Ultra-high-performance Liquid Chromatography Coupled To Mass Spectrometry Research Articles

Structural Studies of Mexican Husk Tomato (Physalis ixocarpa) Fruit Cutin.

Green tomato (Physalis ixocarpa) is a specie native to Mexico, and it is known as "tomatillo" or "husk tomato". The fruit contains vitamins, minerals, phenolic compounds, and steroidal lactones, presenting antimicrobial activity and antinarcotic effects. Therefore, it is not only used in traditional Mexican cuisine, but also in traditional medicine to relieve some discomforts such as fever, cough, and amygdalitis. However, it is a perishable fruit whose shelf life is very short. As a part of the peel, cuticle, and epicuticular waxes represent the most important part in plant protection, and the specific composition and structural characterization are significant to know how this protective biopolymer keeps quality characteristics in fresh fruits. P. ixocarpa cutin was obtained by enzymatic treatments (cellulase, hemicellulose, and pectinase) and different concentrations of TFA, and studied through Cross Polarization Magic Angle Spinning Nuclear Magnetic Resonance (CPMAS 13C NMR), Ultra-High Performance Liquid Chromatography coupled to Mass Spectrometry (UHPLC-MS), and was morphologically characterized by Confocal Laser Scanning Microscopy (CLSM) and Scanning Electron Microscopy (SEM). The main constituents identified under the basis of UHPLC-MS analysis were 9,10,18-trihydroxy-octadecanoic acid and 9,10-epoxy-18-hydroxy-octadecanoic acid with 44.7 and 37.5%, respectively. The C16 absence and low occurrence of phenolic compounds, besides the presence of glandular trichomes, which do not allow a continuous layer on the surface of the fruit, could be related to a lower shelf life compared with other common fruits such as tomato (Solanum lycopersicum).

Simultaneous UHPLC-MS Quantification of Catechins and Untargeted Metabolomic Profiling for Proof-of-Concept Authenticity Determination of Maytenus ssp. Samples.

Due to the widespread use of Maytenus ilicifolia leaves in the treatment of gastric ulcers, herbal medicines derived from such species are distributed by the national health system in Brazil. A related species, Maytenus aquifolium, is also used for the same disorders, and both are popularly known as Espinheira-santa. Due to their popular use, the quality and efficiency of the herbal medicines derived from these species is an important public health issue. The purpose of this study was to develop and test an analytical method that could quantify the content of catechin and epicatechin in dry Maytenus spp. leaves and simultaneously obtain their chemical profile to determine authenticity of the leaf samples. Ultra-high performance liquid chromatography coupled to mass spectrometry (UHPLC-MS) was used to quantify these isomers, in the selected ion monitoring (SIM) mode, while simultaneously analyzing the extract in full-scan mode. This approach was successfully applied to the analysis of commercial and authentic samples of Maytenus spp. Fewer than half the samples presented the minimum epicatechin content of 2.8 mg per g of dry leaf mass, as specified in the 6th Brazilian Pharmacopoeia (2019) for M. ilicifolia. Furthermore, by using untargeted metabolomics, it was observed that the chemical profile of most the samples was not compatible with M. ilicifolia leaves, indicating the need for stricter quality control of this material. The method described herein could be used for this control; moreover, its concept could be adapted and used for an ample variety of medicinal plant products.

Comprehending Cardiac Dysfunction by Oxidative Stress: Untargeted Metabolomics of In Vitro Samples.

Cardiovascular diseases (CVDs) are noncommunicable diseases known for their complex etiology and high mortality rate. Oxidative stress (OS), a condition in which the release of free radical exceeds endogenous antioxidant capacity, is pivotal in CVC, such as myocardial infarction, ischemia/reperfusion, and heart failure. Due to the lack of information about the implications of OS on cardiovascular conditions, several methodologies have been applied to investigate the causes and consequences, and to find new ways of diagnosis and treatment as well. In the present study, cardiac dysfunction was evaluated by analyzing cells’ alterations with untargeted metabolomics, after simulation of an oxidative stress condition using hydrogen peroxide (H2O2) in H9c2 myocytes. Optimizations of H2O2 concentration, cell exposure, and cell recovery times were performed through MTT assays. Intracellular metabolites were analyzed right after the oxidative stress (oxidative stress group) and after 48 h of cell recovery (recovery group) by ultra-high-performance liquid chromatography coupled to mass spectrometry (UHPLC-MS) in positive and negative ESI ionization mode. Significant alterations were found in pathways such as “alanine, aspartate and glutamate metabolism”, “glycolysis”, and “glutathione metabolism”, mostly with increased metabolites (upregulated). Furthermore, our results indicated that the LC-MS method is effective for studying metabolism in cardiomyocytes and generated excellent fit (R2Y > 0.987) and predictability (Q2 > 0.84) values.

