Time-high Resolution Mass Spectrometry Research Articles

Ambient mass spectrometry imaging of food natural products by angled direct analysis in real time high-resolution mass spectrometry

Direct surface analysis in ambient conditions provides information on the position and chemical composition of an object at the time of investigation. An angled sampling probe is developed in this work for direct analysis in real time (DART) ionization high-resolution mass spectrometry. The DART ion source and the interface were modified for improved surface resolution, increased ion transfer efficiency, as well as enabling two-dimensional surface scanning. The angled probe DART-MS system was used for investigating a variety of food samples including fruit peels, ginseng root, plant leaves and sections of radish. Abundant signals and distinct chemical profiles are obtained in seconds, and spatial distribution of different molecules across the sample surfaces can be observed. In addition, the developed system can quickly identify the chemical changes when the surfaces were treated. The method is capable of directly evaluating food sample surfaces with different shapes, hardness, and conditions, without any sample pretreatments.

Analysis of feed additives by DART mass spectrometry: method optimisation and applications for product traceability in the European Union focusing on coccidiostats and carotenoids

In this study, direct analysis in real time high resolution mass spectrometry (DART-HRMS) was used to investigate the accurate characterisation of feed additive formulations containing coccidiostats or carotenoids. The study demonstrates the efficacy of DART-HRMS in identifying the active substances in these formulations and distinguishing between feed additives with the same active substance. The protocol for this method involves two simple steps that are extracting samples with organic solvents and measuring the extracts with DART-HRMS. The study also employs various statistical tools, including a factorial design approach, to optimise the DART-HRMS settings, and multivariate statistics, to establish classification models for feed additive formulations using nominal mass spectra. Our study demonstrates the potential of DART-HRMS in ensuring the correct identification of feed additives containing various coccidiostats or carotenoids and proposes this tool as an additional means for compliance checks with EU regulations.

DART-HRMS allows the detection of toxic alkaloids in animal autopsy specimens and guides the selection of confirmatory methods in accidental plant poisoning

BackgroundIn cases of suspected animal poisonings or intoxications, there is the need for high-throughput, rapid and accurate analytical tools capable of giving rapid answers and, thus, speeding up the early stages of investigations. Conventional analyses are very precise, but do not meet the need for rapid answers capable of orienting the decisions and the choice of appropriate countermeasures. In this context, the use of ambient mass spectrometry (AMS) screening methods in toxicology laboratories could satisfy the requests of forensic toxicology veterinarians in a timely manner. ResultsAs a proof of principle, direct analysis in real time high resolution mass spectrometry (DART-HRMS) was applied to a veterinary forensic case in which 12 of a group of 27 sheep and goats died with an acute neurological onset. Because of evidence in the rumen contents, the veterinarians hypothesized an accidental intoxication after ingestion of vegetable materials.The DART-HRMS results showed abundant signals of the alkaloids calycanthine, folicanthidine and calycanthidine, both in the rumen content and at the liver level. The DART-HRMS phytochemical fingerprinting of detached Chimonanthus praecox seeds was also compared with those acquired from the autopsy specimens. Liver, rumen content and seed extracts were then subjected to LC-HRMS/MS analysis to gather additional insights and confirm the putative assignment of calycanthine anticipated by DART-HRMS.HPLC-HRMS/MS confirmed the presence of calycanthine in both rumen contents and liver specimens and allowed its quantification, ranging from 21.3 to 46.9 mg kg−1 in the latter. This is the first report detailing the quantification of calycanthine in liver after a deadly intoxication event. Significance and noveltyOur study illustrates the potential of DART-HRMS to offer a rapid and complementary alternative to guide the selection of confirmatory chromatography-MSn strategies in the analysis of autopsy specimens from animals with suspected alkaloid intoxication. This method offers the consequent saving of time and resources over those needed for other methods.

Volatility Measurements of Individual Components in Organic Aerosol Mixtures Using Temperature-Programmed Desorption-Direct Analysis in Real Time-High Resolution Mass Spectrometry.

