An active derivatization detection method for inline monitoring the isolation of carbohydrates by preparative liquid chromatography.

Polysaccharides have unique physio-chemical properties and various biological functions and have rapidly expanded interest over the last two decades. The purification of polysaccharides and their degraded oligosaccharides is challenging because carbohydrates have no chromophore and need a proper detector to monitor the chromatographic elution process. This study proposed an active derivatization detection (ADD) method based on active splitting from post-column flow, a microchannel reactor for efficient derivatization of polysaccharide reducing sugars with p-hydroxybenzoic acid hydrazide, and in-line detection by the UV detector of liquid chromatography system. The method and device were validated by the use of 11 monosaccharides, sulfated oligosaccharides (from degraded carrageenan), and polysaccharides (from Zizania latifolia). It has shown much better performance than the traditional phenol-sulfuric acid method (gold standard). Moreover, the ADD module presumes an add-in to the original preparative LC system, independent of the scale of the purification process and type of system. The developed method is versatile for chromatographic separation of carbohydrates and lays the foundation for their subsequent studies.

Facile preparation of embedded polar group-containing pentafluorophenyl stationary phases for highly selective separations of diverse analytes.

Pentafluorophenyl (PFP) stationary phase is one of the most important phases after the C18 phase in terms of its applications. Three embedded polar groups (EPG)-containing stationary phases were newly synthesized to act the EPGs as additional interaction sites. The silica surface was initially modified with (3-aminopropyl)trimethoxysilane (APS). The APS-modified silicas were coupled with 2,3,4,5,6-pentafluorobenzoic acid, 2,3,4,5,6-pentafluorophenylacetic acid, and 2,3,4,5,6-pentafluoro-anilino(oxo)acetic acid to obtain Sil-PFP-BA, Sil-PFP-AA, and Sil-PFP-AN phases, respectively. The new phases were characterized by elemental analysis, ATR-FTIR, scanning electron microscopy (SEM), and thermogravimetric analysis (TGA). The phases were evaluated with the Tanaka and Neue tests in reversed-phase liquid chromatography (RPLC). In addition, they were characterized as hydrophilic phases by the Tanaka test protocol used in hydrophilic interaction chromatography (HILIC) separation mode. The Sil-PFP-AA phase showed the highest molecular shape selectivity in RPLC, while Sil-PFP-AN achieved the highest separability in HILIC compared to the commercial PFP reference column. The Sil-PFP-AA phase was successfully applied for the analysis of capsaicinoids from real samples of fresh chili peppers (Capsicum spp.) in RPLC and the Sil-PFP-AN phase for vitamin C (ascorbic acid) in HILIC.

Loss of Pip4k2c confers liver-metastatic organotropism through insulin-dependent PI3K-AKT pathway activation.

Liver metastasis (LM) confers poor survival and therapy resistance across cancer types, but the mechanisms of liver-metastatic organotropism remain unknown. Here, through in vivo CRISPR-Cas9 screens, we found that Pip4k2c loss conferred LM but had no impact on lung metastasis or primary tumor growth. Pip4k2c-deficient cells were hypersensitized to insulin-mediated PI3K/AKT signaling and exploited the insulin-rich liver milieu for organ-specific metastasis. We observed concordant changes in PIP4K2C expression and distinct metabolic changes in 3,511 patient melanomas, including primary tumors, LMs and lung metastases. We found that systemic PI3K inhibition exacerbated LM burden in mice injected with Pip4k2c-deficient cancer cells through host-mediated increase in hepatic insulin levels; however, this circuit could be broken by concurrent administration of an SGLT2 inhibitor or feeding of a ketogenic diet. Thus, this work demonstrates a rare example of metastatic organotropism through co-optation of physiological metabolic cues and proposes therapeutic avenues to counteract these mechanisms.

Surface Modification of Silica with ß-Alanine Derivatives for Unique Applications in Liquid Chromatography.

