Chromatography-ion Trap Tandem Mass Spectrometry Research Articles

A novel flavonol-polysaccharide from Tamarix chinensis alleviates influenza A virus-induced acute lung injury. Evidences for its mechanism of action

BackgroundTamarix chinensis Lour. is a Chinese medicine used for treating inflammation-related diseases and its crude polysaccharides (MBAP90) exhibited significant anticomplement activities in vitro. PurposeTo obtain anticomplement homogenous polysaccharides from MBAP90 and explore its therapeutic effects and potential mechanism on influenza A virus (IAV)-induced acute lung injury (ALI). MethodsAnticomplement activity-guided fractionation of the water-soluble crude polysaccharides from the leaves and twigs of T. chinensis were performed by diethylaminoethyl-52 (DEAE-52) cellulose and gel permeation columns to yield a homogeneous polysaccharide MBAP-5, which was further characterized using ultra-high-performance liquid chromatography-ion trap tandem mass spectrometry (UPLC-IT-MS) and nuclear magnetic resonance (NMR) analysis. In vitro, the anticomplement activity of MBAP-5 through classical pathway was measured using a hemolytic test. The therapeutic effects of MBAP-5 on ALI were evaluated in H1N1-infected mice. H&E staining, enzyme linked immunosorbent assay (ELISA), immunohistochemistry, and western blot were used to systematically access lung histomorphology, inflammatory cytokines, degree of complement component 3c, 5aR, and 5b-9 (C3c, C5aR, and C5b-9) deposition, and inflammasome signaling pathway protein expressions in lung tissues. ResultsMBAP-5 was a novel flavonol-polysaccharide with the molecular weight (Mw) of 153.6 kDa. Its structure was characterized to process a backbone of →4)-α-D-GlcpA-(1→, →6)-α-D-Glcp-(1→, →3,4)-α-D-Glcp-(1→, →3,4,6)-α-D-Glcp-(1→, and →4,6)-β-D-Glcp-(1→, as well as branches of α-L-Araf-(1→ and β-D-Galp-(1→. Particularly, O-3 of →3,4,6)-α-D-Glcp-(1→ was substituted by quercetin. In vitro assay showed that MBAP-5 had a potent anticomplement activity with a CH50 value of 102 ± 4 µg/ml. Oral administration of MBAP-5 (50 and 100 mg/kg) effectively attenuated the H1N1-induced pulmonary injury in vivo by reducing pulmonary edema, virus replication, and inflammatory responses. Mechanistically, MBAP-5 inhibited the striking deposition and contents of complement activation products (C3c, C5aR, and C5b-9) in the lung. Toll-like receptor 4 (TLR4) /transcription factor nuclear factor κB (NF-κB) signaling pathway was constrained by MBAP-5 treatment. In addition, MBAP-5 could suppress activation of the inflammasome pathways, including Nod‐like receptor pyrin domain 3 (NLRP3), cysteinyl aspartate specific proteinase-1/12 (caspase-1/12), apoptosis‑associated speck‑like protein (ASC), gasdermin D (GSDMD), interleukin (IL)-1β, and IL-18 expressions. ConclusionsA novel flavonol-polysaccharide MBAP-5 isolated from T. chinensis demonstrated a therapeutic effect against ALI induced by IAV attack. The mechanism might be associated with inhibition of complement system and inflammasome pathways activation.

Yokukansan, a Traditional Japanese Medicine, Suppresses Stress-Induced Sympathetic Activation via Central Responses in Rats