Lipidomic signatures from physically frail and robust older adults at hospital admission

Identifying serum biomarkers that can predict physical frailty in older adults would have tremendous clinical value for primary care, as this condition is inherently related to poor quality of life and premature mortality. We compared the serum lipid profile of physically frail and robust older adults to identify specific lipid biomarkers that could be used to assess physical frailty in older patients at hospital admission. Forty-three older adults (58.1% male), mean (range) age 86.4 (78–100 years) years, were classified as physically frail (n = 18) or robust (n = 25) based on scores from the Short Physical Performance Battery (≤ 6 points). Non-targeted metabolomic study by ultra-high performance liquid chromatography coupled to mass spectrometry (UHPLC-MS) analysis with later bioinformatics data analysis. Once the significantly different metabolites were identified, the KEGG database was used on them to establish which were the metabolic pathways mainly involved. Area under receiver-operating curve (AUROC) analysis was used to test the discriminatory ability of lipid biomarkers for frailty based on the Short Physical Performance Battery. We identified a panel of five metabolites including ceramides Cer (40:2), Cer (d18:1/20:0), Cer (d18:1/23:0), cholesterol, and phosphatidylcholine (PC) (14:0/20:4) that were significantly increased in physically frail older adults compared with robust older adults at hospital admission. The most interesting in the physically frail metabolome study found with the KEGG database were the metabolic pathways, vitamin digestion and absorption, AGE-RAGE signaling pathway in diabetic complications, and insulin resistance. In addition, Cer (40:2) (AUROC 0.747), Cer (d18:1/23:0) (AUROC 0.720), and cholesterol (AUROC 0.784) were identified as higher values of physically frail at hospital admission. The non-targeted metabolomic study can open a wide view of the physically frail features changes at the plasma level, which would be linked to the physical frailty phenotype at hospital admission. Also, we propose that metabolome analysis will have a suitable niche in personalized medicine for physically frail older adults.

Ultra-HPLC-MS pseudo-targeted metabolomic profiling reveals metabolites and associated metabolic pathway alterations in Asian plum (Prunus salicina) fruits in response to gummosis disease.

Plum (Prunus spp.) is an economically and nutritionally important stone fruit that is grown worldwide. Gummosis disease (GD) is one of the most common limiting factors that adversely affects the yield and quality of stone fruits such as plum. Elucidating plum fruit metabolomics responses is essential to develop sustainable agricultural practices to combat GD in the future. Herein, an ultra-high-performance liquid chromatography coupled to mass-spectrometry (UHPLC-MS) pseudo-targeted metabolomic profiling was first performed to elucidate the overall metabolic alterations in Asian plum (Prunus salicina Lindl.) fruit in response to GD. The most pivotal differential metabolites, including certain amino acids and proanthocyanidins, in GD and control groups were identified by combining multivariate data analysis with strict statistical criteria. Metabolic pathway enrichment analysis showed that GD induced a series of coordinated defence responses and reprogramming of various metabolic pathways, including glucosinolate biosynthesis, 2-oxocarboxylic acid metabolism, valine, leucine and isoleucine degradation, and isoquinoline alkaloid biosynthesis pathways. Using UHPLC-MS-based pseudo-targeted metabolomic profiling, we systematically evaluated overall metabolic modifications in Asian plum fruits in response to GD for the first time. The identified metabolic pathway alterations helped to better understand the internal relationships and related metabolic networks.