Atmospheric organic aerosols (OA) have profound effects on air quality, visibility, and radiative forcing of climate. Quantitative assessment of gas-particle equilibrium of OA components is critical to understand formation, growth, distribution, and evolution of OA in the atmosphere. This study presents a novel ambient pressure measurement approach developed and tested for untargeted screening of individual components in complex OA mixtures, followed by targeted chemical speciation of identified species and assessment of their physicochemical properties such as saturation vapor pressure and enthalpies of sublimation/evaporation. The method employs temperature-programmed desorption (TPD) experiments coupled to "direct analysis in real time" (DART) ionization source and high resolution mass spectrometry (HRMS) detection. Progression of the mass spectra is acquired in the TPD experiments over a T = 25-350 °C temperature range, and extracted ion chromatograms (EIC) of individual species are used to infer their apparent enthalpies of sublimation/evaporation (ΔHsub*) and saturation vapor pressure (pT*, Pa, or CT*, μg m-3) as a function of T. We validate application of this method for analysis of selected organic compounds with known ΔHsub and CT values, which showed excellent agreement between our results and the existing data. We then extend these experiments to interrogate individual components in complex OA samples generated in the laboratory-controlled ozonolysis of α-pinene, limonene, and β-ocimene monoterpenes. The abundant OA species of interest are distinguished based on their accurate mass measurements, followed by quantitation of their apparent ΔHsub* and CT* values from the corresponding EIC records. Comparison of C298K* values derived from our experiments for the individual OA components with the corresponding estimates based on their elemental composition using a "molecular corridors" (MC) parametrization suggests that the MC calculations tend to overestimate the saturation vapor pressures of OA components. Presented results indicate very promising applicability of the TPD-DART-HRMS method for the untargeted analysis of organic molecules in OA and other environmental mixtures, enabling rapid detection and quantification of organic pollutants in the real-world condensed-phase samples at atmospheric pressure and without sample preparation.

Combined ambient ionization mass spectrometric and chemometric approach for the differentiation of hemp and marijuana varieties of Cannabis sativa

BackgroundHemp and marijuana are the two major varieties of Cannabis sativa. While both contain Δ9-tetrahydrocannabinol (THC), the primary psychoactive component of C. sativa, they differ in the amount of THC that they contain. Presently, U.S. federal laws stipulate that C. sativa containing greater than 0.3% THC is classified as marijuana, while plant material that contains less than or equal to 0.3% THC is hemp. Current methods to determine THC content are chromatography-based, which requires extensive sample preparation to render the materials into extracts suitable for sample injection, for complete separation and differentiation of THC from all other analytes present. This can create problems for forensic laboratories due to the increased workload associated with the need to analyze and quantify THC in all C. sativa materials.MethodThe work presented herein combines direct analysis in real time—high-resolution mass spectrometry (DART-HRMS) and advanced chemometrics to differentiate hemp and marijuana plant materials. Samples were obtained from several sources (e.g., commercial vendors, DEA-registered suppliers, and the recreational Cannabis market). DART-HRMS enabled the interrogation of plant materials with no sample pretreatment. Advanced multivariate data analysis approaches, including random forest and principal component analysis (PCA), were used to optimally differentiate these two varieties with a high level of accuracy.ResultsWhen PCA was applied to the hemp and marijuana data, distinct clustering that enabled their differentiation was observed. Furthermore, within the marijuana class, subclusters between recreational and DEA-supplied marijuana samples were observed. A separate investigation using the silhouette width index to determine the optimal number of clusters for the marijuana and hemp data revealed this number to be two. Internal validation of the model using random forest demonstrated an accuracy of 98%, while external validation samples were classified with 100% accuracy.DiscussionThe results show that the developed approach would significantly aid in the analysis and differentiation of C. sativa plant materials prior to launching painstaking confirmatory testing using chromatography. However, to maintain and/or enhance the accuracy of the prediction model and keep it from becoming outdated, it will be necessary to continue to expand it to include mass spectral data representative of emerging hemp and marijuana strains/cultivars.

Rapid, novel screening of toxicants in poison baits, and autopsy specimens by ambient mass spectrometry.

