Liquid Chromatography Coupled With High-resolution Mass Spectrometry Research Articles

Identification of extractables by liquid chromatography-high resolution mass spectrometry: A case study to understand the extraction profile of different disposable syringes

The current study was undertaken to evaluate clinical-grade disposable syringes of various brands based on the identification of an extraneous impurity leaching out from the rubber gasket of the syringe during sample preparation for liquid chromatography method development. The syringes were evaluated using aqueous and organic solvents and their mixtures to understand susceptibility towards extractables. It was observed that the extraction propensity of different brands of syringes was varying in terms of number and levels of extractables. A total of eight extractables (including the extraneous impurity observed during method development) were identified from the syringe gaskets with the help of liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS), nuclear magnetic resonance (NMR) and/or infrared (IR) spectroscopy. To the best of authors' knowledge, four out of eight proposed extractables are not reported in the literature. A tentative pathway for the formation of the extractables was also proposed.

Application of an Integrated and Open Source Workflow for LC-HRMS Plant Metabolomics Studies. Case-Control Study: Metabolic Changes of Maize in Response to Fusarium verticillioides Infection.

Liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) represents the most powerful metabolomics platform to investigate biological systems. Reproducible and standardized workflows allow obtaining a meaningful biological interpretation. The purpose of this study was to set up and apply an open-source workflow for LC-HRMS plant metabolomics studies. Key steps of the proposed workflow were as follows: (1) experimental design, (2) sample preparation, (3) LC-HRMS analysis, (4) data processing, (5) custom database search, (6) statistical analysis, (7) compound identification, and (8) biochemical interpretation. Its applicability was evaluated through the study of metabolomics changes of two maize recombinant inbred lines with contrasting phenotypes with respect to disease severity after Fusarium verticillioides infection of seedlings. Analysis of data from the case-control study revealed abundance change in metabolites belonging to different metabolic pathways, including two amino acids (L-tryptophan and tyrosine), five flavonoids, and three N-hydroxynnamic acid amides.

Induction of Antibacterial Metabolites by Co-Cultivation of Two Red-Sea-Sponge-Associated Actinomycetes Micromonospora sp. UR56 and Actinokinespora sp. EG49.

Liquid chromatography coupled with high resolution mass spectrometry (LC-HRESMS)-assisted metabolomic profiling of two sponge-associated actinomycetes, Micromonospora sp. UR56 and Actinokineospora sp. EG49, revealed that the co-culture of these two actinomycetes induced the accumulation of metabolites that were not traced in their axenic cultures. Dereplication suggested that phenazine-derived compounds were the main induced metabolites. Hence, following large-scale co-fermentation, the major induced metabolites were isolated and structurally characterized as the already known dimethyl phenazine-1,6-dicarboxylate (1), phenazine-1,6-dicarboxylic acid mono methyl ester (phencomycin; 2), phenazine-1-carboxylic acid (tubermycin; 3), N-(2-hydroxyphenyl)-acetamide (9), and p-anisamide (10). Subsequently, the antibacterial, antibiofilm, and cytotoxic properties of these metabolites (1–3, 9, and 10) were determined in vitro. All the tested compounds except 9 showed high to moderate antibacterial and antibiofilm activities, whereas their cytotoxic effects were modest. Testing against Staphylococcus DNA gyrase-B and pyruvate kinase as possible molecular targets together with binding mode studies showed that compounds 1–3 could exert their bacterial inhibitory activities through the inhibition of both enzymes. Moreover, their structural differences, particularly the substitution at C-1 and C-6, played a crucial role in the determination of their inhibitory spectra and potency. In conclusion, the present study highlighted that microbial co-cultivation is an efficient tool for the discovery of new antimicrobial candidates and indicated phenazines as potential lead compounds for further development as antibiotic scaffold.