A quality control perspective on Devil's claw, Harpagophytum procumbens and H. zeyheri: phytochemical analysis and DNA barcoding

Devil's claw is a popular natural anti-inflammatory phytomedicine. The preparation of Devil's claw products is complicated by the similarity between the morphotypes of Harpagophytum procumbens and H. zeyheri. This study investigated the potential of a validated ultra-high performance liquid chromatography coupled to mass spectrometry (UHPLC-MS) method to investigate the harpagoside content of methanol extracts of the secondary tubers of both species. In addition, DNA barcoding was used to determine the identity of the Harpagophytum species included in commercial Devil's claw products. Herbal reference material of H. procumbens and H. zeyheri, originally harvested in southern Africa, was morphologically authenticated and commercial products containing Harpagophytum were purchased on the internet using the search terms “Harpagophytum” or “Devil's claw.” High-performance thin-layer chromatography (HPTLC) and UHPLC-MS was used for the qualitative and quantitative determination of harpagoside content in commercial products. The UHPLC-MS method developed had a limit of detection (LOD) of 0.165 µg/mL and a limit of quantification (LOQ) of 0.499 µg/mL. Quantitative UHPLC-MS analysis revealed high variability in the harpagoside content for both species; H. procumbens (0.17-4.37%) and H. zeyheri (0.00-3.07%). Only 41% of H. procumbens and 17% of H. zeyheri raw materials contained harpagoside. Using HPTLC, 90% of the commercial products tested were determined to contain harpagoside. The results were congruent with the UHPLC-MS analysis where the harpagoside content was out of specification for 10% of analyzed commercial products. The harpagoside content in commercial products varied from not detected to 3.50%. PCR amplification followed by comparative sequencing analysis of trnL-F, and ycf1 plastid gene amplicons indicated specific sites in trnL-F at positions 118 and 146 and at position 538 in ycf1 amplicons suitable to distinguish the two Harpagophytum species. These sites were used to barcode the herbal products and Character-based Barcode Recognition Obtained with Nucleotide eXposés (BRONX) analyses verified the taxonomic identity of the Harpagophytum in the samples. The results revealed that 76% (16/21) of the commercial products tested were incorrectly labelled as H. procumbens and, instead, contained H. zeyheri. Our study suggests that DNA barcoding in combination with the UHPLC-MS and HPTLC could significantly support herbal product authentication. This study contributed knowledge and methods that can be implemented to enhance the quality of commercial Devil's claw products.

α-Glucosidase Inhibitory Activity of Tannat Grape Phenolic Extracts in Relation to Their Ripening Stages

The present study aimed to screen grape extracts as novel α-glucosidase inhibitors to prevent type-2 diabetes and hyperglycemia. The total polyphenol content (TPC) was measured by Folin-Ciocalteu assay and the stilbene, anthocyanin and flavan-3-ol compounds were measured by Ultra High-Performance Liquid Chromatography coupled to Mass Spectrometry (UHPLC-MS). The α-glucosidase inhibitory of seed and skin Tannat grape extracts at four ripening stages were investigated. The highest TPC values were measured in seeds at the “veraison stage” (65.29 ± 5.33 g of Gallic Acid Equivalent (GAE) per kilogram of Fresh Weight (FW)). This was in accordance with the high flavan-3-ol contents measured for these two extracts (43.22 ± 2.59 and 45.45 ± 6.48 g/kg of seeds FW, respectively). The skin and seed extracts at the first stage of ripening exerted strong α-glucosidase inhibition, exceeding 95% (p < 0.05). A high linear correlation (R = 0.723, p ≤ 0.05) was observed between flavan-3-ol contents and the α-glucosidase inhibitory activity. The stilbene contents and this activity were moderately to strongly anti-correlated (R = –0.828, p ≤ 0.05 for trans-resveratrol). The enzyme kinetic studies revealed a mixed type of inhibition. This study brings promising results for the therapeutic potential of seed and skin Tannat grape extracts as a functional food product with anti-diabetic activity.

A Novel Serum Metabolomic Profile for the Differential Diagnosis of Distal Cholangiocarcinoma and Pancreatic Ductal Adenocarcinoma.

The diagnosis of adenocarcinomas located in the pancreas head, i.e., distal cholangiocarcinoma (dCCA) and pancreatic ductal adenocarcinoma (PDAC), constitutes a clinical challenge because they share many symptoms, are not easily distinguishable using imaging techniques and accurate biomarkers are not available. Searching for biomarkers with potential usefulness in the differential diagnosis of these tumors, we have determined serum metabolomic profiles in healthy controls and patients with dCCA, PDAC or benign pancreatic diseases (BPD). Ultra-high-performance liquid chromatography coupled to mass spectrometry (UHPLC-MS) analysis was performed in serum samples from dCCA (n = 34), PDAC (n = 38), BPD (n = 42) and control (n = 25) individuals, divided into discovery and validation cohorts. This approach permitted 484 metabolites to be determined, mainly lipids and amino acids. The analysis of the results led to the proposal of a logistic regression model able to discriminate patients with dCCA and PDAC (AUC value of 0.888) based on the combination of serum levels of nine metabolites (acylcarnitine AC(16:0), ceramide Cer(d18:1/24:0), phosphatidylcholines PC(20:0/0:0) and PC(O-16:0/20:3), lysophosphatidylcholines PC(20:0/0:0) and PC(0:0/20:0), lysophosphatidylethanolamine PE(P-18:2/0:0), and sphingomyelins SM(d18:2/22:0) and SM(d18:2/23:0)) and CA 19-9. In conclusion, we propose a novel specific panel of serum metabolites that can help in the differential diagnosis of dCCA and PDAC. Further validation of their clinical usefulness in prospective studies is required.