Objectives: Yokukansan (YKS) is a traditional Japanese Kampo medicine approved by the Ministry of Health, Labour and Welfare of Japan. Recently, in addition to its indications as a drug treatment, YKS has been shown to have ameliorative effects on various psychological and behavioral responses such as anxiety, aggression, and stress responses. Stress exposure activates not only the hypothalamic–pituitary–adrenal axis but also the sympathetic nervous system. Sympathetic activation in brain regions such as the paraventricular hypothalamic nucleus (PVN) stimulates elevation of plasma catecholamine (noradrenaline and adrenaline) levels. We previously reported that various stress-related neuropeptides and several kinds of stressors, such as restraint stress (RS), increase plasma catecholamine and that brain prostanoids and their synthases mediate these responses in rats. In the present study, to determine if YKS treatment can affect stress-induced sympathetic activation, we examined the effects of YKS treatment on the RS-induced elevation of plasma catecholamine levels and related prostanoid production in the PVN of rats. Methods: YKS (1000 mg/10 mL/kg, p.o.) or vehicle was administered to rats daily for 14 days, and all rats were exposed to RS for 60 min on day 14. Before and during stress exposure on day 14, blood samples and PVN dialysates were collected and analyzed by high-performance liquid chromatography and liquid chromatography-ion trap tandem mass spectrometry, respectively. Results: Our results showed that repeated administration of YKS suppressed the RS-induced increase in plasma adrenaline but not noradrenaline. Furthermore, YKS administration also suppressed the RS-induced elevation of both prostaglandin E2 and thromboxane B2 levels in the PVN. In addition, we found that repeated administration of YKS suppressed the RS-induced increase in serotonin, gamma-aminobutyric acid, and acetylcholine in the PVN. Conclusion: Our results suggest that YKS can ameliorate stress-induced sympathetic activation via inhibition of stress responses in the brain.

Two Natural Flavonoid Substituted Polysaccharides from Tamarix chinensis: Structural Characterization and Anticomplement Activities.

Two novel natural flavonoid substituted polysaccharides (MBAP-1 and MBAP-2) were obtained from Tamarix chinensis Lour. and characterized by HPGPC, methylation, ultra-high-performance liquid chromatography-ion trap tandem mass spectrometry (UPLC-IT-MSn), and NMR analysis. The results showed that MBAP-1 was a homogenous heteropolysaccharide with a backbone of 4)-β-d-Glcp-(1→ and →3,4,6)-β-d-Glcp-(1→. MBAP-2 was also a homogenous polysaccharide which possessed a backbone of →3)-α-d-Glcp-(1→, →4)-β-d-Glcp-(1→ and →3,4)-β-d-Glcp-2-OMe-(1→. Both the two polysaccharides were substituted by quercetin and exhibited anticomplement activities in vitro. However, MBAP-1 (CH50: 0.075 ± 0.004 mg/mL) was more potent than MBAP-2 (CH50: 0.249 ± 0.006 mg/mL) and its reduced product, MBAP-1R (CH50: 0.207 ± 0.008 mg/mL), indicating that multiple monosaccharides and uronic acids might contribute to the anticomplement activity of the flavonoid substituted polysaccharides of T. chinensis. Furthermore, the antioxidant activity of MBAP-1 was also more potent than that of MBAP-2. In conclusion, these two flavonoid substituted polysaccharides from T. chinensis were found to be potential oxidant and complement inhibitors.

Glucose infusion suppresses acute restraint stress-induced peripheral and central sympathetic responses in rats.

Acute restraint stress (RS) induces sympathetic activation such as elevating plasma catecholamines, resulting in increase in blood glucose. We aimed to investigate whether glucose infusion affects the RS-induced sympathetic responses. Plasma catecholamines were measured by high-performance liquid chromatography with electrochemical detection. Blood glucose levels were measured with a glucometer and a glucose assay kit. Cardiac parameters were measured by echocardiographic and hemodynamic analysis. Prostanoid levels in the paraventricular nucleus of hypothalamus (PVN) microdialysates were measured by liquid chromatography-ion trap tandem mass spectrometry analysis. RS significantly increased plasma noradrenaline and adrenaline. Intravenous infusion of a 5% glucose solution significantly attenuated the RS-induced elevation of plasma adrenaline but did not alter the plasma noradrenaline. Glucose administration during RS suppressed the progression of cardiac impairment by attenuating the decline rates in left ventricular diastolic, end-diastolic volume, stroke volume, fractional shortening, and ejection fraction. Both Intravenous and intracerebroventricular infusion of glucose solution significantly attenuated the RS-induced elevation of thromboxane B2 (TxB2) (a metabolite of TxA2) levels in the PVN but did not alter prostaglandin E2 levels in the PVN. Our results demonstrate that glucose infusion suppresses RS-induced elevation of plasma adrenaline and left ventricular dysfunction. In the brain, glucose infusion suppresses RS-induced production of TxA2 in the PVN.

