2D GC-MS Research Articles

GC/MS and 2D NMR-based approach to evaluate the chemical profile of hydroalcoholic extract from Agaricus blazei Murill and its anti-inflammatory effect on human neutrophils

Ethnopharmacological relevanceAgaricus blazei Murill (AbM) is one of the main mushrooms used for medicinal purposes. The use of AbM in the preparation of teas is widespread mainly in Asian countries, while in Brazil it is used as a functional food to combat inflammatory diseases and cancer. Aim of the studyThe main focus of this study was the characterization of the chemical profile of the hydroalcoholic extract of Agaricus blazei Murill (AbE), as well as the evaluation of its cytotoxic and anti-inflammatory potential using human neutrophils. Materials and methodsThe extract was prepared by dynamic maceration using a mixture of ethanol and water (70/30, v v−1) as solvent. The chemical profile characterization was carried out by 2D NMR and GC-MS techniques. The cytotoxicity of AbE was evaluated through studies of hemolytic potential, cell viability and membrane integrity. The anti-inflammatory activity was analyzed by a PMA-induced neutrophil degranulation assay. ResultsChemical analysis of AbE revealed the presence of 28 metabolites in its composition, with mannitol as the major compound. AbE at 1–200 μg mL−1 and mannitol at 4–160 μg mL−1, showed low hemolytic and cytotoxic potential against human red blood cells and neutrophils. Furthermore, both were able to significantly reduce the release of myeloperoxidase. ConclusionsThese results indicate that AbE is a promising natural product to be incorporated into pharmaceutical dosage forms intended for the adjuvant treatment of inflammatory diseases.

3-Cyclohexyl-6-phenyl-1-(p-tolyl)pyrimidine-2,4(1H,3H)-dione

The synthesis of a novel uracil derivative, 3-cyclohexyl-6-phenyl-1-(p-tolyl)pyrimidine-2,4(1H,3H)-dione (4), is reported via a four-component reaction involving an α-chloroketone (1), an aliphatic isocyanate (2), a primary aromatic amine (3) and carbon monoxide. The proposed reaction mechanism involves a Pd-catalyzed carbonylation of 2-chloro-1-phenylethan-1-one (1), leading to a β-ketoacylpalladium key intermediate, and, at the same time, in situ formation of non-symmetrical urea deriving from cyclohexyl isocyanate (2) and p-toluidine (3). After a chemo-selective acylation of the non-symmetrical urea and the subsequent cyclization of the acylated intermediate, 3-cyclohexyl-6-phenyl-1-(p-tolyl)pyrimidine-2,4(1H,3H)-dione (4) is formed. Uracil derivative 4 was isolated in good yield (73%) and fully characterized by 1H, 13C, 2D 1H-13C HSQC and 2D 1H-13C HMBC NMR, FT-IR spectroscopy and GC-MS spectrometry.

Isolation and Characterization of Impurities in Commercially Marketed Δ8-THC Products.

Qualitative analysis of several commercial products containing Δ8-tetrahydrocannabinol (Δ8-THC) as a major component using GC-MS resulted in the identification of several impurities along with Δ8-THC. In an attempt to isolate and identify these impurities, a commercial Δ8-THC distillate was selected for the isolation work. Eleven impurities were isolated using a variety of chromatographic techniques, and their chemical structures were determined. These include Δ4,8-iso-tetrahydrocannabinol (1), Δ4-iso-tetrahydrocannabinol (2), Δ8-cis-iso-tetrahydrocannabinol (3), 4,8-epoxy-iso-tetrahydrocannabinol (4), 8-hydroxy-iso-tetrahydrocannabinol (5), 9β-hydroxyhexahydrocannabinol (6), 9α-hydroxyhexa-hydrocannabinol (7), iso-tetrahydrocannabifuran (8), cannabicitran (CBT, 9), olivetol (10), and Δ9-THC (11). The chemical structures of the purified compounds were determined using several spectroscopic methods, including 1D (1H, 13C, and DEPT-135) and 2D (COSY, HMQC, HMBC, and NOESY) NMR, LC-MS, and GC-MS. Other naturally occurring cannabinoids and impurities were also identified in GC-MS chromatograms but were not isolated. These were cannabidiol (CBD, 12), cannabinol (CBN, 13), hexahydrocannabinol (HHC, 14), and Δ8-tetrahydrocannabivarin (Δ8-THCV, 15). The chemical structure of Δ8-THCV (15), for which a standard was not available, was confirmed by partial synthesis and NMR analysis. This is the first report for many of the above compounds as well as Δ8-THCV as impurities in Δ8-THC products.

