Search results for: tandem mass spectrometry data.

Authors: Temesgen Sidamo, Prakruti S. Rao, Eleni Akllilu, Workineh Shibeshi, Yumi Park, Yong-Soon Cho, Jae-Gook Shin, Scott K. Heysell, Stellah G. Mpagama, Ephrem Engidawork

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The fluoroquinolones (FQs) are used off-label for the treatment of multidrug-resistant tuberculosis (MDR-TB), and for evaluation in shortening the duration of drug-susceptible TB in recently prioritized regimens. Within the class, levofloxacin (LFX) and moxifloxacin (MXF) play a substantial role in ensuring success in treatment outcomes. However, sub-therapeutic plasma concentrations of either LFX or MXF may drive unfavorable treatment outcomes. To the best of our knowledge, the pharmacokinetics of LFX and MXF in Ethiopian patients with MDR-TB have not yet been investigated. Therefore, the aim of this study was to develop a population pharmacokinetic (PopPK) model of levofloxacin (LFX) and moxifloxacin (MXF) and assess the percent probability of target attainment (PTA) as defined by the ratio of the area under the plasma concentration-time curve over 24-h (AUC0-24) and the in vitro minimum inhibitory concentration (MIC) (AUC0-24/MIC) in Ethiopian MDR-TB patients. Steady-state plasma was collected from 39 MDR-TB patients enrolled in the programmatic treatment course and the drug concentrations were determined using optimized liquid chromatography-tandem mass spectrometry. In addition, the in vitro MIC of the patients' pretreatment clinical isolates was determined. PopPK and simulations were run at various doses, and PK parameters were estimated. The effect of covariates on the PK parameters and the PTA for maximum mycobacterial kill and resistance prevention was also investigated. LFX and MXF both fit in a one-compartment model with adjustments. The apparent volume of distribution (V) and clearance (CL) of LFX were influenced by serum creatinine (Scr), whereas the absorption constant (Ka) and V of MXF were influenced by Scr and BMI, respectively. The PTA for LFX maximal mycobacterial kill at the critical MIC of 0.5 mg/L was 29%, 62%, and 95% with the simulated 750 mg, 1000 mg, and 1500 mg doses, respectively, whereas the PTA for resistance prevention at 1500 mg was only 4.8%, with none of the lower doses achieving this target. At the critical MIC of 0.25 mg/L, there was no difference in the PTA (94.4%) for maximum bacterial kill among the simulated doses of MXF (600 mg, 800 mg, and 1000 mg), but the PTA for resistance prevention improved proportionately with dose. Standard LFX and MXF doses may not provide adequate drug exposure. LFX PopPK is more predictable for maximum mycobacterial kill, whereas MXF's resistance prevention target increases with dose. Scr and BMI are likely to be important covariates in dose optimization or therapeutic drug monitoring (TDM) studies in Ethiopian patients.

Keywords: population PK, PTA, moxifloxacin, levofloxacin, MDR-TB patients, ethiopia

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Authors: Mustafa M. Donma, Orkide Donma

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Metabolic syndrome (MetS) is a disease associated with obesity. It is a complicated clinical problem possibly affecting body composition as well as macrominerals. These parameters gain further attention, particularly in the pediatric population. The aim of this study is to investigate the amount of discrete body composition fractions in groups that differ in the severity of obesity. Also, the possible associations with calcium (Ca), phosphorus (P), magnesium (Mg) will be examined. The study population was divided into four groups. Twenty-eight, 29, 34, and 34 children were involved in Group 1 (healthy), 2 (obese), 3 (morbid obese), and 4 (MetS), respectively. Institutional Ethical Committee approved the study protocol. Informed consent forms were obtained from the participants. The classification of obese groups was performed based upon the recommendations of the World Health Organization. Metabolic syndrome components were defined. Serum Ca, P, Mg concentrations were measured. Within the scope of body composition, fat mass, fat-free mass, protein mass, mineral mass were determined by a body composition monitor using bioelectrical impedance analysis technology. Weight, height, waist circumference, hip circumference, head circumference, and neck circumference values were recorded. Body mass index, diagnostic obesity notation model assessment index, fat mass index, and fat-free mass index values were calculated. Data were statistically evaluated and interpreted. There was no statistically significant difference among the groups in terms of Ca and P concentrations. Magnesium concentrations differed between Group 1 and Group 4. Strong negative correlations were detected between P as well as Mg and fat mass index as well as diagnostic obesity notation model assessment index in Group 4, the group, which comprised morbid obese children with MetS. This study emphasized unique associations of P and Mg minerals with diagnostic obesity notation model assessment index and fat mass index during the evaluation of morbid obese children with MetS. It was also concluded that diagnostic obesity notation model assessment index and fat mass index were more proper indices in comparison with body mass index and fat-free mass index for the purpose of defining body composition in children.