A simple, selective and eco-friendly method for quantification of polychlorinated biphenyls in sediment samples at ultra-trace levels by headspace solid-phase microextraction-gas chromatography-ion trap tandem mass spectrometry

ABSTRACT An analytical method based on headspace solid-phase microextraction gas chromatography- ion trap tandem mass spectrometry (HS-SPME-GC-ITMS/MS) was optimised and developed for estimating 20 predominant polychlorinated biphenyl (PCB) concentrations in sediment samples. The effect of experimental parameters involving fibre materials, amount of water added, temperature effect, sorption time, stirring effect, and the addition of solvents were examined with gas chromatography-electron capture detector (GC-ECD). The analytical performance of association of HS-SPME-GC-ITMS/MS proposed methodology was examined by spiked sediment with the selected PCB congeners. The repeatability and reproducibility of measurements (n = 6), expressed with relative standard deviation (RSD), was below 13% and 16%, respectively. Good linearity was observed within the concentration range studied and the LODs were between 0.02 and 0.82 ng g−1. Accuracy was assessed by analysing a reference sediment sample with certified concentrations of several PCBs. Standard addition calibrations were done and concentrations of PCBs with the proposed method were in good agreement with the certified PCB concentrations. The method was also efficiently applied to real sample sediment from Bizerte Harbour, revealing the existence of several congeners at level less than 1.1 ng g−1. These obtained results were also on concordance with analysis of this real sample using a conventional soxhlet extraction method. The developed procedure is highly effective and selective, free of organic solvents, relatively simple and fast, and become a suitable alternative for routine monitoring of residues of PCB from sediments at ultra-trace levels.

Magnetic hollow bimetallic zinc/cobalt zeolitic imidazolate framework as sorbent for efficiently enriching aflatoxins combined with UHPLC-IT-MSn determination.

A novel magnetic hollow bimetallic zinc/cobalt-based zeolitic imidazolate framework (MHB-Zn/Co-ZIF-8) was prepared via a microwave-assisted chemical etching in methanol. The structure, morphology, and specific surface area were characterized by X-ray diffraction and FTIR spectroscopy, scanning and transmission electron microscopy, and N2 adsorption. The hollow nanostructures with high internal specific surface area, abundant active sites, and reduced aggregation of nanoparticles endow the hollow zeolitic imidazolate framework (ZIF) nanoparticle with high chemical stability, desirable durability, and excellent adsorption abilities. The MHB-Zn/Co-ZIF-8 nanoparticle was used as an effective sorbent for magnetic solid-phase extraction (MSPE) of trace aflatoxins B1, B2, G1, and G2 from fruit juice and fruit samples. The main parameters affecting the efficiency of MSPE procedures were investigated and optimized. The results show that, under optimized conditions, enrichment factors ranging from 67- to 355-fold are obtained for the target analytes. The method is linear in the range 1.0 to 100.0ngmL-1 with correlation coefficients (R2) from 0.9960 to 0.9992. The limits of detection of four aflatoxins are in the range 0.18 to 1.50ngmL-1 and the average recoveries range from 75.1 to 102.4%, with relative standard deviations (RSDs) less than 13.6%. This work presents the excellent extraction performance of aflatoxins B1, B2, G1, and G2 on MHB-Zn/Co-ZIF-8. In addition, the applicability of the MSPE coupling with ultrahigh-performance liquid chromatography-ion trap tandem mass spectrometry (UHPLC-IT-MSn) for trace analysis in complex matrices is shown. Graphical abstract Schematic presentation of magnetic hollow bimetallic zinc/cobalt zeolitic imidazolate framework as sorbent for efficiently enriching aflatoxins B1, B2, G1, and G2 from fruit juice samples prior to ultrahigh-performance liquid chromatography-ion trap tandem mass spectrometry (UHPLC-IT-MSn) determination.

A 96-Monolithic inorganic hollow fiber array as a new geometry for high throughput solid-phase microextraction of doxorubicin in water and human urine samples coupled with liquid chromatography-tandem mass spectrometry.