Column purchasing cost is an important issue for an analyst to analyze complex sample matrices. Here, we report the development of an amino acid (ß-alanine)-derived stationary phase (Sil-Ala-C12) with strategic and effective interaction sites (amide and urea as embedded polar groups with C12 alkyl chain) able to separate various kinds of analytes. Owing to the balanced hydrophobicity and hydrophilicity of the phase, it showed exceptional separation abilities in both reversed-phase high-performance liquid chromatography (RP-HPLC) as a hydrophobic phase and hydrophilic interaction chromatography (HILIC) as a hydrophilic phase. Remarkably, the baseline separation was achieved for the challenging ß- and γ-isomers of tocopherol. Usually, three columns such as pentafluorophenyl or C30, C18, and sulfobetaine HILIC are required for the analysis of vitamin E, capsaicinoids, and vitamin C in chili peppers (Capsicum spp.), respectively. However, only Sil-Ala-C12 was able to separate these analytes. A single column can serve 3-4 purposes, which suggests that Sil-Ala-C12 had the potential to reduce column purchasing costs.

Design of ß-Alanine-Derived Novel C18 Stationary Phase Containing Embedded Urea and Amide Groups for the Separation of Versatile Analytes.

Novel stationary phases have been emerging recently. A ß-alanine-derived embedded urea and amide group-containing C18 phase (Sil-Ala-C18) was prepared for the first time. The media were packed into a 150 × 2.1 mm HPLC column, and the newly designed column was evaluated with the Tanaka and Neue test protocols in reversed-phase liquid chromatography (RPLC) separation mode. Moreover, it was characterized by the Tanaka test protocol in hydrophilic interaction chromatography (HILIC) separation mode. The new phase was characterized by elemental analysis, attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and solid-state 13C cross-polarization magic angle spinning (CP/MAS) NMR spectroscopy at variable temperatures. The chromatographic evaluation involved very good separation of nonpolar shape-constrained isomers, polar and basic compounds in RPLC, and highly polar compounds in HILIC compared to the commercial reference columns. The Sil-Ala-C18 phase was able to separate the challenging ß- and γ-isomers of tocopherol. The phase was also successfully applied for the separation of the isomers of tocopherol (vitamin E) and capsaicinoids from real samples of chili peppers (Capsicum spp.) in RPLC and ascorbic acid (vitamin C) in HILIC.

Comprehensive two-dimensional gas chromatography with flow modulator coupled via tube plasma ionization to an atmospheric pressure high-resolution mass spectrometer for the analysis of vermouth volatile profile.

The analysis of complex samples is a big analytical challenge due to the vast number of compounds present in these samples as well as the influence matrix components could cause in the methodology. In this way, comprehensive two-dimensional gas chromatography coupled to mass spectrometry (GC × GC-MS) is a very powerful tool to achieve the characterization of complex samples. Nevertheless, due to possible coelutions occurring in these matrices, mixed spectra are generally obtained with electron ionization (EI) which could extremely complicate the identification of the analytes. Thereby, new methodology setups are required to improve the confidence on the identification in non-targeted determinations. Here, we present a high-throughput methodology consisting of GC × GC with flow modulation coupled to high-resolution atmospheric pressure mass spectrometry (HRMS) via a novel tube plasma ion source (TPI). The flow modulator allows to easily automate the GC × GC method compared to traditional cryo-modulators, while the soft ionization provided by TPI helps to preserve the [M]+• or [M+H]+ ions, thus increasing the confidence in the identification. Additionally, the combination of a flow modulation with an atmospheric pressure mass spectrometer significantly improves the sensitivity over flow modulated GC × GC-EI-MS methods because no split is required. This methodology was applied to the analysis of a complex sample such as vermouth where the volatile profile is usually considered by consumers as a product quality indicator since it raises the first sensations produced during its consumption. Using this approach, different classes of compounds were tentatively identified in the sample, including monoterpenes, terpenoids, sesquiterpenoids and carboxylic acid, and carboxylate esters among others, showing the great potential of a GC × GC-TPI-qTOF-MS platform for improving the confidence of the identifications in non-targeted applications.