The issue of eosin fading: A combined spectroscopic and mass spectrometric approach applied to historical lakes

The huge number of complex formulations of synthetic dyes and pigments commercialized in the 19th century makes their comprehensive characterization both difficult and fundamental for their successful identifications in artworks. The description of the exact starting composition of a wide collection of reference materials is preliminary to the study of the challenging photo-degradation processes in which most of the early synthetic dyes and pigments are involved.In this paper, four eosin-based lakes, all containing eosin Y, and one also containing eosin B, but differing in the organic and inorganic materials used as fillers, were fully characterized. These formulations were reconstructed on the base of historical recipes from the extensive Wiesel's collection, dated to the 19th century and bought by Dr Kremer (Kremer Pigmente, Germany) in the 1990s. The composition of each formulation was characterized by Fourier transform infrared spectroscopy (FT-IR) and luminescence spectroscopy was used as a probe for their inert-distinction. Reference paint layers were then prepared by casting the lakes in linseed oil, and their ageing and impact on color change was monitored for one year by analyzing two sets of paint model systems by colorimetry. For each system, two sets were prepared and one was stored in the dark while the other exposed to natural light. The material distribution and homogeneity of the samples were tested by Synchrotron Radiation based micro infrared spectroscopy (SR-μ-FT-IR). Finally, an ultra-sensitive method based on Liquid Chromatography coupled with High Resolution Mass Spectrometry (LC-HRMS) allowed us to characterize eosin degradation products detected but never identified before, and to evaluate their kinetics of formation.

Mitigation of lead neurotoxicity by the ethanolic extract of Laurus leaf in rats.

The present study was conducted in order to assess the chemical composition of Laurus, its antioxidant activities, and benefit from the Laurus extract effect on neurotoxicity caused by lead acetate (Pb). Chemical profile was assayed by using liquid chromatography coupled with high-resolution mass spectrometry (LC-HR-MS). In this study, 40 male rats were divided into four groups (10 rats per each group): (1) control group, (2) Laurus group: rats treated with 250mg/kg b. wt. of Laurus leaves extract, (3) Pb group: rats treated with 100mg/kg b. wt. of lead acetate, (4) Pb+Laurus group: rats treated with 250mg/kg b. wt. of Laurus leaves extract in addition to lead acetate for 30 days. At the end of experiment, some estimates were calculated from blood samples, brain tissue, and histological examination. The results showed that the extract is highly affluent in total flavonoids, total phenolic, and also has antioxidant activity. The LC-MS appeared a wide range of compounds in the extract. The oxidative stress resulted from exposure to lead acetate has been reported to cause reduction in body and brain weights, levels of RBCs, acetylcholinesterase (AChE), GSH, SOD, and CAT in addition to increase in levels of WBCs and MAD. Moreover, Laurus leaves extract notably lessened the biochemical changes caused by lead acetate in the blood, homogenate, and brain tissue (P<0.05). The current study indicates the antioxidant activity of Laurus leaves extract and assumes that it has a defensive role against the oxidative damage caused by lead in a rat's brain.

Lipidomic profiling of amniotic fluid and its application in fetal lung maturity prediction.

BackgroundThe pulmonary surfactant especially lipids in amniotic fluid can reflect the development stage of fetal lung maturity (FLM). However, the conventional lecithin/sphingomyelin (L/S) ratio method by thin layer chromatography (TLC) is insufficient and inconvenient for FLM prediction in clinical practice.MethodsThe amniotic fluid samples were collected from the pregnant women in labor or undergoing amniocentesis and analyzed for its lipid contents with the liquid chromatography coupled with high‐resolution mass spectrometry (LC‐HRMS) method and the lamellar body count (LBC) method. To reveal the lipidomic profiling of different FLM stages, three groups of amniotic fluid samples including 8 from premature group (gestational week (GW) < 37), 10 from mature group (GW < 37), and 10 from mature group (GW > 38) were compared with the control group (n = 6) of 18 GWs separately.ResultsIn the FLM prediction study, the sensitivity of the LC‐HRMS method and LBC method was 91% and 73%, respectively; the specificity was 100% and 95%, respectively. The most significant metabolic pathway was linoleic acid metabolism between the premature group and the control group. Both glycerophospholipid metabolism and glycosylphosphatidylinositol‐anchor biosynthesis were enriched in the mature groups. In search of potential FLM prediction markers in amniotic fluid, 8 phosphatidylcholines, 1 sphingomyelin, and 1 phosphatidylethanolamine were significantly increased in the mature groups compared with the premature group.ConclusionAn efficient LC‐HRMS method for L/S ratio in predicting FLM was established. The linoleic acid metabolism may play an important role in the fetal lung development.