Innovations in extraction phases, extraction modes and hyphenated instrument configurations, are the most important issues to address for progress in the solid phase microextraction (SPME) methodology. In this regard, we have embarked on the development of a novel biocompatible 96-monolithic inorganic hollow fiber (96-MIHF) array as a new configuration for high-throughput SPME on a 96-well plate system. An arrangement of highly ordered 96 titania/Hydroxyapatite (TiO2/HAP) nanocomposite hollow fibers and corresponding stainless-steel needles on a Teflon plate holder were used as the extraction module. The inorganic hollow fibers were prepared via a rapid and reproducible template approach (Polypropylene hollow fiber) in combination with a sol-gel method in the presence of polyvinyl alcohol (PVA), as a network maker. The hollow fiber-shape sorbents were obtained with excellent precision by weight (RSD%=4.98, n=10) and length (RSD%=1.08, n=10) criteria. The proposed design can overcome a number of geometrically dependent drawbacks of conventional high-throughput SPME methods, mainly the ones related to sorbent amount and surface area due to possessing inner/outer surfaces without additional internal supports. The SPME platform, for the first time, was successfully applied for the extraction and preconcentration of doxorubicin from urine and water media without requiring sample preparation and free from significant matrix effect. The extracted analyte was analyzed by liquid chromatography-ion trap tandem mass spectrometry (LC-MS/MS). Highly satisfactory analytical figures of merit were obtained under optimized conditions. The limit of detection (LOD), limit of quantification (LOQ) and linearity of determination were 0.1ngmL-1, 0.25ngmL-1 and 0.25 to 4000ngmL-1, respectively. The interday, intraday and inter sorbent precisions for three concentration levels ranged from 2.01 to 8.09 % (n=3), 1.02 to 8.65 % (n=5) and 0.99 to 1.02% (n=15), respectively. The mean intra-well RSD value for 96 individual wells in 96-MIHF-SPME-LC-MS/MS (n=3) at the medium concentration level was 7.81%.

Determination of atmospheric particle-bound polycyclic aromatic hydrocarbons using subcritical water extraction coupled with membrane microextraction

A green analytical methodology for the determination of 16 priority polycyclic aromatic hydrocarbons (PAHs) and 8 related compounds in air particulate matter was developed and validated. The method was based on pressurized hot water extraction (PHWE) followed by miniaturized membrane-assisted solvent extraction (MASE) and programmed temperature vaporization-gas chromatography-ion trap tandem mass spectrometry detection (PTV-GC–MS/MS). The parameters studied for PHWE were percentage of organic modifier (25% MeOH), temperature (200 °C) and static extraction time (5 min). For MASE, extraction temperature (30 °C), time (90 min) and effect of an organic modifier were also optimized. The matrix effect was evaluated and compensated using deuterated labelled standards as surrogates for the quantitation of the target compounds. The analytical performance of the method was satisfactory: relative recoveries varied between 78 and 118% and repeatability and intermediate precision were <22% for all compounds. The Method Quantitation Limits (MQL) ranged from 0.9 (TPY) to 75.6 pg m−3 (NAP). Satisfactory results for accuracy and traceability were evidenced by the analysis of a reference material (SRM 1649b) and comparison with previously published methods. The greenness score was estimated and compared with other techniques widely used for the analysis of PAHs in particulate matter, having a lower relative environmental impact.

Optimization of a DI-SPME-GC–MS/MS method for multi-residue analysis of pesticides in waters

This paper discusses the optimization and validation of a complete and simple method for the simultaneous determination of 48 pesticides in water by combining direct immersion solid-phase micro-extraction (DI-SPME) and gas chromatography-ion trap tandem mass spectrometry (GC-ITMS/MS). For the extraction, the different SPME parameters were tested and optimized. As a result, the polyacrylate fiber (PA 85 μm) was selected in direct immersion mode; the extraction temperature was set to 60 °C with an extraction time of 45 min and a stirring speed of 500 rpm; the thermal desorption time of the fiber in the injector port was fixed to 10 min at 275 °C. Concerning the analysis, MS/MS parameters were optimized and figures of merit were compared. Later, the method was validated and showed good linearity in the concentration ranging from 0.05 to 100 μg L−1. The reproducibility of measurements expressed as the relative standard deviation (% RSD) was found to be satisfactory. Furthermore, the detection limits for all the compounds were between 0.001 and 0.458 μg L−1, except for methomyl and hexachlorobenzene (1.09 and 0.99 μg L−1 respectively). Finally, the proposed DI-SPME-GC–MS/MS method was tested successfully for water samples collected along the watershed of Abou Ali River (North Lebanon).

