered expression was observed in any of the other hop plants tested, except for CBCVd-infected hop plants, in which one DNA methylase was also upregulated.

Overview of the development and application of the hyphenated techniques in nutritional analysis.

The development of some sensitive assays for quantitative nutritional analysis with an emphasis on selected hyphenated analytical techniques is reviewed in the present paper. The majority of work is dedicated to reviewing the development of analytical tools for routine analysis of carbohydrates and lipids in biological samples, many of them introduced in our laboratory. Handling biological matrices, where endogenous compounds can mask the analyte of interest or where the occurrence of the coelution effect of several compounds present in different amounts hinders the analyte's peak integration, is a major challenge. To overcome this challenge, hyphenated techniques have become widespread in laboratory practice. Some of these techniques are reviewed, with special attention given to an effective on-line interface for thin-layer chromatography-mass spectrometry and on-line coupling thin-layer chromatography-gas chromatography. Recently introduced an on-line coupling of ion chromatograph and hybrid RF/DC quadrupole-linear ion trap mass spectrometer represent an analytical tool for the solution of bioanalytical problems. Developed methods using ion chromatography-pulsed amperometric detection and ion chromatography-mass spectrometry techniques for the quantitative evaluation of sugars are presented. This paper represents basic contributions of our research work connected with some of modern hyphenated techniques. However, this review is restricted to the published papers to be significant developments or improvements during the last three decades.

Bioavailability of water-soluble CoQ10 in beagle dogs.

The bioavailability of a novel water-soluble inclusion complex of CoQ10, prepared in our laboratory was determined and compared with the bioavailability of commercially available oil-based form of CoQ10. Experimental work consisted of single dose comparative bioavailability study on seven beagle dogs, with a 14-day washout period between treatments. Identification and quantification of CoQ10 was done with HPLC-MS method using positive APCI ionization and SIM mode, M+ m/z 863.4. The bioavailability results confirm that the water-soluble formulation has nearly three times higher AUC(0-48 h), two times higher Cmax, and Tmax is shortened from 6 to 4 h.

Determination of derivatized amino acids in human embryo culture media by gas chromatography.

An adequate analytical method for determination of amino acids can provide a better insight in the metabolism of in vitro human embryo cultures, increasing the success rate of embryo implantation. Since individual amino acid amounts per embryo occur in the nanogram range, GC was the technique of choice, due to its inherent sensitivity and high sample throughput. Amino acids were analyzed as alkyl formate derivatives. The limits of detection (LOD) of all amino acids involved were in the sub-nmol range. The high risk of sample contamination proved to be the major analytical issue, but it could be overcome. For an extended method sensitivity, a simple preconcentration step could also be used.

Determination of phenolic compounds in fennel by HPLC and HPLC-MS using a monolithic reversed-phase column.

A reversed-phase high-performance liquid chromatography (HPLC) method for analyzing phenolic compounds in fennel (Foeniculum vulgare) has been developed. The use of a monolithic column with short dimensions in combination with optimized chromatographic conditions allows over 100 samples per day to be analyzed. Chromatographic parameters such as column temperature and injection volume, were found to be crucial in obtaining adequate selectivity and resolution, consequently allowing short run times. The method was validated for the major phenolic compounds present in fennel plant material: 3-O-caffeoylquinic acid (3-CQA), chlorogenic acid, 4-O-caffeoylquinic acid (4-CQA), eriocitrin, rutin, miquelianin, 1,3-O-dicaffeoylquinic acid (1,3-diCQA), 1,5-O-dicaffeoylquinic acid (1,5-diCQA), 1,4-O-dicaffeoylquinic acid (1,4-diCQA) and rosmarinic acid. The limits of detection (LOD) and the limits of quantitation (LOQ) ranged from 0.05 to 1.0 microg/mL and from 0.15 to 2.5 microg/mL, respectively. With some adaptation, the extraction procedure could be even less invasive, which is useful in screening work.