Keywords: children, fat mass, fat-free mass, macrominerals, obesity

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Authors: Anshuman Srivastava, Shiv Singh, Sheelendra Pratap Singh

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A simple, sensitive and effective gas chromatography tandem mass spectrometry (GC-MS/MS) method was developed for simultaneous analysis of multi pesticide residues (organophosphate, organochlorines, synthetic pyrethroids and herbicides) in food commodities using phenolic resin based activated carbon fibers (ACFs) as reversed-dispersive solid phase extraction (r-DSPE) sorbent in modified QuEChERS (Quick Easy Cheap Effective Rugged Safe) method. The acetonitrile-based QuEChERS technique was used for the extraction of the analytes from food matrices followed by sample cleanup with ACFs instead of traditionally used primary secondary amine (PSA). Different physico-chemical characterization techniques such as Fourier transform infrared spectroscopy, scanning electron microscopy, X-ray diffraction and Brunauer-Emmet-Teller surface area analysis were employed to investigate the engineering and structural properties of ACFs. The recovery of pesticides and herbicides was tested at concentration levels of 0.02 and 0.2 mg/kg in different commodities such as cauliflower, cucumber, banana, apple, wheat and black gram. The recoveries of all twenty-six pesticides and herbicides were found in acceptable limit (70-120%) according to SANCO guideline with relative standard deviation value < 15%. The limit of detection and limit of quantification of the method was in the range of 0.38-3.69 ng/mL and 1.26 -12.19 ng/mL, respectively. In traditional QuEChERS method, PSA used as r-DSPE sorbent plays a vital role in sample clean-up process and demonstrates good recoveries for multiclass pesticides. This study reports that ACFs are better in terms of removal of co-extractives in comparison of PSA without compromising the recoveries of multi pesticides from food matrices. Further, ACF replaces the need of charcoal in addition to the PSA from traditional QuEChERS method which is used to remove pigments. The developed method will be cost effective because the ACFs are significantly cheaper than the PSA. So the proposed modified QuEChERS method is more robust, effective and has better sample cleanup efficiency for multiclass multi pesticide residues analysis in different food matrices such as vegetables, grains and fruits.

Keywords: QuEChERS, activated carbon fibers, primary secondary amine, pesticides, sample preparation, carbon nanomaterials

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Authors: Jelka Pleadin, Nina Kudumija, Ana Vulic, Manuela Zadravec, Tina Lesic, Mario Skrivanko, Irena Perkovic, Nada Vahcic

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Products of animal origin, such as meat and meat products, can contribute to human mycotoxins’ intake coming as a result of either indirect transfer from farm animals exposed to naturally contaminated grains and feed (carry-over effects) or direct contamination with moulds or naturally contaminated spice mixtures used in meat production. Ochratoxin A (OTA) is mycotoxin considered to be of the outermost importance from the public health standpoint in connection with meat products. The aim of this study was to investigate the occurrence of OTA in different traditional meat products circulating on Croatian markets during 2018, produced by a large number of households situated in eastern and north Croatian regions using a variety of technologies. Concentrations of OTA were determined in traditional meat products (n = 70), including dry fermented sausages (Slavonian kulen, Slavonian sausage, Istrian sausage and domestic sausage; n = 28), dry-cured meat products (pancetta, pork rack and ham; n = 22) and cooked sausages (liver sausages, black pudding sausages and pate; n = 20). OTA was analyzed by use of quantitative screening immunoassay method (ELISA) and confirmed for positive samples (higher than the limit of detection) by liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Whereas the bacon samples contaminated with OTA were not found, its level in dry fermented sausages ranged from 0.22 to 2.17 µg/kg and in dry-cured meat products from 0.47 to 5.35 µg/kg, with in total 9% of positive samples. Besides possible primary contamination of these products arising due to improper manufacturing or/and storage conditions, observed OTA contamination could also be the consequence of secondary contamination that comes as a result of contaminated feed the animals were fed on. OTA levels obtained in cooked sausages ranged from 0.32 to 4.12 µg/kg (5% of positives) and could probably be linked to the contaminated raw materials (liver, kidney and spices) used in the sausages production. The results showed an occasional OTA contamination of traditional meat products, pointing that to avoid such contamination on households these products should be produced and processed under standardized and well-controlled conditions. Further investigations should be performed in order to identify mycotoxin-producing moulds on the surface of the products and to define preventative measures that can reduce the contamination of traditional meat products during their production on households and period of storage.