2-Dimensional Gas Chromatography-Mass Spectroscopy: A Review

Gas Chromatography separates components in time. Mass spectroscopy provides information that aids in the identification and structural elucidation of each component. Comprehensive 2-Dimensional gas chromatography (GC×GC) is a novel technique. The revolutionary aspect of GC x GC, with respect to classical multidimensional chromatography, is that the entire sample is subjected to two distinct analytical separations. The resulting enhanced separating capacity makes this approach a prime choice when GC analysts are challenged with highly complex mixtures. The combination of a third mass spectrometric dimension to a GC x GC system generates the most powerful analytical tool today for volatile and semi-volatile analytes. The present review is focused on the instrumentation of 2D GC-MS, the difference between GC and 2d GC, application of 2d GC-MS have been discussed.

Utility of comprehensive GC×GC-TOFMS in elucidation of aromatic hydrocarbon biomarkers

Conventional one-dimensional gas chromatography-mass spectrometry (GC–MS) has been employed for the organic compound analysis since the 1960s. In traditional GC–MS analysis, co-elution can be intense in the chromatographic region occupied by triterpenoids and aromatic steroids and these can form an unresolved complex mixture (UCM) in the total ion chromatogram (TIC). The UCM in routine GC–MS analysis alters the baseline and adds a substantial noise to mass spectra. Here we demonstrate that comprehensive GC×GC-TOFMS with higher peak capacity helps in an improved characterisation of aromatic hydrocarbon biomarkers by resolving many individual peaks. The use of GC×GC-TOFMS achieved exceptional chromatographic separation of higher plant triterpenoids, triaromatic steroids and methylated 2-methyl-2-(4,8,12-trimethyltridecyl) chromans (MTTC) in a complex aromatic fraction of a Cenozoic crude oil from the Tarapur field of the Cambay Basin. Compounds (e.g., des-A-arbora-5,7,9-triene) having scarce abundance that could not be detected with 1D GC–MS analysis were identified by GC×GC-TOFMS. Thus, this approach offers a new avenue to palaeobiological studies, hydrocarbon exploration and evaluation of the distinct palaeodepositonal environments of organic matter.

Pennelliisides A-C, 2,3,4-Trisubstituted Acyl Glucoses Isolated from Solanum pennellii.

Solanum species accumulate a variety of secondary metabolites in their trichomes, and it is well known that acyl sugars are specialized metabolites secreted by the trichomes. However, very few reports provide detailed information on the chemical structure of polyacylated glucose derivatives, due to the α and β isomerization that can occur at the C-1 position. In this study, a strategy was established to isolate polyacylated glucose derivatives. According to the developed strategy, hydroxy groups were derivatized to a benzyloxy group using TriBOT. After isolation of the compounds in pure form and deprotection of the benzyloxy group, the chemical structures of pennelliisides A-C were determined as 2,3,4-O-triisobutyryl-d-glucose, 3-O-(8-methylnonanoyl)-2,4-O-diisobutyryl-d-glucose, and 3-O-decanoyl-2,4-O-diisobutyryl-d-glucose, respectively. Structural elucidation was performed using spectroscopic techniques, including 1D and 2D NMR, FD-MS, and GC-MS. It was also found that the fatty acid moiety contributes to the allelopathic properties of the isolated compounds.