A pilot investigation of a urinary metabolic biomarker discovery in renal cell carcinoma.

Renal cell carcinoma (RCC) is the most common and lethal malignancy of the kidney. Distinguishing RCC from benign renal tumors and healthy controls is still a clinical challenge. Urine metabolomics has been used to identify biomarkers of clinical diseases. In the present study, we explored the urine metabolomes of a cohort of 61 patients with renal tumors (39 RCC and 22 benign renal tumors) and 68 healthy controls using liquid chromatography coupled with high-resolution mass spectrometry (LC-MS). Metabolic profiling of urine could significantly differentiate RCC from healthy controls and benign renal tumors. Metabolic pathways, including lysine metabolism and phenylalanine metabolism, were found to be disturbed in the RCC group. Steroid hormone biosynthesis was significantly different between the benign tumor group and the RCC group. RCC biomarkers were further explored. A metabolite panel consisting of cortolone, testosterone and L-2-aminoadipate adenylate was discovered to have good ability of distinguishing RCC from benign tumors, with an AUC of 0.868 for tenfold cross-validation and 0.873 for the validation group. In addition, the panel of aminoadipic acid, 2-(formamido)-N1-(5-phospho-D-ribosyl) acetamidine and alpha-N-phenylacetyl-L-glutamine could distinguish the RCC group from the healthy control group, with an AUC of 0.841 for tenfold cross-validation and 0.894 for the validation group. This pilot study suggests that urine metabolomics may be useful in differentiating RCC from healthy controls and benign renal tumors.

Lipid Profile Characterization and Lipoprotein Comparison of Extracellular Vesicles from Human Plasma and Serum.

Extracellular vesicles (EVs) consist of lipid bilayers, occur in various biofluids, and are invaluable in biomarker screening. Liquid chromatography coupled with high-resolution mass spectrometry (LC-MS) was recently used to study comprehensive EV lipid profiles in vitro. The aim of this study was to establish a lipidomics platform for human plasma and serum EVs for comprehensive characterization of their lipid profiles, and to compare them with those of other lipid-containing particles, such as high-density lipoproteins (HDL), and low/very low-density lipoproteins (LDL/VLDL). Isolation was validated by specific protein markers; CD9 and MHC class I for EVs, apoA-I for HDL, and apoB-100 for LDL/VLDL. Lipidomics identified 264 lipids from isolated plasma EVs, HDL, and LDL/VLDL. The absolute lipid levels per unit protein content in the EVs were more than eight times lower than those of the lipoproteins. Moreover, the EVs had higher lysoglycerophospholipid levels than HDL or LDL/VLDL. Similar profiles were also determined for human serum. The present study found that the lipid profiles of EVs are unique and distinctly different from those of lipoproteins. The lipidomics platform applied to human plasma and serum EVs could generate important information for the exploration and qualification of biomarkers in disease diagnosis.

Development of an ammonium chloride-enhanced thermal-assisted-ESI LC-HRMS method for the characterization of chlorinated paraffins

Simultaneous quantification of short-, medium-, and long-chain chlorinated paraffins (CPs) in environmental matrices is challenging and has received much attention from environmental chemists. In this study, ammonium-chloride-enhanced liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) was developed for the first time to quantify CPs in sediments and aqueous samples. Three ionization sources, including atmospheric pressure chemical ionization (APCI), electrospray ionization (ESI), and thermal-assisted-ESI, were employed to examine the performance of ammonium chloride as the chloride ion supply reagent in comparison with traditional chloride ion supply reagent, dichloromethane. Ammonium chloride can be easily used with reversed-phase liquid chromatography (LC), whereas dichloromethane is not compatible with aqueous LC mobile phase. Furthermore, other anion-supply reagents, such as ammonium formate, ammonium acetate, and ammonium bromide, were also tested. It was concluded that the adducts of the CPs with the anions were reversible and could partially dissociate into deprotonated CP ions. The yield of deprotonated CP ions was associated with the gas-phase basicity of the deprotonated CP ions and the corresponding anions. Furthermore, collision-induced dissociation curves were drawn to quantify the stability of anionic CP adducts. The ammonium-chloride-enhanced LC-HRMS was further employed for identifying CPs in sediment samples and coupled with an online SPE method for detecting CPs in aqueous samples. This study may significantly contribute to the qualification and quantification of CPs in environmental matrices.