Multi-2D LC × LC as a Novel and Powerful Implement for the Maximum Separation of Complex Samples.

Achieving complete information about the chemical composition of complex samples requires the use of multianalytical platforms able to maximize the acquisition of high-quality data for unequivocal identification. However, this process requires long analysis times and several instruments. Food analysis is one of the analytical fields where the analysis of very complex samples has a huge impact. One of these complex samples is vermouth, a fortified wine based on the maceration of a large number of herbs, fruits, barks, seeds, and leaves. The application of conventional or even advanced analytical techniques like comprehensive two-dimensional (2D) liquid chromatography (LC × LC) does not provide enough separation power to resolve the complete profile of this sample. In this work, a novel 2DLC strategy called multi-2D LC × LC is developed. This new setup consists of the use of two different columns with different separation properties in the second dimension (2D) that can be selected during the LC × LC analysis accordingly to the chemical nature of the compounds eluted from the first dimension (1D). The vermouth sample was analyzed using a 1D-PFP and a combination of HILIC (from 0 to 30 min) and C18 (from 30 to the end) columns in the 2D. This setup increased both the peak capacity and the orthogonality of the analysis in comparison to the use of only one of the columns in the 2D. Multi-2D LC × LC is presented as an integrated 2DLC tool that maximizes the separation capacity for very complex samples.

Enhancing the compatibility of normal-phase chromatography x reversed-phase chromatography by combination of low-temperature sensitive aqueous-phase compatible normal- phase chromatography and at-column dilution modulation.

The nearly opposite retention mechanism in the two-dimensional liquid chromatography (2D-LC), which combines normal phase liquid chromatography (NPLC) and reversed phase liquid chromatography (RPLC), shows extremely high orthogonality and theoretical peak capacity. However, peak breakthrough and peak distortion caused by the highly incompatible 2D mobile phases counteracts the advantages offered by high orthogonality. To address this difficulty, this study proposes a comprehensive two-dimensional NPLC × RPLC integrating temperature-sensitive aqueous-phase compatible normal-phase chromatography (TSACNPLC) and at-column dilution modulation (ACDM). The proposed 2D-LC system uses an aqueous-miscible acetonitrile/methanol eluent in the 1st D NPLC, instead of an aqueous-phase immiscible eluent, such as n-hexane/methanol, to increase the miscibility with the RP mobile phase system. Additionally, the system exploits temperature-sensitive retention behavior to enhance the retention ability of aqueous-phase compatible NPLC. To verify the feasibility of the proposed 2D-LC, this study selected three multi-component samples with mid-to-low polarity, including ethoxylated (n ≈ 6) bisphenol A (BPA-6EO), ethoxylated (n ≈ 6) tristearylphenol (TSP-6EO), and safflower methanol extract. Next, the effectiveness of the constructed 2D-LC was systematically investigated, including low temperature-induced retention enhancement of NPLC, overcoming solvent incompatibility by ACDM, and optimization of 2 D separation conditions, was systematically investigated.

Post-fast refeeding enhances intestinal stem cell-mediated regeneration and tumourigenesis through mTORC1-dependent polyamine synthesis.

For more than a century, fasting regimens have improved health, lifespan, and tissue regeneration in diverse organisms, including humans. However, how fasting and post-fast refeeding impact adult stem cells and tumour formation has yet to be explored in depth. Here, we demonstrate that post-fast refeeding increases intestinal stem cell (ISC) proliferation and tumour formation: Post-fast refeeding augments the regenerative capacity of Lgr5+ intestinal stem cells (ISCs), and loss of the tumour suppressor Apc in ISCs under post-fast refeeding leads to a higher tumour incidence in the small intestine and colon than in the fasted or ad libitum (AL) fed states. This demonstrates that post-fast refeeding is a distinct state. Mechanistically, we discovered that robust induction of mTORC1 in post-fast-refed ISCs increases protein synthesis via polyamine metabolism to drive these changes, as inhibition of mTORC1, polyamine metabolite production, or protein synthesis abrogates the regenerative or tumourigenic effects of post-fast refeeding. Thus, fast-refeeding cycles must be carefully considered when planning diet-based strategies for regeneration without increasing cancer risk, as post-fast refeeding leads to a burst not only in stem cell-driven regeneration but also in tumourigenicity.