Complementary chemical characterization of distillates obtained from industrial hemp hurds by thermal processing

This study focused on spectroscopic characterization of slow pyrolysis distillates from three different hemp hurd variates of industrial hemp (Cannabis sativa L). Raw distillates were collected at drying, torrefaction and pyrolysis stage of the slow pyrolysis process starting from room temperature up to the maximum operating temperature of 350 °C. Complementary chemical characterization of the hemp hurd distillate fractions was achieved using Fourier transform infrared (FTIR) spectroscopy, 1D and 2D NMR spectroscopy, comprehensive two-dimensional gas chromatography-mass spectrometry (2D GC–MS) and liquid chromatography–high resolution mass spectrometry (LC–HRMS). These analyses provided information on the concentration of main chemical constituents of distillates, detected valuable, volatile, semivolatile and nonvolatile compounds and their chemical nature as indicated by functional groups. This study reveals the possibility of optimizing the use of hemp hurds as high-value products by utilizing slow pyrolysis for biochar and further processing for distillates.

Biomarkers of photosynthetic sulfur bacteria in Lower Cretaceous crude oils, East Brazilian marginal basin

Diagenetic aromatic carotenoids and degradation products, which are derived from the carotenoids of photosynthetic sulfur bacteria, were characterized in the aromatic fraction of Brazilian Cretaceous crude oils (NE Brazil, Sergipe-Alagoas Basin) by high-temperature comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (HT-GC × GC–TOFMS). Unresolved complex mixtures (UCM), coeluting compounds, and limited mass spectrometric identification were noted by traditional one-dimensional (1D) GC–MS screening of aromatic compounds. Group-type separation on a two-dimensional chromatographic plane, first- and second-dimension retention times (1tR and 2tR, respectively), and mass spectra enabled the assignment of alkylated mono-, di-, tri-, and tetra-aryl hydrocarbons with confidence. A series of diagenetic C40 carotenoids and degraded products, which are likely indicators for green-brown sulfur bacteria, were detected. This finding corroborates previous results, and isorenieratane was relatively more abundant than 2,3,6-trimethylaryl isoprenoids. Moreover, atropisomeric biphenyls (m/z 237), which coeluted using 1D GC–MS analyses, were resolved by GC × GC and identified by mass spectrometry. The identification of minor abundances of chlorobactane, diagenetic biphenyl chlorobactane, and related compounds, for the first time in these Brazilian Cretaceous oils, may indicate a contribution of green sulfur bacteria (Chlorobiaceae) and photic zone euxinia in shallow waters.

Analysis of defensive secretion of a milkweed bug Lygaeus equestris by 1D GC-MS and GC\xd7GC-MS: sex differences and host-plant effect

The composition of defensive secretion produced by metathoracic scent glands was analysed in males and females of the milkweed bug Lygaeus equestris (Heteroptera) using gas chromatography with mass spectrometric detection (GC-MS). The bugs were raised either on cardenolide-containing Adonis vernalis or on control sunflower seeds in order to determine whether the possibility to sequester cardenolides from their host plants would affect the composition of defensive scent-gland secretion. Profiles of the composition of defensive secretions of males and females raised on sunflower were closely similar, with predominant presence of (E)-2-octenal, (E)-2-octen-1-ol, decanal and 3-octen-1-ol acetate. The secretion of bugs raised on A. vernalis was more sexually dimorphic, and some chemicals e.g. (E,E)-2,4-hexadienyl acetate and 2-phenylethyl acetate were dominant in males, but absent in females. Compared to bugs from sunflower, the scent-gland secretion of bugs raised on A. vernalis was characterized by lower overall intensity of the peaks obtained for detected chemicals and by absence of some chemicals that have supposedly antipredatory function ((E)-2-hexenal, (E)-4-oxo-hex-2-enal, 2,4-octadienal). The results suggest that there might be a trade-off between the sequestration of defensive chemicals from host plants and their synthesis in metathoracic scent-glands.