Animal poisoning and dissemination of baits in the environment have public health and ethological implications, which can be followed by criminal sanctions for those responsible. The reference methods for the analysis of suspect baits and autopsy specimens are founded on chromatographic-based techniques. They are extremely robust and sensitive, but also very expensive and laborious. For this reason, we developed an ambient mass spectrometry (AMS) method able to screen for 40 toxicants including carbamates, organophosphate and chlorinated pesticides, coumarins, metaldehyde, and strychnine. Spiked samples were firstly purified and extracted by dispersive solid phase extraction (QuEChERS) and then analyzed by direct analysis in real time high-resolution mass spectrometry (DART-HRMS). To verify the performance of this new approach, 115 authentic baits (n = 59) and necropsy specimens (gastrointestinal content and liver, n = 56) were assessed by the official reference methods and combined QuEChERS-DART-HRMS. The agreement between the results allowed evaluation of the performances of the new screening method for a variety of analytes and calculation of the resultant statistical indicators (the new method had overall accuracy 89.57%, sensitivity of 88.24%, and a specificity of 91.49%). Taking into account only the baits, 96.61% of overall accuracy was achieved with 57/59 samples correctly identified (statistical sensitivity 97.50%, statistical specificity 94.74%). Successful identification of the bitter compound, denatonium benzoate, in all the samples that contained rodenticides (28/28) was also achieved. We believe initial screening of suspect poison baits could guide the choice of reference confirmatory methods, reduce the load in official laboratories, and help the early stages of investigations into cases of animal poisoning.

Direct analysis in real time high-resolution mass spectrometry for authenticity assessment of lamb

In comparison to more traditional methods of determining food authenticity, such as gas chromatography analysis, the primary advantages of DART-HRMS include its high speed and throughput of analysis. This study used a non-targeted metabolomics method based on real-time high-resolution mass spectrometry combined with chemometric analysis to distinguish lamb samples from four regions. Orthogonal least squares-discriminant analysis revealed a distinct difference between these four lamb regions. The potential markers were chosen based on the variable's importance in projection values, variance, and fold change. A total of 79 markers were identified using the matching chemistry database. These markers differed significantly between lambs in four regions according to heatmap analysis. The linear discriminatory analysis model had an initial classification rate of 100.0% and a cross-validation accuracy of 82.50% on the identified markers. The research demonstrates that DART-HRMS can perform a rapid authentication evaluation of lamb samples.

Ambient mass spectrometry for rapid authentication of milk from Alpine or lowland forage.

Metabolomics approaches, such as direct analysis in real time-high resolution mass spectrometry (DART-HRMS), allow characterising many polar and non-polar compounds useful as authentication biomarkers of dairy chains. By using both a partial least squares discriminant analysis (PLS-DA) and a linear discriminant analysis (LDA), this study aimed to assess the capability of DART-HRMS, coupled with a low-level data fusion, discriminate among milk samples from lowland (silages vs. hay) and Alpine (grazing; APS) systems and identify the most informative biomarkers associated with the main dietary forage. As confirmed also by the LDA performed against the test set, DART-HRMS analysis provided an accurate discrimination of Alpine samples; meanwhile, there was a limited capacity to correctly recognise silage- vs. hay-milks. Supervised multivariate statistics followed by metabolomics hierarchical cluster analysis allowed extrapolating the most significant metabolites. Lowland milk was characterised by a pool of energetic compounds, ketoacid derivates, amines and organic acids. Seven informative DART-HRMS molecular features, mainly monoacylglycerols, could strongly explain the metabolomic variation of Alpine grazing milk and contributed to its classification. The misclassification between the two lowland groups confirmed that the intensive dairy systems would be characterised by a small variation in milk composition.

New strategies for the differentiation of fresh and frozen/thawed fish: A rapid and accurate non-targeted method by ambient mass spectrometry and data fusion (part A)