Review on atmospheric pressure ionization sources for gas chromatography-mass spectrometry. Part I: Current ion source developments and improvements in ionization strategies.

The use of atmospheric pressure ionization (API) sources has become very popular for gas chromatography-mass spectrometry (GC-MS) determinations. GC-API-MS shows important advantages over traditional vacuum ionization sources such as a lower fragmentation preserving the molecular or quasi-molecular ion, the combination of GC separations with advanced mass spectrometers typically developed for liquid chromatography (LC) systems, a significantly higher sensitivity, or the reduction of costs due to the capability to use the same MS for both LC- and GC- couplings. For these reasons, the development of new API sources and GC-API-MS platforms has exponentially increased during the last years. Thus, this review is mainly focused on the last advances in GC-API-MS instrumentation. New setups and couplings on extensively reported API techniques as well as the development of new API sources for GC-MS coupling are thoroughly discussed. Moreover, novel ionization strategies have been reviewed to overcome some of the drawbacks of GC-API-MS methodologies.

Review on atmospheric pressure ionization sources for gas chromatography-mass spectrometry. Part II: Current applications.

The application of atmospheric pressure ionization (API) sources in gas chromatography-mass spectrometry (GC-MS) determinations is becoming more popular since they have shown great capabilities to sort out the main drawbacks of vacuum ionization techniques like electron ionization (EI) and chemical ionization (CI). The development of new API techniques and set-ups have grown in the last decades, opening the field of GC-MS to new applications and facing some of the major issues in current analytical methodologies such as the requirement of a compromise between sensitivity and selectivity. Thus, this review is mainly focused on the use of GC-API-MS in different application fields such as food analysis (food safety and food metabolomics), environmental analysis, clinical analysis, drug and pharmaceutical analysis, and petroleomics, among others. The methodologies have been critically reviewed to compare the performance of different API sources and approaches, highlighting the main contributions to overcoming some of the major limitations of the current methodologies as well as the new perspectives that GC-API-MS might open in the different fields.

Exopolysaccharide composition and size in Sulfolobus acidocaldarius biofilms.

Extracellular polymeric substances (EPS) comprise mainly carbohydrates, proteins and extracellular DNA (eDNA) in biofilms formed by the thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius. However, detailed information on the carbohydrates in the S. acidocaldarius biofilm EPS, i.e., the exopolysaccharides (PS), in terms of identity, composition and size were missing. In this study, a set of methods was developed and applied to study the PS in S. acidocaldarius biofilms. It was initially shown that addition of sugars, most significantly of glucose, to the basal N-Z-amine-based growth medium enhanced biofilm formation. For the generation of sufficient amounts of biomass suitable for chemical analyses, biofilm growth was established and optimized on the surface of membrane filters. EPS were isolated and the contents of carbohydrates, proteins and eDNA were determined. PS purification was achieved by enzymatic digestion of other EPS components (nucleic acids and proteins). After trifluoroacetic acid-mediated hydrolysis of the PS fraction, the monosaccharide composition was analyzed by reversed-phase liquid chromatography (RP-LC) coupled to mass spectrometry (MS). Main sugar constituents detected were mannose, glucose and ribose, as well as minor proportions of rhamnose, N-acetylglucosamine, glucosamine and galactosamine. Size exclusion chromatography (SEC) revealed the presence of one single PS fraction with a molecular mass of 4-9 × 104 Da. This study provides detailed information on the PS composition and size of S. acidocaldarius MW001 biofilms and methodological tools for future studies on PS biosynthesis and secretion.

Quantification and identification of bile acids in saliva by liquid chromatography-mass spectrometry: Possible non-invasive diagnostics of Barrett's esophagus?