Exercise training counteracts urothelial carcinoma-induced alterations in skeletal muscle mitochondria phospholipidome in an animal model

Cancer associated body wasting is the cause of physical disability, reduced tolerance to anticancer therapy and reduced survival of cancer patients and, similarly to cancer, its incidence is increasing. There is no cure for this clinical condition, and the pathophysiological process involved is largely unknown. Exercise training appears as the gold standard non-pharmacological therapy for the management of this wasting syndrome. Herein we used a lipidomics approach based on liquid chromatography coupled with high-resolution mass spectrometry (LC-HR-MS) to study the effect of exercise in the modulation of phospholipids profile of mitochondria isolated from gastrocnemius muscle of a pre-clinical model of urothelial carcinoma-related body wasting (BBN induced), submitted to 13 weeks of treadmill exercise after diagnosis. Multivariate analysis showed a close relationship between the BBN exercise group and both control groups (control sedentary and control exercise), while the BBN sedentary group was significantly separated from the control groups and the BBN exercise group. Univariate statistical analysis revealed differences mainly in phosphatidylserine (PS) and cardiolipin (CL), although some differences were also observed in phosphatidylinositol (PI, LPI) and phosphatidylcholine (PC) phospholipids. PS with shorter fatty acyl chains were up-regulated in the BBN sedentary group, while the other species of PS with longer FA and a higher degree of unsaturation were down-regulated, but the BBN exercise group was mostly similar to control groups. Remarkably, exercise training prevented these alterations and had a positive impact on the ability of mitochondria to produce ATP, restoring the healthy phospholipid profile. The remodelling of mitochondria phospholipid profile in rats with urothelial carcinoma allowed confirming the importance of the lipid metabolism in mitochondria dysfunction in cancer-induced skeletal muscle remodelling. The regulation of phospholipid biosynthetic pathways observed in the BBN exercise group supported the current perspective that exercise is an adequate therapeutic approach for the management of cancer-related muscle remodeling.

Formation of new disinfection by-products of priority substances (Directive 2013/39/UE and Watch List) in drinking water treatment.

The degradation of priority substances (Directive 2013/39/UE and Watch List) by chlorine dioxide (ClO2) and the formation of disinfection by-products (DBPs) in a drinking water treatment plant (DWTP) located near Barcelona (NE Spain) were investigated. For the first time, the reactivity with ClO2 of several compounds frequently found at the entrance of the DWTP such as erythromycin, clarithromycin, chlorpyrifos, and imidacloprid was evaluated in both simulated and real conditions. To identify potential DBPs, experiments were performed at laboratory scale by simulating the operational disinfection conditions in the DWTP. Liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) working in full scan and target-MS/HRMS modes was used for the identification of the generated DBPs. Several new DBPs were found, three from erythromycin, one from clarithromycin, two from chlorpyrifos, and one from imidacloprid. Then, the presence and behavior through DWTP treatment of priority substances and their DBPs were investigated in order to evaluate their generation in real working conditions. Two of the potential DBPs, anhydroerythromycin, and N-desmethyl clarithromycin were already identified in the raw water of DWTP, but N-desmethyl clarithromycin was also generated after the chlorine dioxide treatment step. Both compounds were eliminated by the treatments applied in the DWTP; anhydroerythromycin was eliminated after ozonation in the upgraded conventional treatment and after reverse osmosis in the advanced treatment while N-desmethyl clarithromycin is recalcitrant in the upgraded conventional treatment, but it was eliminated by reverse osmosis.