Cost-effective FTIR and 1H NMR spectrometry used to screen valuable molecules extracted from selected West African trees by a sustainable biochar process

This study focused on wood extractives by a slow pyrolysis thermal extraction technique and screening of potentially valuable compounds from three African trees: Lophira lanceolata Tiegh. ex Keay., Dialium guineense Willd. and Afzelia africana Sm. The primary objective of the thermal treatment was biochar production but also liquids were collected and analyzed. Raw materials were cut into sizes of about 25 cm in length and then processed with slow pyrolysis from 20 °C up to 400 °C; the slow pyrolysis process included two stages, drying and pyrolysis. Five end-point temperatures were used in the range of 250 °C–400 °C. Raw distillates were collected at three temperatures (120 °C, 60 °C and 25 °C) and their composition screened by either cost-effective FTIR or benchtop 80 MHz 1H NMR methods. HR-NMR and 2D GC–MS were used to confirm some findings. Results from the drying stage indicated that all distillates contained mostly water >90%, acetic acid, methanol and some other compounds at very low levels. The percentages of acetic acid were 3.5% for L. lanceolate, 2.8% for D. guineensis and 3.8% for A. africana (% with respect to the identified peaks). Results from the pyrolysis stage revealed the presence of bioactive compounds such as ketones, methanol, hydrocarbons, acetic acid, esters, alkenes and phenolic compounds at higher quantities than in the drying stage. The quantity of phenols in certain runs and distillate fractions using NMR was 1%–20% for L. lanceolata, 7–17% for D. guineensis and 10%–17% for A. africana (% with respect to the composition of the identified peaks). The percentage of hydrocarbons was 1%–48% for L. lanceolata, 1%–34% for D. guineensis and 2%–33% for A. africana. The percentage of ketones was 7%–19% for L. lanceolate, 4%–27% for D. guineensis and 19%–22% for A. africana. In general, the results indicated the presence of potential valuable compounds at relatively high concentrations. By optimizing the slow pyrolysis process parameters, it is possible to collect distillates with high concentrations of valuable biomolecules.

Constituintes Químicos e Atividade Antimicrobiana de Ramos e Folhas de Cordia insignis (Boraginaceae)

Phytochemical investigation of the branches and leaves of the species Cordia insignis led to the isolation and identification of β-sitosterol (1), stigmasterol (2), campesterol (3), 3-O-β-D-glucopyranosyl sitosterol (4), 3-O-β-D- glucopyranosyl stigmasterol (5), scoparone (6), α-amyrin (7), β-amyrin (8), methyl caffeate (9), allantoin (10), scopoletin (11), caffeic acid (12), methyl rosmarinate (13). These substances had their structures determined by analysis of their 1D and 2D NMR and GC-MS spectra and by comparison with data reported in the literature. The hydroethanolic extracts of the branches and leaves and their respective fractions were submitted to antibacterial and antifungal evaluation. The extracts and fractions showed good antifungal activity but did not present antibacterial activity. This study is the first work with C. insignis.

Optimization and Isolation of 4,8,12,16-Tetramethylheptadecan-4-olide from Deinbollia pinnata

Manual agitation on ultrasonic assisted extraction process, fractionation, isolation and purification afforded 4,8,12,16-tetramethylheptadecan-4-olide (1) along with 11 compounds 2-12 i.e., squalene (2), phytyl palmitate (3), lupeol (4), taraxasterol (5), myristic acid (6), palmitic acid (7), campesterol (8), stigmasterol (9), λ-sitosterol (10), stigmastan-3, 5-diene (11) and stigmasta-5,22-diene-3-ol acetate (12). Their structures were elucidated spectroscopically (2D NMR, IR, GC-MS and 1D NMR). The optimal conditions for high yield of extracts were obtained at 45 °C, after 35 min and solvent ratio 50:50 mL for 83.01 % yield; which was applied on agita-sonication process for bulk sample extraction of D. pinnata leaves with single solvent at a time. Thus, this work provides alternative method to overcome large sample extraction for phyto-constituents of isolation of 4,8,12,16-tetramethylheptadecan-4-olide (1) with ten other compounds from this specie pinnata and genius Deinbollia except stigmasterol (9).

Reactions of Vinylcyclopropane and Bicyclopropyl Compounds With Maleic Anhydride

Vinylcyclopropane and bicyclopropyl C6 compounds are studied as diene precursors in the Diels-Alder reaction. Their rearrangement under different thermal and microwave conditions leads to specific isomeric hexa-, penta- or butadienes, condensed with maleic anhydride as a dienophile. Adduct stereochemistries were compared to those of previously-synthesized model compounds, and are fully characterized with 2D NMR and GC-MS spectroscopies. Some unsaturated terpenes bearing vinylcyclopropane moieties were also condensed, and their adduct structures assigned.