Oleamide, a Bioactive Compound, Unwittingly Introduced into the Human Body through Some Plastic Food/Beverages and Medicine Containers.

The purpose of the study was to investigate the migration of oleamide, a polymer lubricant, and a bioactive compound, from various plastic, marketed containers for food/beverages and medicines into polymer contact liquid. Methanol, food/medicine simulants or real samples were used to extract polymer leachables and extractables. Migrated oleamide into polymer contact liquids was determined by ultra-high performance liquid chromatography coupled to mass spectrometry (UHPLC-MS). The concentration of oleamide in the extracts of medicinal and insulin syringes was 7351 ng mL−1 and 21,984 ng mL−1, respectively. The leachates of intravenous (i.v.) infusion bottle, medicinal and insulin syringes contained 17 ng mL−1, 12 ng mL−1 and 152 ng mL−1, respectively. Oleamide in the extracts of dummies ranged from 30 to 39 ng mL−1, while in the leachates of baby bottles, from 12 to 23 ng mL−1. Leachates of soft drink bottles contained from 6 to 15 ng mL−1 oleamide, milk bottles from 3 to 9 ng mL−1, liquid yogurt bottles 17 ng mL−1 and water bottles from 11 to 18 ng mL−1. Bottled real matrices of oil and milk contained oleamide in the range from 217 to 293 ng mL−1. Moreover, the source of migrated oleamide (e.g., containers, caps, other parts) was identified. Oleamide is listed in the current EU regulations without a specific migration limit. Accordingly, these values are considered of no concern, unless future toxicological studies prove the opposite.

Combination of stable isotope ratio data and chromatographic impurity signatures as a comprehensive concept for the profiling of highly prevalent synthetic cannabinoids and their precursors

In this study, we utilized elemental analyser (EA) and gas-chromatography (GC) isotope ratio mass spectrometry (IRMS) and ultra-high-performance liquid chromatography coupled to mass spectrometry (UHPLC-MS) in a comprehensive profiling approach assessing the chromatographic impurity signatures and δ13C and δ15N isotope ratios of synthetic cannabinoids from police seizures and internet test purchases. Main target of this study was the highly prevalent synthetic cannabinoid MDMB-CHMICA (methyl (2S)-2-([1-(cyclohexylmethyl)-1H-indol-3-yl]formamido)-3,3-dimethylbutaoate). Overall, 61 powder and 118 herbal blend (also called “Spice-Products”) samples were analysed using both analytical techniques and evaluated in a joint model to link samples from a common source. As a key finding, three agglomerates of Spice-product samples with similar dates of purchase were identified in the IRMS data, possibly representing larger shipments of MDMB-CHMICA, each produced with the same precursor material, successively delivered to the European market. The three agglomerates were refined into multiple sub-clusters based on the impurity profiling data, each representing an individual synthesis batch. One of the agglomerates identified in the IRMS data was found to consist two groups of four sub-clusters, respectively, with majorly different impurity profiles, demonstrating the necessity for both analytical techniques to extract the maximum amount of information from a limited sample pool. Additionally, 31 samples containing the recently surfaced synthetic cannabinoid Cumyl-PeGaClone (5-pentyl-2-(2-phenylpropan-2-yl)-2,5-dihydro-1H-pyrido[4,3-b]indol-1-one) were analysed for their and δ13C and δ15N isotope ratios to put the isotopic data recorded for MDMB-CHMNICA in a more global perspective. Three building blocks of precursor chemicals (indole, tert-leucine, cumylamine) potentially used for the synthesis of the two named synthetic cannabinoids were acquired from different global vendors and measured for their δ13C and δ15N isotope ratios to better understand variations in the isotopic composition of the synthetic cannabinoids and to trace their origin.