Determination of 16 polycyclic aromatic hydrocarbons (PAHs) in surface and groundwater in North Lebanon by using SPME followed by GC–ITMS/MS

This paper discusses the development of a gas chromatography–ion trap tandem mass spectrometry (GC–ITMS/MS) coupled to solid phase micro-extraction for the quantitative determination of 16 polycyclic aromatic hydrocarbons (PAHs) in water samples. In this study, the factors influencing the GC–ITMS/MS method were investigated and optimized, and then a calibrating program was developed for the quantification of the PAHs under study. The method showed linear calibration curves for the 16 studied PAHs in the range of 0.1–100 ng mL−1 with the correlation coefficient (r2) between 0.92 and 0.999. The precisions (relative standard deviation, RSD) were below 15% and recoveries were between 76 and 105%. The limits of detection and limits of quantification were in the low ng mL−1 level. Therefore, these limits correlated with the standard method 8270D set by the United States Environmental Protection Agency. The optimized method was then effectively applied for the analysis of the 16 PAHs in surface and groundwater samples collected from 30 locations along the Basin of Abou Ali River in Lebanon. So far, no or very low PAHs concentrations were found in groundwater sampling locations while considerable concentrations (ranging from 0 to 28.72 ng mL−1) were detected in surface water of some locations. Consequently, the method was demonstrated to be applicable for the analysis of PAHs in the sampled waters.

Simultaneous accelerated solvent extraction and purification for the determination of 13 organophosphate esters in soils by gas chromatography-tandem mass spectrometry.

Through the optimization of extraction, purification, and determination parameters, a reliable and convenient analytical method for the simultaneous determination of 13 organophosphate esters in soils was developed. The method is based on one-step extraction and purification by accelerated solvent extraction and analysis by gas chromatography-ion trap tandem mass spectrometry. Under the optimal conditions, the method detection limits and method quantitation limits ranged from 0.10 to 0.22ng/g and from 0.33 to 0.72ng/g, respectively. The recoveries obtained were in the range of 81.7-107%, and the relative standard deviations were less than 12%. As compared to other methods, this proposed method was simple and time and solvent saving. The developed method was successfully applied to analyze organophosphate esters in soil samples collected from Liaohe estuarine wetland, China. Thirteen organophosphate esters were detected in all of the soil samples which indicated that Liaohe estuarine wetland has polluted by organophosphate esters.

PCBs and PCDD/Fs in Bluefin Tuna: Occurrence and Dietary Intake.

Polychlorinated biphenyls (PCBs) and polychlorinated dibenzo-furans (PCDD/Fs) were measured in Mediterranean bluefin tuna (Thunnus thynnus) to verify the compliance with the EU regulations for food commercialization. The estimated intakes were also evaluated. The analyses were performed by gas chromatography-ion trap tandem mass spectrometry (GC-MS-MS). The PCBs were dominant (1132.0 ng g−1 l.w.), followed by PCDFs (23.2 pg g−1 l.w.) and PCDDs (8.5 pg g−1 l.w.). The pollutant levels (dl-PCBs: 0.7 pg TEQ/g w.w.; PCDD/Fs: 1.9 pg TEQ/g w.w.) and their sum expressed as TEQ values (2.6 pg TEQ/g w.w.) remained below the limits for human consumption proposed by the European Union. On the contrary, the sum of the six indicator non-dioxin-like PCBs (84.2 ng g−1 w.w.) was slightly above the maximum level fixed by the in-force legislation. The estimated dietary intakes for PCDD/Fs plus dl-PCBs were below the toxicological reference values (TRVs) set by various international bodies, while non-cancer and cancer risk assessment revealed a safety concern. Additionally, the estimated intake of ndl-PCBs exceeded the maximum levels set by different European countries. These findings suggest caution in tuna consumption together with an active and frequent surveillance of the chemical quality of its flesh.

A fast and accurate isotope dilution GC-IT-MS/MS method for determination of eugenol in different tissues of fish: Application to a depletion study in mandarin fish.