Keywords: Croatian households, ochratoxin-A, traditional cooked sausages, traditional dry-cured meat products

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Authors: S. Ebadzadsahraei, H. Kazemian

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The main objective of this research was to measure particulate matter (PM₂.₅) and Volatile Organic Compound (VOCs) as two classes of air pollutants, at Prince George (PG) neighborhood in warm and cold seasons. To fulfill this objective, analytical protocols were developed for accurate sampling and measurement of the targeted air pollutants. PM₂.₅ samples were analyzed for their chemical composition (i.e., toxic trace elements) in order to assess their potential source of emission. The City of Prince George, widely known as the capital of northern British Columbia (BC), Canada, has been dealing with air pollution challenges for a long time. The city has several local industries including pulp mills, a refinery, and a couple of asphalt plants that are the primary contributors of industrial VOCs. In this research project, which is the first study of this kind in this region it measures physical and chemical properties of particulate air pollutants (PM₂.₅) at the city neighborhood. Furthermore, this study quantifies the percentage of VOCs at the city air samples. One of the outcomes of this project is updated data about PM₂.₅ and VOCs inventory in the selected neighborhoods. For examining PM₂.₅ chemical composition, an elemental analysis methodology was developed to measure major trace elements including but not limited to mercury and lead. The toxicity of inhaled particulates depends on both their physical and chemical properties; thus, an understanding of aerosol properties is essential for the evaluation of such hazards, and the treatment of such respiratory and other related diseases. Mixed cellulose ester (MCE) filters were selected for this research as a suitable filter for PM₂.₅ air sampling. Chemical analyses were conducted using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) for elemental analysis. VOCs measurement of the air samples was performed using a Gas Chromatography-Flame Ionization Detector (GC-FID) and Gas Chromatography-Mass Spectrometry (GC-MS) allowing for quantitative measurement of VOC molecules in sub-ppb levels. In this study, sorbent tube (Anasorb CSC, Coconut Charcoal), 6 x 70-mm size, 2 sections, 50/100 mg sorbent, 20/40 mesh was used for VOCs air sampling followed by using solvent extraction and solid-phase micro extraction (SPME) techniques to prepare samples for measuring by a GC-MS/FID instrument. Air sampling for both PM₂.₅ and VOC were conducted in summer and winter seasons for comparison. Average concentrations of PM₂.₅ are very different between wildfire and daily samples. At wildfire time average of concentration is 83.0 μg/m³ and daily samples are 23.7 μg/m³. Also, higher concentrations of iron, nickel and manganese found at all samples and mercury element is found in some samples. It is able to stay too high doses negative effects.

Keywords: air pollutants, chemical analysis, particulate matter (PM₂.₅), volatile organic compound, VOCs

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Authors: Ayman El Behiry, Rasha Nabil Zahran, Reda Tarabees, Eman Marzouk, Musaad Al-Dubaib

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Mastitis is one of the most economic disease affecting dairy cows worldwide. Its classic diagnosis using bacterial culture and biochemical findings is a difficult and prolonged method. In this research, using of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) permitted identification of different microorganisms with high accuracy and rapidity (only 24 hours for microbial growth and analysis). During the application of MALDI-TOF MS, one hundred twenty strains of Staphylococcus and Streptococcus species isolated from milk of cows affected by clinical and subclinical mastitis were identified, and the results were compared with those obtained by traditional methods as API and VITEK 2 Systems. 37 of totality 39 strains (~95%) of Staphylococcus aureus (S. aureus) were exactly detected by MALDI TOF MS and then confirmed by a nuc-based PCR technique, whereas accurate identification was observed in 100% (50 isolates) of the coagulase negative staphylococci (CNS) and Streptococcus agalactiae (31 isolates). In brief, our results demonstrated that MALDI-TOF MS is a fast and truthful technique which has the capability to replace conventional identification of several bacterial strains usually isolated in clinical laboratories of microbiology.