Multigram scale synthesis of 3,4- and 3,6-dihydro-2H-thiopyran 1,1-dioxides and features of their NMR spectral behavior

A new four-step synthesis of 3,4- and 3,6-dihydro-2H-thiopran-1,1-dioxides from dihydro-2H-thiopyran-3(4H)-one is reported. The title compounds are synthesized starting with oxidation of the ketone with a 30% aqueous solution of hydrogen peroxide in a mixture of AcOH-Ac2O. The keto group is then reduced by sodium borohydride followed by mesylation and elimination of methanesulfonic acid under basic conditions (pyridine for 3,4-isomer and aqueous NaOH for 3,6-isomer). This sequence is simpler, than previously known methods, uses cheaper and more readily available reagents, and leads to 2H-thiopran-1,1-dioxides on multigram scale with 64% and 74% total yields, respectively. The structure and purity of the compounds were confirmed by 2D NMR and GCMS methods. The proposed method expands the means to access functionalized cyclic sulfones as building blocks in the synthesis of combinatorial libraries of new biologically active compounds.

1H NMR, 1H–1H 2D TOCSY and GC-MS analyses for the identification of olive oil in Early Bronze Age pottery from Castelluccio (Noto, Italy)

The development of analytical research in recent decades, at the edge between analytical chemistry and archaeology, provides new methods for the study of organic residues that are usually highly sensitive to natural decay.

Isolation and Structural Characterization of Echinocystic Acid Triterpenoid Saponins from the Australian Medicinal and Food Plant Acacia ligulata.

The Australian plant Acacia ligulata has a number of traditional food and medicinal uses by Australian Aboriginal people, although no bioactive compounds have previously been isolated from this species. Bioassay-guided fractionation of an ethanolic extract of the mature pods of A. ligulata led to the isolation of the two new echinocystic acid triterpenoid saponins, ligulatasides A (1) and B (2), which differ in the fine structure of their glycan substituents. Their structures were elucidated on the basis of 1D and 2D NMR, GC-MS, LC-MS/MS, and saccharide linkage analysis. These are the first isolated compounds from A. ligulata and the first fully elucidated structures of triterpenoid saponins from Acacia sensu stricto having echinocystic acid reported as the aglycone. Compounds 1 and 2 were evaluated for cytotoxic activity against a human melanoma cancer cell line (SK-MEL28) and a diploid fibroblast cell line (HFF), but showed only weak activity.

The Phytochemical Investigation and Biological Activities of <em>Berberis Orthobotrys</em>

<p>The leaf extract of <em>Berberis orthobotrys </em>Bien. ex Aitch.<em> </em>(B.o.)<em> </em>afforded three compounds viz., β-sitosterol (<strong>1</strong>), sesamin (<strong>2</strong>) and 10-eicosanol (<strong>3</strong>) which were identified by spectroscopic methods including 1D and 2D NMR, GC-MS, IR and comparison of their spectral data with the published data. To the best of our knowledge these three compounds are reported here for the first time to be isolated from <em>Berberis orthobotrys </em>Bien. ex Aitch.<em> </em>(B.o.). Moreover the root extract exhibited good antilieshmanial activity and root and fruit extracts demonstrated minor antifungal activity against <em>Fusarium solani</em>.</p>

Flavor and chiral stability of lemon-flavored hard tea during storage

Flavor stability of hard tea beverage was investigated over eight weeks of storage. The volatile compounds were analyzed using solid-phase microextraction-gas chromatography–mass spectrometry (SPME-GC–MS) and two-dimensional GC–MS. Quantitative analysis showed that the concentrations of linalool, citronellol, geranial, neral, geraniol, and nerol decreased dramatically during storage, whereas α-terpineol showed an increasing trend during storage. Heart-cut two-dimensional GC–MS (2D-GC–MS) chirality analysis showed that (R)-(+)-limonene, (R)-(−)-linalool, (S)-(−)-α-terpineol and (S)-(−)-4-terpineol dominated in the fresh hard tea samples, however, the configuration changed during storage for the terpene alcohols. The storage conditions did not change the configuration of limonene. A conversion of (R)-(−)-linalool to (S)-(+) form was observed during storage. Both (S)-α-terpineol and (S)-4-terpineol dominated at beginning of the storage, but (R)-(+)-α-terpineol became dominated after storage, suggested in addition to isomerization from (S)-α-terpineol, other precursors could also generate α-terpineol with (R)-isomer preference.