An accurate, rapid and effective method was established for determination of eugenol in plasma, muscle, skin, liver, kidney and gill of fish using gas chromatography-ion trap tandem mass spectrometry. Samples of muscle, skin, liver, kidney and gill were prepared using the modified QuEChERS (quick, easy, cheap, effective, rugged and safe) procedure, and a plasma sample was prepared by a liquid-liquid extraction procedure. Eugenol was monitored in <7 min using an electron-ionization source in MS/MS mode and quantified by an internal standard of eugenol-d3 . The limit of detection was 5.0 μg/kg, and the limit of quantification was 10.0 μg/kg. The calibration curve was linear in the range of 5-1000 μg/L (R2 = 0.9996). Intra- and inter-day precisions of eugenol expressed as relative standard deviation were within 9.74%, and the accuracy exhibited a relative error ranging from -2.20 to 8.89%. The developed method was successfully used to study the elimination regularity of eugenol in mandarin fish.

Determination of benzothiazoles in seafood species by subcritical water extraction followed by solid-phase microextraction-gas chromatography-tandem mass spectrometry: estimating the dietary intake.

Benzothiazoles are high production volume chemicals widely used in many industrial and household applications. However, information on their occurrence in aquatic organisms is very limited, although a high level of bioaccumulation is expected. In this study and for the first time, a method was developed involving subcritical water extraction followed by solid-phase microextraction coupled to gas chromatography-ion trap-tandem mass spectrometry for the determination of five benzothiazoles in seafood. The repeatability and reproducibility of the method were under 21% (%RSD, n=5, 100ngg-1 (dw)), while method detection limits and method quantification limits were between 0.5 and 10ngg-1 (dw) and 1 and 50ngg-1 (dw), respectively. Ten widely consumed fish and shellfish species from the county of Tarragona (Catalonia, Spain) were selected in order to estimate dietary exposure and to assess the human health risks. The most frequently determined compounds were benzothiazole and 2-(methylthio)-benzothiazole, with squid being the species which showed the highest level of benzothiazole (82ngg-1 (dw)). In terms of human exposure, the current concentrations of benzothiazoles found in fish and shellfish could not be compared to threshold values because of the lack of toxicological data.

Comparative study of comprehensive gas chromatography-nitrogen chemiluminescence detection and gas chromatography-ion trap-tandem mass spectrometry for determining nicotine and carcinogen organic nitrogen compounds in thirdhand tobacco smoke

Thirdhand tobacco smoke (THS) constitutes a poorly understood pathway of exposure of non-smokers, especially toddlers, to tobacco-related carcinogens. However, to date most of the carcinogens present in tobacco smoke have not been detected in THS and, therefore, the significance of THS health risk is still unknown. In this study, we have compared the performance of two analytical methods – one based on gas chromatography coupled to ion trap mass spectrometry detection (GC-IT-MS) and the other on comprehensive two-dimensional gas chromatography coupled to a nitrogen chemiluminescence detector (GC×GC-NCD) – for simultaneously determining, in settled house dust, the presence of 16 organic nitrogen carcinogens already detected in tobacco smoke. The target compounds included four aromatic amines, two nitrocompounds, eight N-nitrosamines and two tobacco-specific nitrosamines, as well as nicotine as a tobacco marker. Dust samples were extracted using in-cell clean up pressurized liquid extraction with silica as clean up sorbent and ethyl acetate as the organic solvent, with average recovery of 89%. Although GC-IT-MS, using chemical ionization with methanol and tandem MS, performed well, the optimized GC×GC-NCD gave lower limits of detection (from 4 to 22ngg−1) and better repeatability and reproducibility a low concentration levels (%RSD<8%) and, therefore, was applicable for determining these different groups of carcinogens without the need for derivatization prior to the GC analysis.The performance of the optimized PLE/GC×GC-NCD method was tested by quantifying the target compounds in house dust samples from smokers’ and non-smokers’ homes. The median carcinogen compounds detected was 3.8μgg−1 and 1.1μgg−1 in smokers’ and non-smokers’ house dust, respectively. In this study, we have detected highly carcinogenic aromatic amines and nitro compounds for the first time in settled house dust complementing the state of knowledge of THS composition and providing fresh evidence about THS health risks.