Keywords: identification, mastitis pathogens, mass spectral, phenotypical

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Authors: Maged El-Sayed Mohamed

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Eucalyptus camaldulensis is one on the most well-known species of the genus Eucalyptus in the Middle east, its importance relay on the high production of its unique volatile constituents which exhibits many medicinal and pharmacological activities. The gall-forming wasp (Leptocybe invasa) has recently come into sight as the main pest attacking E. camaldulensis and causing severe injury. The wasp lays its eggs in the petiole and midrib of leaves and stems of young shoots of E. camaldulensis, which leads to gall formation. Gall formation by L. invasa damages growing shoot and leaves of Eucalyptus, resulting in abscission of leaves and drying. AIM: This study is an attempt to investigate the effect of the gall wasp (Leptocybe invasa) attack on the volatile constitutes of E. camaldulensis. This could help in the control of this wasp through stimulating plant defenses or production of a new allelochemicals or insecticide. The study of volatile constitutes of Eucalyptus before and after attack by the wasp can help the re-use and recycle of the infected Eucalyptus trees for new pharmacological and medicinal activities. Methodology: The fresh gall wasp-attacked and healthy leaves (100 g each) were cut and immediately subjected to hydrodistillation using Clevenger-type apparatus for 3 hours. The volatile fractions isolated were analyzed using Gas chromatography/mass spectrometry (GC/MS). Kovat’s retention indices (RI) were calculated with respect to a set of co-injected standard hydrocarbons (C10-C28). Compounds were identified by comparing their spectral data and retention indices with Wiley Registry of Mass Spectral Data 10th edition (April 2013), NIST 11 Mass Spectral Library (NIST11/2011/EPA/NIH) and literature data. Results: Fifty-nine components representing 89.13 and 88.60% of the total volatile fraction content respectively were quantitatively analyzed. Twenty-six major compounds at an average concentration greater than 0.1 ± 0.02% have been used for the statistical comparison. From those major components, twenty-one were found in both the attacked and healthy Eucalyptus leaves’ fractions in different concentration and five components, mono terpene p-Mentha-2-4(8) diene and the sesquiterpenes δ-elemene, β-elemene, E-caryophyllene and Bicyclogermacrene, were unique and only produced in the attacked-leaves’ fraction. CONCLUSION: Newly produced components or those commonly found in the volatile fraction and changed in concentration could represent a part of the plant defense mechanisms or might be an element of the plant allelopathic and communication mechanisms. Identification of the components of the gall wasp-damaged leaves can help in their recycling for different physiological, pharmacological and medicinal uses.

Keywords: Eucalyptus camaldulensis, eucalyptus recycling, gall wasp, Leptocybe invasa, plant defense mechanisms, Terpene fraction

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Authors: Chinnawat Satsananan

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The amount of heavy metals in our environment has been of great concern because of their toxicity when their concentration is more than the permissible level. These metals enter the environment by different ways such as industrial activities, soil pollution. We have used flame atomic absorption spectrometry technique for determination of the concentration of Co, Cd and Pb in different tissues of five samples of seafoods (mackerel, squid, mussels, scallops and shrimp). The concentrations of Co, Cd and Pb in all examined seafoods were less than the reported literature values (WHO). The results mentioned that the seafoods obtained from Thewet Market were safety to consumption and make the quality of life of people in the community look better.

Keywords: heavy metals, seafood, atomic absorption spectrometry, Bangkok

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Authors: Shamim Mushtaq, Moazzam Shahid

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Breast cancer (BC) claims the lives of half a million women every year and is the most common cause of death in the developing world. In 2019, it was estimated that BC alone accounts for 15% of all cancer deaths in younger women (aged < 45 years old) with advanced-stage lung metastasis. According to the World Health Organization & International Union against Cancer, in Asia, a high number of cancer-related deaths will be observed in 2020, whereas the burden will be reduced in Western countries due to awareness about the disease, better health facilities and advanced treatments. In the last 15 years, it has been reported that the incidence of BC has increased by 1.1% among Asian compared to the US population from 2003 to 2012. To date, several BC biological subtypes have been reported so far, which are associated with different treatment responses. The heterogeneity and diversity of BC reflected these different subtypes, including Luminal A (23.7% prevalence) and B (38.8% prevalence) that have pathological estrogen receptor (ER+)-positive tumors, the human epidermal growth factor receptor 2 (HER2) (11.2% prevalence) and triple-negative breast cancer (TNBC) (25% prevalence). According to Shaukat Khanum Memorial Cancer Hospital and Research Centre – Pakistan, ten years of data showed that among 636 BC patients, 30.5% had TNBC who were <40 years of age, which is an extremely alarming situation. Therefore, there is a dire need to explore and develop therapeutic targets for the treatment of early TNBC. Since the last decade, unfortunately, there has been little success in understanding the complexity of TNBC and in discovering new biological therapeutic targets. However, conventional chemotherapy is the only choice of treatment for TNBC patients. Many investigators revealed advances in multi-omics (multiple "omes", e.g., genome, proteome, transcriptome, epigenome, and microbiome) which were later identified as actionable targets and increased prevalence in TNBC patients. However, various drugs have been identified so far which are related to a particular diagnostic and prognostic biomarker. For example, Epidermal growth factor receptor ( EGFR or ErbB-1), HER-2/neu (ErbB-2), HER-3 (ErbB-3), and HER-4 (ErbB-4). Protein Transglin-2 (TAGLN 2 ) and Profilins-1 (Pfn-1 ) are the ubiquitously expressed large family of proteins present in all eukaryotes, enabling actin cytoskeletal reorganization. It is known that the oncogenic transformation of cells is accompanied by alteration in the actin cytoskeleton. There are causal connections between altered expression of actin cytoskeletal regulators and cancer progression. Our case-control study identified TAGLN-2 and Pfn-1 proteins in TNBC blood by mass spectrometry. Both TAGLN-2 and Pfn-1 proteins are differentially expressed in early and advanced stages of TNBS patients, which could be potential predictors or therapeutic targets for TNBC.