Ambient mass spectrometry (AMS) was applied for the first time to differentiate fresh and frozen/thawed Dicentrarchus labrax (European sea bass). One hundred and twenty samples were submitted to two extraction procedures and analyzed in positive and negative ion modes by direct analysis in real time-high resolution mass spectrometry (DART-HRMS). The four DART-HRMS datasets were concatenated with a low-level data fusion approach, and the resultant unique block of data was submitted to multivariate statistical analysis to tease out the most informative m/z values capable of codifying fresh and defrosted D. labrax . The statistical significance (p-value) and fold change (FC) of these informative signals were then evaluated with univariate analyses and tentatively assigned. The 25 features were then used to build a support vector machine (SVM) model capable of classifying D. labrax samples according to whether they were fresh or previously frozen. The SVM model has accuracy, sensitivity and specificity of 100%, both in training and test set. The SVM model was used to successfully classify an independent set of twenty fresh and frozen/thawed Salmo salar (Atlantic salmon). The concentrations of the most significant molecular features were quantified by gas-chromatography-mass spectrometry. • (+/−)DART-HRMS of polar and non-polar extracts of sea bass was performed. • The four DART-HRMS datasets were concatenated by low-level data fusion. • PLS-DA was performed on the training set to extrapolate the informative signals. • SVM classification model was constructed with the selected signals. • The built-in SVM model was validated with on hold-out subset with high accuracy.

Coral Genus Differentiation Based on Direct Analysis in Real Time-High Resolution Mass Spectrometry-Derived Chemical Fingerprints.

Coral reefs are one of the most biologically diverse ecosystems, and the accurate identification of the species is essential for diversity assessment and conservation. Current genus determination approaches are time-consuming and resource-intensive and can be highly subjective. To explore the hypothesis that the small-molecule profiles of coral are genus-specific and can be used as a rapid tool to catalogue and distinguish between coral genera, the small-molecule chemical fingerprints of the species Acanthastrea echinata, Catalaphyllia jardinei, Duncanopsammia axifuga, Echinopora lamellosa, Euphyllia divisa, Euphyllia paraancora, Euphyllia paradivisa, Galaxea fascicularis, Herpolitha limax, Montipora confusa, Monitpora digitata, Montipora setosa, Pachyseris rugosa, Pavona cactus, Plerogyra sinuosa, Pocillopora acuta, Seriatopora hystrix, Sinularia dura, Turbinaria peltata, Turbinaria reniformis, Xenia elongata, and Xenia umbellata were generated using direct analysis in real time-high resolution mass spectrometry (DART-HRMS). It is demonstrated here that the mass spectrum-derived small-molecule profiles for coral of different genera are distinct. Multivariate statistical analysis processing of the DART-HRMS data enabled rapid genus-level differentiation based on the chemical composition of the coral. Coral samples were analyzed with no sample preparation required, making the approach rapid and efficient. The resulting spectra were subjected to kernel discriminant analysis (KDA), which furnished accurate genus differentiation of the coral. Leave-one-out cross-validation (LOOCV) was carried out to determine the classification accuracy of each model and confirm that this approach can be used for coral genus attribution with prediction accuracies ranging from 86.67 to 97.33%. The advantages and application of the statistical analysis to DART-HRMS-derived coral chemical signatures for genus-level differentiation are discussed.

Rapid Screening of Antibiotic Residues in Milk Powder Using Direct Analysis in Real Time High Resolution Mass Spectrometry

Rapid Screening of Antibiotic Residues in Milk Powder Using Direct Analysis in Real Time High Resolution Mass Spectrometry

A longevity study to determine appropriate storage conditions for sexual lubricants using spectroscopic and spectrometric techniques

There has been an increase in the number of reported cases in which assailants have used a condom or sexual lubricant. Sexual predators understand that DNA from seminal fluid is one of the main forms of evidence used to connect a suspect to a victim or crime scene; therefore, the use of condoms in sexual assault cases has increased. This dilemma has led to research into other types of trace evidence (e.g., sexual lubricants). The study presented herein was performed to determine the optimal storage time length and conditions appropriate for storing trace lubricant residue. This research project used Fourier transform infrared spectroscopy (FTIR), direct analysis in real time-high resolution mass spectrometry (DART-HRMS), and gas chromatography-mass spectrometry (GC-MS) techniques to analyze the stored swabs containing sexual lubricants at 8 timepoints over 18 months. Blank, personal lubricant, and condom swabs were stored under several environmental conditions. Swab storage allowed for the determination of whether lubricants degraded, thus adversely affecting the ability to recognize the classification patterns. Resulting spectra from each instrument can be used to identify environmental degradation of lubricant samples and generate investigative leads. Pearson Correlation Coefficients and time plots of the chemical components were used to determine if chemical breakdown occurred for each lubricant and if the pattern changed significantly. Results showed that the strongest correlations occur between the initial timepoint (T 0) and one month (T1m), with correlations decreasing consistently up to eighteen months (T18m). The best storage conditions were determined to be 22 °C at both 16% and 50% humidity. The ability to understand the effects of environment and time to the analysis of sexual lubricants may offer helpful information when analyzing sexual assault kits.