Profiling of synthesis-related impurities of the synthetic cannabinoid Cumyl-5F-PINACA in seized samples of e-liquids via multivariate analysis of UHPLC-MSn data.

Vaping of synthetic cannabinoids via e-cigarettes is growing in popularity. In the present study, we tentatively identified 12 by-products found in a pure sample of the synthetic cannabinoid Cumyl-5F-PINACA (1-(5-fluoropentyl)-N-(2-phenylpropan-2-yl)-1H-indazole-3-carboxamide), a prevalent new psychoactive substance (NPS) in e-liquids, via high-resolution mass spectrometry fragmentation experiments (HRMS/MS). Furthermore, we developed a procedure to reproducibly extract this synthetic cannabinoid and related by-products from an e-liquid matrix via chloroform and water. The extracts were submitted to flash chromatography (F-LC) to isolate the by-products from the main component. The chromatographic impurity signature was subsequently assessed by ultra-high-performance liquid chromatography coupled to mass spectrometry (UHPLC-MS) and evaluated by automated integration. The complete sample preparation sequence (F-LC+UHPLC-MS) was validated by comparing the semi-quantitative signal integrals of the chromatographic impurity signatures of five self-made e-liquids with varying concentrations of Cumyl-5F-PINACA [0.1, 0.2, 0.5, 0.7 and 1.0% (w/w)], giving an average relative standard deviation of 6.2% for triplicate measurements of preparations of the same concentration and 10.5% between the measurements of the five preparations with different concentrations. Lastly, the chromatographic signatures of 14 e-liquid samples containing Cumyl-5F-PINACA from police seizures and Internet test purchases were evaluated via hierarchical cluster analysis for potential links. For the e-liquid samples originating from test purchases, it was found that the date of purchase, the identity of the online shop, and the brand name are the critical factors for clustering of samples.

Deciphering the Resistance Mechanism of Tomato Plants Against Whitefly-Mediated Tomato Curly Stunt Virus Infection through Ultra-High-Performance Liquid Chromatography Coupled to Mass Spectrometry (UHPLC-MS)-Based Metabolomics Approaches.

Begomoviruses, such as the Tomato curly stunt virus (ToCSV), pose serious economic consequences due to severe crop losses. Therefore, the development and screening of possible resistance markers is imperative. While some tomato cultivars exhibit differential resistance to different begomovirus species, in most cases, the mechanism of resistance is not fully understood. In this study, the response of two near-isogenic lines of tomato (Solanum lycopersicum), differing in resistance against whitefly-mediated ToCSV infection were investigated using untargeted ultra-high-performance liquid chromatography coupled to mass spectrometry (UHPLC-MS)-based metabolomics. The responses of the two lines were deciphered using multivariate statistics models. Principal component analysis (PCA) scores plots from various time intervals revealed that the resistant line responded more rapidly with changes to the metabolome than the susceptible counterpart. Moreover, the metabolic reprogramming of chemically diverse metabolites that span a range of metabolic pathways was associated with the defence response. Biomarkers primarily included hydroxycinnamic acids conjugated to quinic acid, galactaric acid, and glucose. Minor constituents included benzenoids, flavonoids, and steroidal glycoalkaloids. Interestingly, when reduced to the level of metabolites, the phytochemistry of the infected plants’ responses was very similar. However, the resistant phenotype was strongly associated with the hydroxycinnamic acid derivatives deployed in response to infection. In addition, the resistant line was able to mount a stronger and quicker response.

Amphetamine-type stimulants analysis in oral fluid based on molecularly imprinting extraction