Online eluent-switching technique coupled anion-exchange liquid chromatography–ion trap tandem mass spectrometry for analysis of non-steroidal anti-inflammatory drugs in pig serum

A novel method for online extraction, pH-gradient separation, and analysis of nine non-steroidal anti-inflammatory drugs (NSAIDs) was developed by coupling online eluent-switching technique to single anion-exchange chromatographic column/ion trap mass spectrometer (MS) and used for monitoring NSAIDs residues in pig serum. A neutral eluent and a pH-gradient eluent were used for extraction and separation of NSAIDs, respectively. Each of nine NSAIDs has an MS precursor ion of either [M−H]− or [M−Na]−. The extracted ion chromatogram for a specific product ion of each NSAID was used for its quantitative analysis. The dynamic linear ranges of calibration curves were all 0–200ngmL−1 (R2>0.9950). The analysis accuracies estimated by spiking standard concentrations at 20, 100, and 200ngmL−1 were 80.5–99.9%. The corresponding intra-day and inter-day precisions (RSD%) were 2.5–14.5% and 2.9–15.2%, respectively. The limit of detection/limit of quantitation of NSAIDs were 1.3/4.3, 0.5/1.6, 0.2/0.5, 2.5/8.2, 1.5/4.9, 0.6/2.1, 0.6/2.0, 0.5/1.7, and 0.6/2.1ngmL−1 for carprofen, diclofenac, flunixin, ibuprofen, ketoprofen, meclofenamic acid sodium, mefenamic acid, niflumic acid, and tolfenamic acid, respectively. After 1h injection of a dose containing 2mgkg−1 weight pig of flunixin and tolfenamic acid to the pigs, a residue amount of 3480±36ngmL−1 and 431±13ngmL−1, respectively, was reached for the incurred pig serum specimens and both residues were reduced to about 20ngmL−1 at the time of 24h.

Occurrence of β-lactam and polyether ionophore antibiotics in surface water, urban wastewater, and sediment

The occurrence and fate of antibiotic compounds in the aquatic environment has been recognized as an emerging area of interest in environmental chemistry. An analytical method was developed and validated for determining β-lactam and polyether ionophore antibiotics in surface water, urban wastewater, and sediment based on solid-phase extraction (SPE) and liquid chromatography–ion trap tandem mass spectrometry (LC-MS-MS). The average recovery of β-lactams and polyether ionophores from all the fortified samples was generally higher than 80% (except for amoxicillin and ampicillin). A total of 400 actual water and sediment samples were analysed using SPE-LC-MS-MS to evaluate the occurrence and fate of these antibiotic compounds in a river and a wastewater treatment plant in northern Colorado. Most samples were found to be negative for β-lactam compounds. In those samples with positive results, β-lactam compounds were found at 15–17 ng/L in influent wastewater samples, 9–11 ng/L in surface water samples, and 0.13–0.36 μg/kg in sediment samples. No β-lactam compounds were detected in any effluent wastewater samples. The β-lactams are expected to be easily eliminated in wastewater treatment plants due to the lability of the β-lactam ring towards chemical and microbial degradation. The average concentration of ionophores ranged 0.03–0.05 μg/L in surface water samples and 0.5–3.1 μg/kg in sediment samples. These results confirm that β-lactam and ionophore antibiotics are adsorbed by sediment.

Changes in hypothalamic neurotransmitter and prostanoid levels in response to NMDA, CRF, and GLP-1 stimulation.

Determination of neuroactive substances, such as neurotransmitters and prostanoids, in the extracellular space of the living brain is a very important technique in neuroscience. The hypothalamic paraventricular nucleus (PVN) is one of the most important autonomic control centers in the brain. Recently, we demonstrated that thromboxane (Tx) A2 in the PVN may play an important role in adrenomedullary outflow evoked by N-methyl-D-aspartate (NMDA), corticotrophin-releasing factor (CRF), and glucagon-like peptide-1 (GLP-1) stimulation using microdialysis sampling and liquid chromatography-ion trap tandem mass spectrometry (LC-ITMS(n)). In the present study, we investigated whether centrally administered NMDA, CRF, and GLP-1 can release five neurotransmitters, acetylcholine (ACh), histamine, glutamate (Glu), γ-aminobutyric acid (GABA), and serotonin (5-HT), along with six prostanoids, TxB2, prostaglandin (PG) E2, PGD2, 15-deoxy-∆(12,14) (15d)-PGJ2, 6-keto-PGF1α, and PGF2α in rat PVN microdialysates after optimization of LC-ITMS(n) conditions . All stimulations increased the levels of 5-HT, TxB2, PGE2, and PGF2α (except for 5-HT stimulated with GLP-1). Only NMDA increased the levels of ACh, Glu, and GABA. Treatment with dizocilpine maleate (MK-801), a noncompetitive NMDA receptor antagonist, attenuated the NMDA-induced increase in the levels of neuroactive substances except for Glu. This is the first study to use LC-ITMS(n) to analyze neurotransmitters and prostanoids in the same microdialysates from rat PVN.