Bile acids are a group of steroid compounds essential for lipid digestion. However, when bile acids are refluxed into the stomach and the esophagus, during the so called duodenogastroesophageal reflux, they can have a detrimental effect on the esophageal epithelium and cause pathological changes of esophageal tissue, e.g., Barrett's esophagus (BE). The levels of bile acids in saliva could therefore serve as possible biomarkers for the diagnostics of BE. In this work, we focused on optimization of sample collection and preparation by solid-phase extraction and subsequent quantification of 11 bile acids (unconjugated, glycine-conjugated) in saliva from healthy volunteers and BE patients by ultra-high-performance liquid chromatography coupled to triple-quadrupole tandem mass spectrometry. Moreover, high resolution MS (Orbitrap-MS) was utilized for identification of new bile acids in saliva. Methods for saliva collection including simple spitting and the Salivette® saliva collection system were compared; the latter was found to be unsuitable due to excessive retention of bile acids in the cotton swab. Methanol with 0.1% formic acid were selected for protein precipitation and bile acid extraction prior to SPE. Separation was performed in gradient elution of methanol and 0.1% formic acid in less than 10 min. Saliva from BE patients contained higher levels of almost all bile acids, and the tested groups could be distinguished by principal component analysis. In untargeted analysis by high resolution MS, taurine-conjugated bile acids and glycine-conjugated dihydroxy-bile acid sulfate were identified in saliva from healthy volunteers. We propose that analysis of salivary bile acids including taurine conjugates could be applicable in diagnostics of BE, following a larger clinical study.

Aliphatic Aldehydes in the Earth's Crust-Remains of Prebiotic Chemistry?

The origin of life is a mystery that has not yet been solved in the natural sciences. Some promising interpretative approaches are related to hydrothermal activities. Hydrothermal environments contain all necessary elements for the development of precursor molecules. There are surfaces with possible catalytic activity, and wide ranges of pressure and temperature conditions. The chemical composition of hydrothermal fluids together with periodically fluctuating physical conditions should open up multiple pathways towards prebiotic molecules. In 2017, we detected potentially prebiotic organic substances, including a homologous series of aldehydes in Archean quartz crystals from Western Australia, more than 3 billion years old. In order to approach the question of whether the transformation of inorganic into organic substances is an ongoing process, we investigated a drill core from the geologically young Wehr caldera in Germany at a depth of 1000 m. Here, we show the existence of a similar homologous series of aldehydes (C8 to C16) in the fluid inclusions of the drill core calcites, a finding that supports the thesis that hydrothermal environments could possibly be the material source for the origin of life.

Downregulation of eNOS and preserved endothelial function in endothelial-specific arginase 1-deficient mice.

Arginase 1 (Arg1) is a ubiquitous enzyme belonging to the urea cycle that catalyzes the conversion of l-arginine into l-ornithine and urea. In endothelial cells (ECs), Arg1 was proposed to limit the availability of l-arginine for the endothelial nitric oxide synthase (eNOS) and thereby reduce nitric oxide (NO) production, thus promoting endothelial dysfunction and vascular disease. The role of EC Arg1 under homeostatic conditions is in vivo less understood. The aim of this study was to investigate the role of EC Arg1 on the regulation of eNOS, vascular tone, and endothelial function under normal homeostatic conditions in vivo and ex vivo. By using a tamoxifen-inducible EC-specific gene-targeting approach, we generated EC Arg1 KO mice. Efficiency and specificity of the gene targeting strategy was demonstrated by DNA recombination and loss of Arg1 expression measured after tamoxifen treatment in EC only. In EC Arg1 KO mice we found a significant decrease in Arg1 expression in heart and lung ECs and in the aorta, however, vascular enzymatic activity was preserved likely due to the presence of high levels of Arg1 in smooth muscle cells. Moreover, we found a downregulation of eNOS expression in the aorta, and a fully preserved systemic l-arginine and NO bioavailability, as demonstrated by the levels of l-arginine, l-ornithine, and l-citrulline as well as nitrite, nitrate, and nitroso-species. Lung and liver tissues from EC Arg1 KO mice showed respectively increase or decrease in nitrosyl-heme species, indicating that the lack of endothelial Arg1 affects NO bioavailability in these organs. In addition, EC Arg1 KO mice showed fully preserved acetylcholine-mediated vascular relaxation in both conductance and resistant vessels but increased phenylephrine-induced vasoconstriction. Systolic, diastolic, and mean arterial pressure and cardiac performance in EC Arg1 KO mice were not different from the wild-type littermate controls. In conclusion, under normal homeostatic conditions, lack of EC Arg1 expression is associated with a down-regulation of eNOS expression but a preserved NO bioavailability and vascular endothelial function. These results suggest that a cross-talk exists between Arg1 and eNOS to control NO production in ECs, which depends on both L-Arg availability and EC Arg1-dependent eNOS expression.