Evaluation of a rinsing-based cleaning process for pipes.

Pipes of various designs were constructed. Pipes were filled with a model solution resembling a dermal solution product. After the removal of the model solution, pipes were rinsed several times with ethanol and rinsing solutions of each step analyzed by gas chromatography. The results gave the information about the dependency between the configuration of the pipe and the efficiency of the cleaning operation. From concentrations measured in the reactor, expected concentrations in rinsing solutions from pipes were predicted. The obtained results confirm that the amount of residues per surface area increases when a pipe includes bends and valves. In terms of extra contamination, each bend was equal to 25 cm, while each valve was equal to 100 cm of pipe length when pipes of 1.8 cm in diameter were used. It was proven that the contributions of individual valves and bends in the pipe are additive in the calculation. The validity of the proposed model was confirmed by experimental data.

Comprehensive thin layer chromatography×gas chromatography using headspace sampling modulation-A case study on fatty acid composition analysis.

The recently developed comprehensive TLC×GC technique using headspace sampling is presented. The main advantage of this approach, as demonstrated in lipid analysis, is the possibility to include a transesterification step of glycerides into fatty acid methyl ester derivatives (FAME) because no particular constraints in terms of operational time between TLC and GC are present. Besides being a relatively low-cost solution, TLC×GC by means of headspace sampling provides many benefits in terms of flexibility of separation conditions and modulation sampling width. The technique provides over two orders of magnitude of linear range with TLC sample loads of about 1mg with good reproducibility and accuracy, as demonstrated by multiple headspace extraction (MHE) tests. The technique is a viable alternative to the established but more expensive HPLC×GC technique. The useful range of TLC×GC in terms of analyte volatility can be further extended with a future development of devices based on thermal desorption.

Direct analysis of carbohydrates in animal plasma by ion chromatography coupled with mass spectrometry and pulsed amperometric detection for use as a non-invasive diagnostic tool.

The present paper demonstrates that electrochemical detection (ECD) coupled to ion chromatography and electrospray ionization tandem mass spectrometry (IC-ECD-ESI/MS/MS) can be used to rapidly estimate some indications of the health status of organisms. The lactulose to mannitol ratio (L/M) is used as a non-invasive assay to investigate small intestinal absorption pathways and mucosal integrity. In the present study, an evaluation of the negative effects of nonsteroidal anti-inflammatory drug meloxicam perorally administrated to a group of dogs was carried out by determining the lactulose/mannitol index using the IC-ECD-ESI/MS/MS hyphenated technique. According to the results of the study, meloxicam altered gastrointestinal permeability. Coenzyme Q(10) (CoQ(10)) was tested to determine if it could prevent meloxicam induced gastrointestinal damage and it was found that CoQ(10) could be an effective preventive treatment. Furthermore, plasma glucose concentration level was determined to be an indirect indicator of the oxidative state in the blood. To find out the beneficial effects of a double antioxidant combination (α-lipoic acid (ALA) and CoQ(10)) on the total glucose level in chickens, ALA and CoQ(10) were provided as food additives in factory farm raised chicken. The results of the pilot study indicate that the glucose level in the plasma of chickens group fed with CoQ(10) and ALA was significantly decreased compared to the control group. Ion chromatography (IC) utilizing pulsed amperometric detection (PAD) was compared to ion chromatography coupled with tandem mass spectrometry (MS/MS) as an analytical tool for monitoring the carbohydrate level in biological fluids. In electrochemical detection, the newly developed two-pulse waveform successfully withstands matrix effects in biological samples. Continuous on-line desalting of the high salt concentrations used as the eluent for carbohydrate separation from the anion-exchange column allows coupling of IC and MS techniques. A make-up solution (0.5mM LiCl) was delivered prior to MS detection for efficient ionization of eluted carbohydrates. Method validation showed that both used techniques are practically comparable and some advantages of each are presented.