Implementation of Chemometric Tools To Improve Data Mining and Prioritization in LC-HRMS for Nontarget Screening of Organic Micropollutants in Complex Water Matrixes.

One of the most critical steps in nontarget screening of organic micropollutants (OMP) in complex environmental samples is handling of massive data obtained from liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS). Multivariate chemometric methods have brought about great progress in processing big data obtained from high-dimensional chromatographic systems. This work aimed at a comprehensive evaluation of two LC-Q-Orbitrap mass spectrometry full-scan data sets for target and nontarget screening of OMPs in drinking and wastewater samples, respectively. For each data set, following segmentation in the chromatographic dimension, at first multivariate curve resolution alternating least-squares (MCR-ALS) was employed for simultaneous resolution of global matrices. The chromatographic peaks and the corresponding mass spectra of OMP were fully resolved in the presence of highly co-eluting irrelevant and interfering peaks. Then partial least-squares-discriminant analysis was conducted to investigate the behavior of MCR-ALS components in different water classes and selection of most relevant components. Further prioritization of features in wastewater before and after ozonation and their reduction to 24 micropollutants were then obtained by univariate statistics. Two-way information retrieved from MCR-ALS of LC-MS1 data was also used to choose common precursor ions between recoveredand measured data through data-dependent acquisition. MS1 and MS2 spectral features were used for tentative identification of prioritized OMPs. This study indicates that the described strategy can be used as a promising tool to facilitate both feature selection through a reliable classification and interference-free identification of micropollutants in nontargeted and class-wise environmental studies.

Supporting non-target identification by adding hydrogen deuterium exchange MS/MS capabilities to MetFrag

Liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) is increasingly popular for the non-targeted exploration of complex samples, where tandem mass spectrometry (MS/MS) is used to characterize the structure of unknown compounds. However, mass spectra do not always contain sufficient information to unequivocally identify the correct structure. This study investigated how much additional information can be gained using hydrogen deuterium exchange (HDX) experiments. The exchange of “easily exchangeable” hydrogen atoms (connected to heteroatoms), with predominantly [M+D]+ ions in positive mode and [M-D]− in negative mode was observed. To enable high-throughput processing, new scoring terms were incorporated into the in silico fragmenter MetFrag. These were initially developed on small datasets and then tested on 762 compounds of environmental interest. Pairs of spectra (normal and deuterated) were found for 593 of these substances (506 positive mode, 155 negative mode spectra). The new scoring terms resulted in 29 additional correct identifications (78 vs 49) for positive mode and an increase in top 10 rankings from 80 to 106 in negative mode. Compounds with dual functionality (polar head group, long apolar tail) exhibited dramatic retention time (RT) shifts of up to several minutes, compared with an average 0.04 min RT shift. For a smaller dataset of 80 metabolites, top 10 rankings improved from 13 to 24 (positive mode, 57 spectra) and from 14 to 31 (negative mode, 63 spectra) when including HDX information. The results of standard measurements were confirmed using targets and tentatively identified surfactant species in an environmental sample collected from the river Danube near Novi Sad (Serbia). The changes to MetFrag have been integrated into the command line version available at http://c-ruttkies.github.io/MetFrag and all resulting spectra and compounds are available in online resources and in the Electronic Supplementary Material (ESM).Graphical abstract

Metabolomic profiling and anti-infective potential of Zinnia elegans and Gazania rigens (Family Asteraceae)

The present study evaluates the chemical composition of Zinnia elegans and Gazania rigens based on their metabolomic profiles using liquid chromatography coupled with high-resolution mass spectrometry (LC‐HR-MS), alongside with the anti-infective activities of their ethanol extracts, as well as, different fractions. A significant difference was observed between the LC-MS profiles of the two plants such as, coumarins, sesquiterpene lactones and phenylethanoids which were characteristic for Z. elegans, while amides and phenolic acid derivatives were characteristic for G. rigens. These results highlight the chemical potential of Z. elegans and G. rigens. Furthermore, the ethyl acetate fraction of Z. elegans showed a significant antimalarial activity with IC50 values of 21.03 and 13.72 µg/mL against Plasmodium falciparum D6 and P. falciparum W2, respectively.