Development of a Tube Plasma Ion Source for Gas Chromatography-Mass Spectrometry Analysis and Comparison with Other Atmospheric Pressure Ionization Techniques.

A tube plasma ionization (TPI) open-air source for gas chromatography-mass spectrometry (GC-MS) was developed. This source is based on an inverse low temperature plasma configuration where the pin inner electrode is applying the high voltage and the grounded electrode is the housing itself. The ionization possibilities were tested by using an EPA mix of priority contaminants, showing that 68% of the analytes could undergo both proton-transfer and charge-exchange reactions. The potential of using different discharge gases (He and Ar) to ionize the analytes and auxiliary gases (He, N2, O2, and synthetic air) to transport the ions toward the MS was carefully investigated. Additionally, the addition of water was also tested to show the different ionization trends in the TPI source. Finally, the ionization by TPI under both dry and wet conditions was compared with other gas-phase atmospheric pressure ionization sources showing TPI could ionize a wider range of compounds (97%) than atmospheric pressure chemical ionization (APCI, 95%) and atmospheric pressure photoionization (APPI, 87%). Besides, the detection capability of TPI was better than APCI and APPI, achieving instrumental limits of detection down to 3 fg on column, which demonstrates the great potential of this ionization source for GC-MS determinations.

Characterization of the Extracellular Volatile Metabolome of Pseudomonas Aeruginosa Applying an in vitro Biofilm Model under Cystic Fibrosis-Like Conditions.

BACKGROUND: Cystic fibrosis (CF) is an autosomal recessive hereditary disease that leads to the production of thickened mucus in the lungs, favouring polymicrobial infections, such as chronic lung infections with the bacterial opportunistic pathogen Pseudomonas aeruginosa. METHOD: A biofilm model in combination with an adapted sampling and GC-MS analysis method were applied to in vitro studies on different variables influencing the composition of the extracellular volatile metabolome of P. aeruginosa. RESULTS: A significant influence on the metabolome could be demonstrated for the culture medium as well as the atmosphere during cultivation (aerobic or anaerobic). Furthermore, a significant influence of the mucoid (alginate-overproducing) phenotype of the bacterium on quantity and composition of volatile organic compounds could be observed. Based on the results a solid culture medium was developed to simulate the nutrient conditions in the lungs of a CF patient. The extracellular volatile metabolome of bacterial strains P. aeruginosa ATCC 10145, PAO1 and FRD1 was characterized under CF-like conditions. CONCLUSIONS: Bacterial strain-dependent metabolites were identified. When P. aeruginosa PAO1 and FRD1 clinical isolates were compared, 36 metabolites showed significant variations in intensities. When the clinical isolates were compared with the reference strain (P. aeruginosa ATCC 10145), 28 metabolites (P. aeruginosa PAO1) and 70 metabolites (P. aeruginosa FRD1) were determined whose peaks showed significant deviation (p > 95%) in intensity. Furthermore, the bacterial strains could be differentiated from each other by means of two principal components.

Ultrafast cold-brewing of coffee by picosecond-pulsed laser extraction.