High-performance thin-layer chromatography and direct analysis in real time-high resolution mass spectrometry of non-extractable polyphenols from tropical fruit peels

Passiflora species, mangosteen, and cherimoya peels are a source of bioactive phenolic compounds. Nevertheless, a significant fraction of polyphenols, called non-extractable polyphenols (NEPs), are retained in the extraction residue after a conventional extraction. Thus, alkaline, acid, and enzymatic-assisted extractions to recover high contents of antioxidant NEPs from the extraction residue of fruit peels, were compared in this work. A high-performance thin-layer chromatography method with UV/Vis detection was developed in order to obtain the phenolic profile for the extracts. The most intense bands were further analyzed by direct analysis in real-time-high-resolution mass spectrometry to tentatively identified NEPs in fruit peel extracts. Total phenolic and proanthocyanidin contents and antioxidant capacity of the extracts were measured to carry out a multivariate statistical analysis. Alkaline hydrolysis was the most efficient treatment to recover NEPs from fruit peels as well as a promising treatment to obtain antioxidant extracts along with EAE. Cherimoya peel extracts were the richest in antioxidant NEPs. This work highlights that many NEPs remain on the extraction residue of fruit peels after conventional extraction and are not usually taken into account.

Oregano authentication by mid-level data fusion of chemical fingerprint signatures acquired by ambient mass spectrometry

Economically motivated adulteration (EMA) of herbs and spices is very frequent and a major cause of concern to consumers, manufacturers and legislators. Rapid, non-targeted methods capable of distinguishing authentic herbs from those mixed with low cost materials are desirable. To this aim, ambient mass spectrometry (AMS) was applied to authenticate dried oregano leaves. Authentic and adulterated oregano samples were submitted to two extraction procedures and analysed in positive and negative ion modes by direct analysis in real time-high resolution mass spectrometry (DART-HRMS). Mid-level data fusion of the four blocks of DART-HRMS data was performed, and the resultant unique dataset was submitted to supervised statistical analysis to ascertain the signals that discriminate authentic from adulterated oregano. The fourteen most informative signals of authenticity were tentatively assigned and validated by support vector machine (SVM). In the final model, accuracy, sensitivity and specificity of >90% were obtained. Independent authentic and adulterated oregano samples were used to validate the discriminative m/z values and evaluate the classification capability of the model. To the best of our knowledge this is the first application of AMS coupled to mid-level data fusion for oregano authentication.

Bioprofiling of Mexican Plectranthus amboinicus (Lour.) essential oil via planar chromatography–effect-directed analysis combined with direct analysis in real time high-resolution mass spectrometry

Aerial parts of Plectranthus amboinicus (Lour.) were collected in the Northern Mexico City for a comprehensive effect-directed profiling. Its hydrodistilled P. amboinicus essential oil (PAEO) was separated on HPTLC silica gel plates with n-hexane—ethyl acetate—ethanol, 95:3:2, followed by derivatization with the anisaldehyde – sulfuric acid reagent. The UV/Vis/FLD detection was expanded by a biological and biochemical detection. Eight different effect-directed assays were performed including the 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay as well as biochemical assays for inhibition of acetylcholinesterase, α- and β-glucosidases, α-amylase and tyrosinase as well as antimicrobial assays against Gram-negative Aliivibrio fischeri and Gram-positive Bacillus subtilis bacteria. The bioprofiling, subsequent direct analysis in real time mass spectrometry of the detected five bioactive compound zones and comparison with literature data resulted in the tentative assignment of caryophyllene oxide (hRF 20), α-humulene (hRF 26), carvacrol (hRF 40), methyl carvacrol ether (hRF 76) and caryophyllene (hRF 84). The antimicrobial activity against Gram-negative bacteria of PAEO was elicited by a mixture of the multi-potent compounds, whereas α-humulene was strongly acting against Gram-positive bacteria. The antioxidant effect was related to carvacrol and the inhibitory effect over AChE, tyrosinase and glucosidases to α-humulene.