Keywords: TNBC, blood biomarkers, mass spectrometry, qPCR, ELISA

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Authors: Amanina Iymia Jeffree, Reena Thriumani, Mohammad Iqbal Omar, Ammar Zakaria, Yumi Zuhanis Has-Yun Hashim, Ali Yeon Md Shakaff

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Metabolite profiling is a strategy to be approached in the pattern recognition method focused on three types of cancer cell line that driving the most to death specifically lung, breast, and colon cancer. The purpose of this study was to discriminate the VOCs pattern among cancerous and control group based on metabolite profiling. The sampling was executed utilizing the cell culture technique. All culture flasks were incubated till 72 hours and data collection started after 24 hours. Every running sample took 24 minutes to be completed accordingly. The comparative metabolite patterns were identified by the implementation of headspace-solid phase micro-extraction (HS-SPME) sampling coupled with gas chromatography-mass spectrometry (GCMS). The optimizations of the main experimental variables such as oven temperature and time were evaluated by response surface methodology (RSM) to get the optimal condition. Volatiles were acknowledged through the National Institute of Standards and Technology (NIST) mass spectral database and retention time libraries. To improve the reliability of significance, it is of crucial importance to eliminate background noise which data from 3rd minutes to 17th minutes were selected for statistical analysis. Targeted metabolites, of which were annotated as known compounds with the peak area greater than 0.5 percent were highlighted and subsequently treated statistically. Volatiles produced contain hundreds to thousands of compounds; therefore, it will be optimized by chemometric analysis, such as principal component analysis (PCA) as a preliminary analysis before subjected to a pattern classifier for identification of VOC samples. The volatile organic compound profiling has shown to be significantly distinguished among cancerous and control group based on metabolite profiling.

Keywords: in vitro cancer cell line, metabolite profiling, pattern recognition, volatile organic compounds

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Authors: Constantinos Vayenas, Athanasios Fokas, Dimitrios Grigoriou

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We use special relativity to compute the inertial and thus gravitational mass of relativistic electron and muon neutrinos, and we find that, for neutrino kinetic energies above 150 MeV/c2, these masses are in the Planck mass range. Consequently, we develop a simple Bohr-type model using gravitational rather than electrostatic forces between the rotating neutrinos as the centripetal force in order to examine the bound rotational states formed by two or three such relativistic neutrinos. We find that the masses of the composite rotational structures formed, are in the meson and baryon mass ranges, respectively. These models contain no adjustable parameters and by comparing their predictions with the experimental values of the masses of protons and pions, we compute a mass of 0.0437 eV/c2 for the heaviest electron neutrino mass and of 1.1 x10-3 eV/c2 for the heaviest muon neutrino mass.

Keywords: geons, gravitational confinement, neutrino masses, special relativity

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Authors: Methaq Algabr, Nabil Al-Hajj, Ameerh Jaber, Amtellah Alshotobi, Shaima'a Al-suryhi, Gadah Whaban, Nawal Alshehari

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Steam distillation of the essential oil of P. jaubertii was performed using a Clevenger apparatus. Essential oils were analyzed by gas chromatography-flame ionization detector (GC-FID) and gas chromatography coupled to chromatography–mass spectrometry (GC-MS). The major chemical components identified in P. jaubertii essential oil include carvotanacetone (63.975%), 1-methyl-1,2-propanedione (5.887%), 2,5-dimethoxy-para-cymene (3.303%) and ar-curcumene (3.276%). The antimicrobial activity of the essential oil of P. jaubertii was evaluated against all tested microorganisms. P. jaubertii essential oil inhibited all tested microorganisms except Escherichia coli with a minimum inhibitory concentration (MIC) of 5.0 μg/mL against Staphylococcus aureus.