Coffee is typically brewed by extracting roasted and milled beans with hot water, but alternative methods such as cold brewing became increasingly popular over the past years. Cold-brewed coffee is attributed to health benefits, fewer acids, and bitter substances. But the preparation of cold brew typically needs several hours or even days. To create a cold-brew coffee within a few minutes, we present an approach in which an ultrashort-pulsed laser system is applied at the brewing entity without heating the powder suspension in water, efficiently extracting caffeine and aromatic substances from the powder. Already 3 min irradiation at room temperature leads to a caffeine concentration of 25 mg caffeine per 100 ml, comparable to the concentrations achieved by traditional hot brewing methods but comes without heating the suspension. Furthermore, the liquid phase's alkaloid content, analyzed by reversed-phase liquid chromatography coupled to high-resolution mass spectrometry, is dominated by caffeine and trigonelline and is comparable to traditional cold-brewed coffee rather than hot-brewed coffee. Furthermore, analyzing the head-space of the prepared coffee variants, using in-tube extraction dynamic head-space followed by gas chromatography coupled to mass spectrometry, gives evidence that the lack of heating leads to the preservation of more (semi-)volatile substances like pyridine, which provide cold-brew coffee its unique taste. This pioneering study may give the impetus to investigate further the possibility of cold-brewing coffee, accelerated by more than one order of magnitude, using ultrafast laser systems.

Differentiation of industrial hemp strains by their cannabinoid and phenolic compounds using LC × LC-HRMS.

Cannabis is an ancient plant that has been used for therapeutic and recreational purposes. Nowadays, industrial hemp, a variety with low concentration of the psychoactive cannabinoid Δ9-tetrahydrocannabinol (THC) and high concentration of non-psychoactive cannabinoids, is getting more and more interest in the food, pharmaceutical, and cosmetic industry. However, cannabis not only contains cannabinoids as bioactive components but also other metabolites like terpenes and phenolic compounds, and the content of these interesting secondary metabolites greatly differs with the genetic variety of the plant. Due to the huge complexity of composition of the cannabis matrix, in this work, a comprehensive two-dimensional liquid chromatography (LC × LC) method has been developed as a very power separation technique coupling a pentafluorophenyl (PFP) and a C18 in the first and second dimensions. Two industrial hemp strains (cookie and gelato) were analyzed to determine the difference in their content of cannabinoids and phenolic compounds. To do this, a new demodulation process was applied for the first time to transform 2D raw data into 1D data which allowed carrying out the chemometric analysis needed to determine the statistical differences between the hemp strains. The cookie strain presented a total of 41 cannabinoid markers, while the gelato strain presented more representative phenolic compounds, in total 24 phenolic compounds were detected as potential markers of this sample. These differences in the chemical composition could determine the industrial destiny of the different hemp strains.

Comparative study for analysis of carbohydrates in biological samples.

This work presents a comparative study for the analysis of carbohydrates for four common chromatographic methods, each coupled to mass spectrometry. Supercritical fluid chromatography (SFC), hydrophilic interaction liquid chromatography (HILIC), reversed-phase liquid chromatography (RP-LC) and gas chromatography (GC) with detection by triple quadrupole mass spectrometer (QqQ-MS) are compared. It is shown that gas chromatography and reversed-phase liquid chromatography, each after derivatisation, are superior to the other two methods in terms of separation performance. Furthermore, comparing the different working modes of the mass spectrometer, it can be determined that a targeted analysis, i.e. moving from full scan to single ion monitoring (SIM) and multiple reaction monitoring (MRM), results in an improvement in the sensitivity as well as the repeatability of the method, which has deficiencies especially in the analysis using HILIC. Overall, RP-LC-MS in MRM after derivatisation with 1-phenyl-3-methyl-5-pyrazolone (PMP) proved to be the most suitable method in terms of separation performance, sensitivity and repeatability for the analysis of monosaccharides. Detection limits in the nanomolar range were achieved, which corresponds to a mass concentration in the low µg/L range. The applicability of this method to different biological samples was investigated with various herbal liquors, pectins and a human glycoprotein.