Rapid fingerprinting of source and environmental microplastics using direct analysis in real time-high resolution mass spectrometry

Microplastics are ubiquitous in the aquatic and terrestrial environment. To prevent further contamination, methods to determine their sources are needed. Techniques to quantify and characterize microplastics in the environment are still evolving for polymers and the additives and leachable substances embedded therein, which constitute the “chemical fingerprint” of an environmental microplastic. There is a critical need for analytical methods that yield such diagnostic information on environmental microplastics that enables identification of their composition and sources of pollution. This study reports on a novel approach for rapid fingerprinting of environmental microplastics and the screening of additives using Direct Analysis in Real Time (DART)-high resolution mass spectrometry. A variety of plastic samples were investigated, including virgin pre-production pellets, microbeads from personal care products, microplastics found in the aquatic environment, and synthetic fibers. The resulting mass spectra display ∼10,000 discrete peaks, corresponding to plastic additives released by thermal desorption and polymer degradation products generated by pyrolysis. These were used to characterize differences among plastic types, microplastic source materials, and environmental samples. Multivariate statistics and elemental composition analysis approaches were applied to analyze fingerprints from the mass spectra. This promising analytical approach is sensitive, (potentially) high-throughput, and can aid in the elucidation of possible sources of microplastics and perhaps eventually to the analysis of bulk environmental samples for plastics.

Rapid detection and validated quantification of psychoactive compounds in complex plant matrices by direct analysis in real time-high resolution mass spectrometry - Application to "Kava" psychoactive pepper products.

Classified by the UNODC as a top 20 plant of concern, Piper methysticum (also known as Kava) is being increasingly abused recreationally for its mind-altering effects. It is of significant forensic relevance to establish methods to rapidly identifyand quantify psychoactive compounds, especially those yet to be scheduled ascontrolled substances and which have exhibited various noteworthy health concerns. Direct analysis in real time high-resolution mass spectrometry (DART-HRMS) demonstrated the ability to detect a range of kavalactones in Pipermethysticum derived products and plant material with no sample preparation. Inaddition, a validated method using calibration curves developed with a deuteratedinternal standard was used for the quantification of the psychoactive moleculeyangonin in various products. DART-HRMS detected the protonated masses of six major kavalactonesand three flavokavains in 18 commercial Kava products. A method consistent withFDA validation guidelines was established for the quantification of yangonin in thevarious complex matrices. Implementation of this method, with an LLOQ of 5 mg/mL, enabled successful quantification of yangonin in 16 Kava products.Concentrations for solid products ranged from 2.71 to 8.99 mg/g, while that forliquid products ranged from 1.03 to 4.59 mg/mL. Rapid identification and quantification of psychoactive smallmolecules in plant material can be accomplished using a validated DART-HRMSprotocol. This work illustrates an approach to qualitative and quantitative analysesof a wide variety of complex matrices derived from plants, and demonstrates thatthe commercially available products analyzed are P. methysticum derived and docontain psychoactive yangonin at quantifiable levels.

Call it a “nightshade”—A hierarchical classification approach to identification of hallucinogenic Solanaceae spp. using DART-HRMS-derived chemical signatures

Plants that produce atropine and scopolamine fall under several genera within the nightshade family. Both atropine and scopolamine are used clinically, but they are also important in a forensics context because they are abused recreationally for their psychoactive properties. The accurate species attribution of these plants, which are related taxonomically, and which all contain the same characteristic biomarkers, is a challenging problem in both forensics and horticulture, as the plants are not only mind-altering, but are also important in landscaping as ornamentals. Ambient ionization mass spectrometry in combination with a hierarchical classification workflow is shown to enable species identification of these plants. The hierarchical classification simplifies the classification problem to primarily consider the subset of models that account for the hierarchy taxonomy, instead of having it be based on discrimination between species using a single flat classification model. Accordingly, the seeds of 24 nightshade plant species spanning 5 genera (i.e. Atropa, Brugmansia, Datura, Hyocyamus and Mandragora), were analyzed by direct analysis in real time-high resolution mass spectrometry (DART-HRMS) with minimal sample preparation required. During the training phase using a top-down hierarchical classification algorithm, the best set of discriminating features were selected and evaluated with a partial least square-discriminant analysis (PLS-DA) classifier to discriminate and visualize the data. The method yields species identity through a class hierarchy, and reveals the most significant markers for differentiation. The overall accuracy of the approach for species identification was 95% and 96% using 100X bootstrapping validation and test samples respectively. The method can be extended for the rapid identification of an infinite number of plant species.