Centrally administered isoproterenol induces sympathetic outflow via brain prostaglandin E2-mediated mechanisms in rats.

Brain β-adrenoceptor stimulation can induce elevations of plasma levels of noradrenaline. However, there have been no detailed studies related to signaling pathways downstream of β-adrenoceptors responsible for central sympathetic outflow. In the present study, we pharmacologically examined the possibility that centrally administered isoproterenol can induce elevations of plasma noradrenaline levels in a brain prostaglandin-dependent manner. In addition, we also examined whether or not intracerebroventricular administration of isoproterenol could release endogenously synthesized prostaglandin (PG) E2 in the hypothalamic paraventricular nucleus (PVN) by using the brain microdialysis technique combined with liquid chromatography-ion trap tandem mass spectrometry (LC-ITMS(n)). Under urethane anesthesia, a femoral venous line was inserted for infusion of saline and a femoral arterial line was inserted for collecting blood samples. Next, animals were placed in a stereotaxic apparatus for application of test agents. Catecholamines in the plasma were extracted by alumina absorption and were assayed by high-performance liquid chromatography with electrochemical detection. Quantification of PGE2 in rat PVN microdialysates was performed by the LC-ITMS(n) method. We demonstrated that centrally administered isoproterenol-induced elevations of plasma noradrenaline could be mediated via activation of β-adrenoceptors and the downstream phospholipase A2-cyclooxygenase pathway. Furthermore, PGE2 in the PVN and the PGE2 receptor EP3 subtype appear to play an important role in the process. Our results suggest that central isoproterenol-induced sympathetic outflow is mediated via brain PGE2 in a PGE2 receptor EP3 subtype-dependent manner.

Influence of pre-treatment process on matrix effect for the determination of musk fragrances in fish and mussel

Musk compounds are widely used as fragrances in personal care products. On account of their widespread use and their low biodegradation, they can be found in environmental samples. In our study two extraction methodologies were compared and different clean-up strategies were also studied in order to develop a reliable analytical method, with minimum matrix effect and good detection limits, to determine synthetic musk fragrances- six polycyclic musks, three nitro musks and the degradation product of one polycyclic musk- in fish and mussel samples. The first extraction technique involves a QuEChERS extraction, a consolidate extraction methodology in the field of food analysis of growing interest over recent years, followed by a dispersive solid-phase extraction (dSPE) as clean-up strategy. The second extraction technique consists of a conventional pressurised liquid extraction (PLE) with dichloromethane and an in-cell clean-up to decrease the matrix effect and remove the undesired components⁎present in PLE extracts. Large volume injection (LVI) followed by gas chromatography-ion trap-tandem mass spectrometry (GC-IT-MS/MS) was chosen as the separation and detection technique. Validation parameters, such as method detection limits and method quantification limits were found at ng g−1 levels for both fish and mussel matrices. Good levels of intra-day and inter-day repeatabilities were obtained analysing fish and mussel samples spiked at 50ng g−1 (d.w.) (n=5, RSDs<17%). The developed PLE/GC-IT-MS/MS method was successfully applied to determine the target musk fragrances present in fish and mussel samples from the local market in Tarragona and fish samples from the Ebro River. The results showed the presence of galaxolide (2.97–18.04ng g−1 (d.w.)) and tonalide (1.17–8.42ng g−1 (d.w.)) in all the samples analysed, while the remaining polycyclic musks such as cashmeran, celestolide and phantolide, were only detected in some of the fish samples analysed. None of the samples analysed contained detectable traces of the nitro musks studied.