Validation of a global quantitative analysis methodology of tryptophan metabolites in mice using LC-MS

In this study, we validated a method for quantifying 20 tryptophan (Trp) catabolites by liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) in 4 different matrices (urine, serum, intestinal contents and liver). The detection limit for all metabolites ranged between 0.015 and 11.25 nmol/L and the dynamic range of the calibration curves were adjusted to allow quantification of metabolites at endogenous levels. Matrix effects were evaluated using isotope labeled internal standards. Reproducibility in the 4 matrices was characterized by CV = 6.2% with an accuracy of 6.6%. Our method has been applied to the determination and quantification of 20 metabolites concentrations in 5 different mouse compartments (plus cecal contents). Our results show that our approach allows for a global exploration of the Trp metabolism by quantifying a large number of Trp metabolites, at the individual level by multi-matrix approach.

Characterization of natural bioactive compounds produced by isolated bacteria from compost of aromatic plants.

This study aimed to highlight the importance of compost from aromatic plants as a stunning source for several bio active compounds generated from their inhabited thermophilic bacteria. Some of the isolated compounds could have a potential role in the treatment of microbial infections. A total of forty different thermophilic bacteria were isolated from compost samples during their thermophilic stage. These isolates were tested for their antimicrobial capabilities against different Gram-positive and -negative bacteria using agar diffusion and double layer agar methods. The potential isolates were further identified based on morphological, biochemical and 16S rRNA gene sequencing methods. They were subjected to submerged state fermentation and the total crude metabolites were recovered using ethyl acetate (EtOAc) extraction. All bioactive metabolites were identified using liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS). It was observed that 2 out of 40 isolates were remarkably active against Gram-positive bacteria. These isolates were genetically identified as Bacillus species and their different active metabolites were characterized in the EtOAc extracts using LC-HRMS. Liquid chromatography coupled with high-resolution mass spectrometry analysis of EtOAc extracts revealed the presence of active metabolites that are responsible for antimicrobial activities. To the best of our knowledge, this is the first time to identify bioactive antimicrobial metabolites from retrieved compost micro-organisms in Egypt. So, compost could be a beneficial area for research as a reliable and continuous natural source for different uncountable communities of bacteria.

Machine learning combined with non-targeted LC-HRMS analysis for a risk warning system of chemical hazards in drinking water: A proof of concept

Guaranteeing clean drinking water to the global population is becoming more challenging, because of the cases of water scarcity across the globe, growing population, and increased chemical footprint of this population. Existing targeted strategies for hazard monitoring in drinking water are not adequate to handle such diverse and multidimensional stressors. In the current study, we have developed, validated, and tested a machine learning algorithm based on the data produced via non-targeted liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) for the identification of potential chemical hazards in drinking water. The machine learning algorithm consisted of a composite statistical model including an unsupervised component (i.e. principal component analysis PCA) and a supervised one (i.e. partial least square discrimination analysis PLS-DA). This model was trained using a training set of 20 drinking water samples previously tested via conventional suspect screening. The developed model was validated using a validation set of 20 drinking water samples of which 4 were spiked with 15 labeled standards at four different concentration levels. The model successfully detected all of the added analytes in the four spiked samples without producing any cases of false detection. The same validation set was processed via conventional trend analysis in order to cross validate the composite model. The results of cross validation showed that even though the conventional trend analysis approach produced a false positive detection rate of ≤5% the composite model outperformed that approach by producing zero cases of false detection. Additionally, the validated model went through an additional test with 42 extra drinking water samples from the same source for an unbiased examination of the model. Finally, the potentials and limitations of this approach were further discussed.

Screening and Identification of Cardioprotective Compounds From Wenxin Keli by Activity Index Approach and in vivo Zebrafish Model.