Keywords: Pulicaria jaubertii, essential oil, antimicrobial, Carvotancetone

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Authors: Timothy L. Porter, T. Randy Dillingham

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Vegetation growth and decomposition, along with soil microbial activity play a complex role in the production of greenhouse gases originating in forest soils. The absorption or emission (respiration) of these gases is a function of many factors relating to the soils themselves, the plants, and the environment in which the plants are growing. For this study, we have constructed a battery-powered, portable field mass spectrometer for use in analyzing gases in the soils surrounding trees, plants, and other areas. We have used the instrument to sample in real-time the greenhouse gases carbon dioxide and methane in soils where plant life may be contributing to the production of gases such as methane. Gases such as isoprene, which may help correlate gas respiration to microbial activity have also been measured. The instrument is composed of a quadrupole mass spectrometer with part per billion or better sensitivity, coupled to battery-powered turbo and diaphragm pumps. A unique ambient air pressure differentially pumped intake apparatus allows for the real-time sampling of gases in the soils from the surface to several inches below the surface. Results show that this instrument is capable of instant, part-per-billion sensitivity measurement of carbon dioxide and methane in the near surface region of various forest soils. We have measured differences in soil respiration resulting from forest thinning, forest burning, and forest logging as compared to pristine, untouched forests. Further studies will include measurements of greenhouse gas respiration as a function of temperature, microbial activity as measured by isoprene production, and forest restoration after fire.

Keywords: forest, soil, greenhouse, quadrupole

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Authors: Marin Senila, Oana Cadar, Thorsten Janisch, Patrick Lacroix-Desmazes

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The study presents the analytical capability and validation of a method based on microwave-assisted acid digestion for quantitative determination of platinum and palladium in catalysts using inductively coupled plasma optical emission spectrometry (ICP-OES). In order to validate the method, the main figures of merit such as limit of detection and limit of quantification, precision and accuracy were considered and the measurement uncertainty was estimated based on the bottom-up approach according to the international guidelines of ISO/IEC 17025. Limit of detections, estimated from blank signal using 3 s criterion, were 3.0 mg/kg for Pt and respectively 3.6 mg/kg for Pd, while limits of quantification were 9.0 mg/kg for Pt and respectively 10.8 mg/kg for Pd. Precisions, evaluated as standard deviations of repeatability (n=5 parallel samples), were less than 10% for both precious metals. Accuracies of the method, verified by recovery estimation certified reference material NIST SRM 2557 - pulverized recycled monolith, were 99.4 % for Pt and 101% for Pd. The obtained limit of quantifications and accuracy were satisfactory for the intended purpose. The paper offers all the steps necessary to validate the determination method for Pt and Pd in catalysts using inductively coupled plasma optical emission spectrometry.

Keywords: catalyst analysis, ICP-OES, method validation, platinum, palladium

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Authors: Qinghua Xing, Noha M. Mesbah, Haisheng Wang, Jun Li, Baisuo Zhao

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Droplet digital PCR (ddPCR) is being increasingly adopted for gene detection and quantification because of its higher sensitivity and specificity. According to previous observations and our lab data, it is essential to use endogenous reference genes (RGs) when investigating gene expression at the mRNA level under salt stress. This study aimed to select and validate suitable RGs for gene expression under salt stress using ddPCR. Six candidate RGs were selected based on the tandem mass tag (TMT)-labeled quantitative proteomics of Alkalicoccus halolimnae at four salinities. The expression stability of these candidate genes was evaluated using statistical algorithms (geNorm, NormFinder, BestKeeper and RefFinder). There was a small fluctuation in cycle threshold (Ct) value and copy number of the pdp gene. Its expression stability was ranked in the vanguard of all algorithms, and was the most suitable RG for quantification of expression by both qPCR and ddPCR of A. halolimnae under salt stress. Single RG pdp and RG combinations were used to normalize the expression of ectA, ectB, ectC, and ectD under four salinities. The present study constitutes the first systematic analysis of endogenous RG selection for halophiles responding to salt stress. This work provides a valuable theory and an approach reference of internal control identification for ddPCR-based stress response models.