Comparison of high-performance thin-layer with overpressured layer chromatography combined with direct bioautography and direct analysis in real time mass spectrometry for tansy root

Bioactive polyacetylenes in tansy (Tanacetum vulgare L.) root extract were separated by overpressured layer chromatography (OPLC) with better resolutions than achievable by capillary flow high-performance thin-layer chromatography (HPTLC). OPLC enabled a longer development distance in a shorter time (16 cm, 7 min) in comparison to conventional HPTLC (7 cm, 16 min). OPLC separations on HPTLC plates with spherical particles resulted in higher resolutions than on such with irregular particles. A slight distortion of zones sometimes occurred that could be eliminated by sonication of the mobile phase through removal of the dissolved air. However, zone distortion did not diminish the meaning of the qualitative outcome. The combination of OPLC with direct bioautography and direct analysis in real time-high resolution mass spectrometry (DART-HRMS) led to the determination of the individual polyacetylenes responsible for the diverse effects. In each recorded mass spectrum, the basepeak was the respective protonated molecule. The assignments confirmed the results of a previous study, however, compounds TR5a (tetradeca-2,4,6-triine-8-en-12-one) and TR5b (trans-2-(2,4-hexadiynylidene)-1,6-dioxaspiro[4,5]dec-3-ene) that were not separable by HPTLC were partially separated by OPLC. Thus, it was proven that TR5b was not an oxidation product of TR5a and thus not formed as artefact during the HPTLC-DART-HRMS analysis, but originally present in the sample.

Bioassay-Guided Isolation and Structure Elucidation of Fungicidal and Herbicidal Compounds from Ambrosia salsola (Asteraceae).

The discovery of potent natural and ecofriendly pesticides is one of the focuses of the agrochemical industry, and plant species are a source of many potentially active compounds. We describe the bioassay-guided isolation of antifungal and phytotoxic compounds from the ethyl acetate extract of Ambrosia salsola twigs and leaves. With this methodology, we isolated and identified twelve compounds (four chalcones, six flavonols and two pseudoguaianolide sesquiterpene lactones). Three new chalcones were elucidated as (S)-β-Hydroxy-2′,3,4,6′-tetrahydroxy-5-methoxydihydrochalcone (salsolol A), (S)-β-Hydroxy-2′,4,4′,6′-tetrahydroxy-3-methoxydihydrochalcone (salsolol B), and (R)-α, (R)-β-Dihydroxy-2′,3,4,4′,6′-pentahydroxydihydrochalcone (salsolol C) together with nine known compounds: balanochalcone, six quercetin derivatives, confertin, and neoambrosin. Chemical structures were determined based on comprehensive direct analysis in real time-high resolution mass spectrometry (HR-DART-MS), as well as 1D and 2D NMR experiments: Cosy Double Quantum Filter (DQFCOSY), Heteronuclear Multiple Quantum Coherence (HMQC) and Heteronuclear Multiple Bond Coherence (HMBC), and the absolute configurations of the chalcones were confirmed by CD spectra analysis. Crystal structure of confertin was determined by X-ray diffraction. The phytotoxicity of purified compounds was evaluated, and neoambrosim was active against Agrostis stolonifera at 1 mM, while confertin was active against both, Lactuca sativa and A. stolonifera at 1 mM and 100 µM, respectively. Confertin and salsolol A and B had IC50 values of 261, 275, and 251 µM, respectively, against Lemna pausicotata (duckweed). The antifungal activity was also tested against Colletotrichum fragariae Brooks using a thin layer chromatography bioautography assay. Both confertin and neoambrosin were antifungal at 100 µM, with a higher confertin activity than that of neoambrosin at this concentration.