Wenxin Keli (WXKL) is a widely used Chinese botanical drug for the treatment of arrhythmia, which is consisted of four herbs and amber. In the present study, we analyzed the chemical composition of WXKL using liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) to tentatively identify 71 compounds. Through typical separate procession, the total extract of WXKL was divided into fractions for further bioassays. Cardiomyocytes and zebrafish larvae were applied for assessment. In vivo arrhythmia model in Cmlc2-GFP transgenic zebrafish was induced by terfenadine, which exhibited obvious reduction of heart rate and occurrence of atrioventricular block. Dynamic beating of heart was recorded by fluorescent microscope and sensitive camera to automatically recognize the rhythm of heartbeat in zebrafish larvae. By integrating the chemical information of WXKL and corresponding bioactivities of these fractions, activity index (AI) of each identified compound was calculated to screen potential active compounds. The results showed that dozens of compounds including ginsenoside Rg1, ginsenoside Re, notoginsenoside R1, lobetyolin, and lobetyolinin were contributed to cardioprotective effects of WXKL. The anti-arrhythmic activities of five compounds were further validated in larvae model and mature zebrafish by measuring electrocardiogram (ECG). Our findings provide a successful example for rapid discovery of bioactive compounds from traditional Chinese medicine (TCM) by activity index based approach coupled with in vivo zebrafish model.

LC-HRMS Biomarker Suspect Screening in Low Dose Studies

Exposure biomarker development is a time consuming procedure, starting with identification of presumably appropriate metabolites of the xenobiotic in question. Investing into unsuitable biomarker candidates (i.e. metabolites which are formed in low proportion or are not formed at all) can be costly. Accordingly, efficient strategies for the identification of promising biomarker candidates are needed. For this purpose, liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) is often applied for metabolite screening in in vitro and animal in vivo screening approaches, typically involving high doses/concentrations of the xenobiotic.Given the limitations of in vitro models and interspecies differences in metabolism, we sought to improve the certainty of metabolite screening by analyzing urine samples from human studies with low oral dosing (i.e. toxicologically uncritical). Metabolite screening was performed by LC-Q-Orbitrap-MS combined with a commercial data mining software. Identification criteria were plausible excretion kinetics, product ion spectra, 13C isotopomer ratios, and retention times.We present results of a pilot study, which identified nine metabolites of the plasticizer di (2-ethylhexyl) terephthalate (DEHTP). Three of them (5OH-MEHTP, 5Oxo-MEHTP, and 5cx-MEPTP) are already established biomarkers of DEHTP exposure. Five additional, specific metabolites have not been described previously. In another example we successfully identified several tentative metabolites of the UV filter avobenzone. However, we also report examples, in which metabolite identification did not succeed by this approach (e.g. the plasticizer DEHA) and we highlight influencing factors (such as mobile phase pH, in source adduct formation and fragmentation) possibly leading to wrong conclusions.The presented approach is a valuable but not universal tool for biomarker development. In some cases, a more sensitive triple quadrupole screening can be superior.

Extended Targeted and Non-Targeted Strategies for the Analysis of Marine Toxins in Mussels and Oysters by (LC-HRMS)

When considering the geographical expansion of marine toxins, the emergence of new toxins and the associated risk for human health, there is urgent need for versatile and efficient analytical methods that are able to detect a range, as wide as possible, of known or emerging toxins. Current detection methods for marine toxins rely on a priori defined target lists of toxins and are generally inappropriate for the detection and identification of emerging compounds. The authors describe the implementation of a recent approach for the non-targeted analysis of marine toxins in shellfish with a focus on a comprehensive workflow for the acquisition and treatment of the data generated after liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) analysis. First, the study was carried out in targeted mode to assess the performance of the method for known toxins with an extended range of polarities, including lipophilic toxins (okadaic acid, dinophysistoxins, azaspiracids, pectenotoxins, yessotoxins, cyclic imines, brevetoxins) and domoic acid. The targeted method, assessed for 14 toxins, shows good performance both in mussel and oyster extracts. The non-target potential of the method was then challenged via suspects and without a priori screening by blind analyzing mussel and oyster samples spiked with marine toxins. The data processing was optimized and successfully identified the toxins that were spiked in the blind samples.