Target analysis of tert-butyldimethylsilyl derivatives of nerve agent hydrolysis products by selectable one-dimensional or two-dimensional gas chromatography–mass spectrometry

A target analysis method for the sensitive and discriminative determination of the nerve agent hydrolysis products alkyl methylphosphonic acids as their tert-butyldimethylsilyl (TBDMS) derivatives was developed using a combination of selectable one- and two-dimensional (1D/2D) GC–MS, and applied to the analysis of samples with significant interfering matrices. After sample drying, the alkylmethylphosphonic acids and methylphosphonic acid (MPA) were converted to TBDMS derivatives by addition of N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide with heating, and subjected to 1D/2D GC–MS. The apparatus consisted of an initial low thermal mass DB-5 column and a second DB-17 column together with an electron ionization quadrupole mass spectrometer, offering simple and flexible switching between one- and two-dimensional GC–MS analysis in a single GC–MS system. Using 1D/2D GC–MS, analytes that do not co-elute with matrix components can be separated using 1D GC mode alone. Only those parts of the chromatogram that are negatively affected by the co-elution of matrix components need to be transferred and separated with 2D GC. Quantitation can be performed by a combination of both separations and mass spectrometric detection. The TBDMS derivatives of ethyl-, isopropyl-, isobutyl-, pinacolyl-, and cyclohexyl-MPA (cHMPA) and MPA itself were well separated within 3min and determined in 1D GC–MS mode with detection limits of around 10ng/ml of reaction mixture (except for the cHMPA derivative, whose mass spectrum contained noisy background peaks). In 2D-GC–MS mode, where each 0.04min elution window from the 1D GC was subjected to heart-cut (H/C) and transferred to the second column after back-flushing the first column, the peak for the cHMPA TBDMS derivative was isolated and afforded a clean mass spectrum within 6min. The recoveries of all the derivatives on 2D GC from 1D GC were estimated to be over 66%, and the detection limits were around 10ng/ml of reaction mixture. In the presence of urine extract, the target compounds were not detected as separated peaks in 1D GC–MS mode (except for isobutyl-MPA), and quantification based on extracted ion monitoring could not be achieved. However, 2D GC–MS of the H/C fractions of the target derivatives gave single peaks with well-defined mass spectra, and the recoveries of the derivatives were over 70% except for cHMPA (31% at 1.25μg/ml). Phosphonic acids could be detected at less than 60ng/ml. Sulfuric acid and phosphoric acid also negatively affected the determination of alkyl methylphosphonic acid TBDMS derivatives in 1D GC–MS, and the MPA-TBDMS-derivative peak was completely obscured by the large sulfuric-acid-derivative peak. However, under 1D/2D GC–MS conditions, baseline separation of the MPA derivative and sulfuric acid derivative was achieved, enabling highly sensitive MPA detection at 20ng/ml.

NEW DEGRADED QUINONE DITERPENOID FROM THE STEMS OF Byrsonima coccolobifolia Kunth. (Malpighiaceae)

A chemical investigation of two specimens of Byrsonima coccolobifolia collected in the southeast cerrado and from central Brazil was performed. A new degraded diterpenoid, byrsonimaquinone, was isolated from the stems along with known compounds. This is the first study on the roots of B. coccolobifolia, and several triterpenes, such as α-amyrin, β-amyrin, oleanolic acid, and glochidonol, along with a mixture of stigmasterol, β-sitosterol and campesterol, were identified. These compounds were identified by spectroscopic analysis techniques, including 1D and 2D NMR, GC-MS and high-resolution mass spectrometry.