A methamphetamine-based molecularly imprinted polymer (MIP) has been prepared by bulk polymerization to recognize new psychoactive substances (NPS) of the amphetamine, cathinones and 2C families in oral fluid samples, being the first precedent of a synthetized MIP for the extraction and preconcentration 32 NPS including amphetamine type substances and synthetic cathinones from oral fluids. Pre-polymerization complex and resulting materials were appropriately characterized by infrared spectroscopy, scanning electron microscopy, and nitrogen adsorption-desorption isotherms. Appropriateness of the material for the specific recognition of the target analytes was also evaluated through computational calculations and experimentally assessed by solid phase extraction (SPE). The most appropriate SPE conditions were evaluated and recoveries of 32 different NPS were obtained, ranging from 80 to 120% with a relative standard deviation (RSD) in all cases lower than 12%. Amphetamine-related NPS were analyzed by a fast and portable methodology based on ion mobility spectrometry (IMS) and a rearguard procedure based on ultra-high performance liquid chromatography coupled to mass spectrometry (UHPLC-MS) providing limit of detection values from 10 to 80 μg L−1 and from 0.03 to 1.3 μg L−1, respectively. Oral fluid samples, containing different interferents like caffeine, fluticasone and cetirizine, were spiked with 300 μg L−1 amphetamine and subsequently analyzed, showing recoveries ranging from 81 to 115% using both methodologies. Thus, this paper shows preliminary results to demonstrate the applicability of the developed procedure which could be used with minor modifications as screening technique in on-road drug analysis.

Identification of degraded bone and tooth splinters from arid environments using palaeoproteomics

The analysis of the skeletal remains of vertebrates in archaeological contexts provides information about human-animal relationship and their environment. Their taxonomic identification based on macroscopic observation is not always possible due to fragmentation and poor preservation. In recent years, proteomics has emerged as an alternative but there is clearly a lack of data in arid environment where diagenesis rapidly affects the integrity of bone proteins. Here, we report the efficiency of three protocols for protein extraction. The protocols used harsh (1 M HCl and 0.6 M HCl) and soft (Tris-EDTA) decalcification agents and were tested on unidentified splinters from the 2000 years-old site of Toteng, Botswana. The preservation of the organic phase was first estimated using attenuated total reflectance Fourier transform infrared spectroscopy and a set of samples with contrasted collagen contents were selected for palaeoproteomics. The extracted proteins were submitted to a bottom-up proteomic approach involving trypsin digestion followed by ultra-high-performance liquid chromatography coupled to mass spectrometry (UHPLC-MS). Our results identify Tris-EDTA buffer as the most suitable decalcification protocol for poorly preserved bones and propose a collagen content threshold of ~3% weight content for successful detection of peptides. This approach, combined with biogeographical and chronological repartitions of mammals in Africa allows refining taxonomic attributions for four out of nine splinters, leading to species identification. Data are available via ProteomeXchange with identifier PXD010725.

A pressurized liquid extraction approach followed by standard addition method and UPLC-MS/MS for a fast multiclass determination of antibiotics in a complex matrix

In this work a fast analytical method for the determination of macrolides, tetracyclines and fluoroquinolones in a compost originating from a mixture of sewage sludge, palm waste and grass was developed by ultra-high performance liquid chromatography coupled to mass spectrometry (U-HPLC/MS). Antibiotics were extracted from compost by using the accelerated solvent extraction (ASE). The chromatographic separation was carried out on a T3 Cortecs C18 column using a mobile phase gradient mixture of water acidified with 1% of formic acid and acetonitrile. Recoveries of 24–30%, 53–93%, 33–57%, 69–135% and 100–171% were obtained for roxithromycin (ROX), chlortetracycline (CTC), oxytetracycline (OTC), enrofloxacin (ENR) and ciprofloxacin (CIP), respectively. As the most part of antibiotics showed significant matrix effect (ME), the method was validated using the standard addition method (SAM) to correct the observed ME. Instrumental variation, of LC/MS system, showed that 93.75% of the relative standard deviation (RSD %) are below 15%, although the organic load of extracts. This analytical method was applied to assess the fate of antibiotics during composting. Two composting experiments were conducted separately after spiking sludge at 2 different concentrations levels. The resulting elimination rates were of 52–76, 69–100, 100 and 24–50% for ROX, CTC, OTC and CIP, respectively. These results suggest that composting process contributes to the removal of residuals concentrations of macrolides and tetracyclines while the fluoroquinolones persist in the final compost product.

Detection of the Bacterial Quorum-Sensing Signaling Molecules N-Acyl-Homoserine Lactones (HSL) and N-Acyl-Homoserine (HS) with an Enzyme-Linked Immunosorbent Assay (ELISA) and via Ultrahigh-Performance Liquid Chromatography Coupled to Mass Spectrometry (UHPLC-MS).