Keywords: endogenous reference gene, salt stress, ddPCR, RT-qPCR, Alkalicoccus halolimnae

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Authors: Jenson George, Thoa Nguyen, Garth Sanewski, Craig Hardner, Heather Eunice Smyth

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Pineapple (Ananas comosus), with its unique sweet flavour, is one of the most popular tropical, non-climacteric fruits consumed worldwide. It is also the third most important tropical fruit in world production. In Australia, 99% of the pineapple production is from the Queensland state due to the favourable subtropical climatic conditions. The flavourful fruit is known to contain around 500 volatile organic compounds (VOC) at varying concentrations and greatly contribute to the flavour quality of pineapple fruit by providing distinct aroma sensory properties that are sweet, fruity, tropical, pineapple-like, caramel-like, coconut-like, etc. The aroma of pineapple is one of the important factors attracting consumers and strengthening the marketplace. To better understand the aroma of Australian-grown pineapples, the matrix-matched Gas chromatography–mass spectrometry (GC-MS), Head Space - Solid-phase microextraction (HS-SPME), Stable-isotope dilution analysis (SIDA) method was developed and validated. The developed method represents a significant improvement over current methods with the incorporation of multiple external reference standards, multiple isotopes labeled internal standards, and a matching model system of pineapple fruit matrix. This method was employed to quantify 28 key aroma compounds in more than 200 genetically diverse pineapple varieties from a breeding program. The Australian pineapple cultivars varied in content and composition of free volatile compounds, which were predominantly comprised of esters, followed by terpenes, alcohols, aldehydes, and ketones. Using selected commercial cultivars grown in Australia, and by employing the sensorial analysis, the appearance (colour), aroma (intensity, sweet, vinegar/tang, tropical fruits, floral, coconut, green, metallic, vegetal, fresh, peppery, fermented, eggy/sulphurous) and texture (crunchiness, fibrousness, and juiciness) were obtained. Relationships between sensory descriptors and volatiles were explored by applying multivariate analysis (PCA) to the sensorial and chemical data. The key aroma compounds of pineapple exhibited a positive correlation with corresponding sensory properties. The sensory and volatile data were also used to explore genetic diversity in the breeding population. GWAS was employed to unravel the genetic control of the pineapple volatilome and its interplay with fruit sensory characteristics. This study enhances our understanding of pineapple aroma (flavour) compounds, their biosynthetic pathways and expands breeding option for pineapple cultivars. This research provides foundational knowledge to support breeding programs, post-harvest and target market studies, and efforts to optimise the flavour of commercial pineapple varieties and their parent lines to produce better tasting fruits for consumers.

Keywords: Ananas comosus, pineapple, flavour, volatile organic compounds, aroma, Gas chromatography–mass spectrometry (GC-MS), Head Space - Solid-phase microextraction (HS-SPME), Stable-isotope dilution analysis (SIDA).

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Authors: El Kolli Meriem, Laouer Hocine, Sahli Farida, Akkal Salah, El Kolli Hayet

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The aerial parts of Daucus gracilis (Apiaceae) were subjected to hydrodistillation by a Clevenger apparatus to obtain the essential oil (EO) which has been analyzed by Gas Chromatography (GC) and GC coupled with mass spectrometry. The antioxidant properties of this EO and D. gracilis methanolic extract were studied by both of the free diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and the reducing power techniques. The dominant constituents of the EO were the elemicin (35.3 %) and the geranyl acetate (26.8 %). Both of EO and methanolic extract showed important antioxidant properties with respectively IC50 of 0,002 mg/ml and 0.06 mg/ml. They showed also a reducing power dose-dependent.

Keywords: daucus gracilis, apiaceae, essential oil, antioxidant activity

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Authors: Pierre-Louis Lucas, Corinne Loutelier-Bourhis, Narimane Mati-Baouche, Philippe Chan Tchi-Song, Patrice Lerouge, Elodie Mathieu-Rivet, Muriel Bardor

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N-glycosylation is a post-translational modification taking place in the Endoplasmic Reticulum and the Golgi apparatus where defined glycan features are added on protein in a very specific sequence Asn-X-Thr/Ser/Cys were X can be any amino acid except proline. Because it is well-established that those N-glycans play a critical role in protein biological activity, protein half-life and that a different N-glycan structure may induce an immune response, they are very important in Biopharmaceuticals which are mainly glycoproteins bearing N-glycans. From now, most of the biopharmaceuticals are produced by mammalian cells like Chinese Hamster Ovary cells (CHO) for their N-glycosylation similar to the human, but due to the high production costs, several other species are investigated as the possible alternative system. In this purpose, the green microalgae Chlamydomonas reinhardtii was investigated as the potential production system for Biopharmaceuticals. This choice was influenced by the facts that C. reinhardtii is a well-study microalgae which is growing fast with a lot of molecular biology tools available. This organism is also producing N-glycan on its endogenous proteins. However, the analysis of the N-glycan structure of this microalgae has revealed some differences as compared to the human. Rather than in Human where the glycans are processed by key enzymes called N-acetylglucosaminyltransferase I and II (GnTI and GnTII) adding GlcNAc residue to form a GlcNAc₂Man₃GlcNAc₂ core N-glycan, C. reinhardtii lacks those two enzymes and possess a GnTI independent glycosylation pathway. Moreover, some enzymes like xylosyltransferases and methyltransferases not present in human are supposed to act on the glycans of C. reinhardtii. Furthermore, the recent structural study by mass spectrometry shows that the N-glycosylation precursor supposed to be conserved in almost all eukaryotic cells results in a linear Man₅GlcNAc₂ rather than a branched one in C. reinhardtii. In this work, we will discuss the new released MS information upon C. reinhardtii N-glycan structure and their impact on our attempt to modify the glycan in a Human manner. Two strategies will be discussed. The first one consisted in the study of Xylosyltransferase insertional mutants from the CLIP library in order to remove xyloses from the N-glycans. The second will go further in the humanization by transforming the microalgae with the exogenous gene from Toxoplasma gondii having an activity similar to GnTI and GnTII with the aim to synthesize GlcNAc₂Man₃GlcNAc₂ in C. reinhardtii.