Quick and reliable quantitative methods requiring low amounts of sample volume are needed for the detection of N-acyl-homoserine lactones (HSL) and their degradation products N-acyl-homoserines (HS) in order to elucidate the occurrence and dynamics of these prevalent quorum-sensing molecules of Gram-negative bacteria in natural samples and laboratory model experiments. A combination of ELISA and UHPLC-MS is presented here which has proven to meet these requirements. Both methods can not only precisely detect and quantify HSLs but also their degradation products HS and thereby enable studying signaling dynamics in quorum sensing, which have been identified to play an essential role in bacterial communication.

Hyperspectral Imaging and Support Vector Machine: A Powerful Combination to Differentiate Black Cohosh (Actaea racemosa) from Other Cohosh Species.

Actaea racemosa (black cohosh) has a history of traditional use in the treatment of general gynecological problems. However, the plant is known to be vulnerable to adulteration with other cohosh species. This study evaluated the use of shortwave infrared hyperspectral imaging (SWIR-HSI) in tandem with chemometric data analysis as a fast alternative method for the discrimination of four cohosh species (Actaea racemosa, Actaea podocarpa, Actaea pachypoda, Actaea cimicifuga) and 36commercial products labelled as black cohosh. The raw material and commercial products were analyzed using SWIR-HSI and ultra-high-performance liquid chromatography coupled to mass spectrometry (UHPLC-MS) followed by chemometric modeling. From SWIR-HSI data (920 - 2514 nm), the range containing the discriminating information of the four species was identified as 1204 - 1480 nm using Matlab software. After reduction of the data set range, partial least squares discriminant analysis (PLS-DA) and support vector machine discriminant analysis (SVM-DA) models with coefficients of determination (R2 ) of ≥ 0.8 were created. The novel SVM-DA model showed better predictions and was used to predict the commercial product content. Seven out of 36 commercial products were recognized by the SVM-DA model as being true black cohosh while 29 products indicated adulteration. Analysis of the UHPLC-MS data demonstrated that six commercial products could be authentic black cohosh. This was confirmed using the fragmentation patterns of three black cohosh markers (cimiracemoside C; 12-β,21-dihydroxycimigenol-3-O-L-arabinoside; and 24-O-acetylhydroshengmanol-3-O-β-D-xylopyranoside). SWIR-HSI in conjunction with chemometric tools (SVM-DA) could identify 80% adulteration of commercial products labelled as black cohosh.

Integrating analytical resolutions in non-targeted wine metabolomics

Direct injection Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), and ultra-high performance liquid chromatography coupled to mass spectrometry (UPLC/MS) were combined for the non-targeted analysis of wine metabolites. The unrivalled resolution on mass measurement allowed by the former and the separation ability of isomeric and isobaric substances by the latter, clearly increases the scope of detectable unknown metabolites in wines. Such methodology is illustrated through the comparison of chemical spaces of a young and an older Pinot noir wine. RP and HILIC chromatography could reveal up to five isomers for a given mass, throughout the explored mass range. CHO, CHOS and CHONS chemical spaces exhibited higher diversities in the older wine, illustrating a molecular oeno-diagenesis process during wine ageing, whereas nitrogen-containing compounds (CHON chemical space) appeared to be fewer in the older wine, likely due to precipitation. This methodological combination is a promising contribution to the wine metabolomics toolkit.

What the devil is in your phytomedicine? Exploring species substitution in Harpagophytum through chemometric modeling of 1H-NMR and UHPLC-MS datasets

Harpagophytum procumbens (Pedaliaceae) and its close taxonomical ally Harpagophytum zeyheri, indigenous to southern Africa, are being harvested for exportation to Europe where phytomedicines are developed to treat inflammation-related disorders. The phytochemical variation within and between natural populations of H. procumbens (n=241) and H. zeyheri (n=107) was explored using proton nuclear magnetic resonance (1H-NMR) and ultra-high performance liquid chromatography coupled to mass spectrometry (UHPLC-MS) in combination with multivariate data analysis methods. The UHPLC-MS results revealed significant variation in the harpagoside content: H. procumbens (0.17–4.37%); H. zeyheri (0.00–3.07%). Only 41% of the H. procumbens samples and 17% of the H. zeyheri samples met the pharmacopoeial specification of ⩾1.2%. Both principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA) indicated separation based on species (UHPLC-MS data OPLS-DA model statistics: R2X=0.258, R2Y (cum)=0.957 and Q2(cum)=0.934; 1H-NMR data OPLS-DA model statistics: R2X=0.830, R2Y=0.865 (cum) and Q2(cum)=0.829). It was concluded that two species are not chemically equivalent and should not be used interchangeably.