Keywords: Chlamydomonas reinhardtii, N-glycosylation, glycosyltransferase, mass spectrometry, humanization

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Authors: Maged El-Sayed Mohamed

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Plant tissue culture practices are presented nowadays as the most promising substitute to a whole plant in the terms of secondary metabolites production. They offer the advantages of high production, tunability and they have less effect on plant ecosystems. Lantana camara is a weed, which is common all over the world as an ornamental plant. Weeds can adapt to any type of soil and climate due to their rich cellular machinery for secondary metabolites’ production. This characteristic is found in Lantana camara as a plant of very rich diversity of secondary metabolites with no dominant class of compounds. Aim: This trait has encouraged the author to develop tissue culture experiments for Lantana camara to be a platform for production and manipulation of secondary metabolites through biotransformation. Methodology: The plant was collected in its flowering stage in September 2014, from which explants were prepared from shoot tip, auxiliary bud and leaf. Different types of culture media were tried as well as four phytohormones and their combinations; NAA, 2,4-D, BAP and kinetin. Explants were grown in dark or in 12 hours dark and light cycles at 25°C. A metabolic profile for the produced callus was made and then compared to the whole plant profile. The metabolic profile was made using GC-MS for volatile constituents (extracted by n-hexane) and by HPLC-MS and capillary electrophoresis-mass spectrometry (CE-MS) for non-volatile constituents (extracted by ethanol and water). Results: The best conditions for the callus induction was achieved using MS media supplied with 30 gm sucrose and NAA/BAP (1:0.2 mg/L). Initiation of callus was favoured by incubation in dark for 20 day. The callus produced under these conditions showed yellow colour, which changed to brownish after 30 days. The rate of callus growth was high, expressed in the callus diameter, which reached to 1.15±0.2 cm in 30 days; however, the induction of callus delayed for 15 days. The metabolic profile for both volatile and non-volatile constituents of callus showed more simple background metabolites than the whole plant with two new (unresolved) peaks in the callus’ nonvolatile constituents’ chromatogram. Conclusion: Lantana camara callus production can be itself a source of new secondary metabolites and could be used for biotransformation studies due to its simple metabolic background, which allow easy identification of newly formed metabolites. The callus production gathered the simple metabolic background with the rich cellular secondary metabolite machinery of the plant, which could be elicited to produce valuable medicinally active products.

Keywords: capillary electrophoresis-mass spectrometry, gas chromatography, metabolic profile, plant tissue culture

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Authors: Sarah A. Alotaibi, Mohammed A. Almutairi, Abdullah A. Alsayari, Adibah M. Almutairi, Somaiah K. Almubayedh

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Concerns over the presence of acrylamide in food date back to 2002, when Swedish scientists stated that, in carbohydrate-rich foods, amounts of acrylamide were formed when cooked at high temperatures. Similar findings were reported by other researchers which, consequently, caused major international efforts to investigate dietary exposure and the subsequent health complications in order to properly manage this issue. Due to this issue, in this work, we aim to determine the acrylamide level in different foods (coffee, potato chips, biscuits, and baby food) commonly consumed by the Saudi population. In a total of forty-three samples, acrylamide was detected in twenty-three samples at levels of 12.3 to 2850 µg/kg. In reference to the food groups, the highest concentration of acrylamide was found in coffee samples (<12.3-2850 μg/kg), followed by potato chips (655-1310 μg/kg), then biscuits (23.5-449 μg/kg), whereas the lowest acrylamide level was observed in baby food (<14.75 – 126 μg/kg). Most coffee, biscuits and potato chips products contain high amount of acrylamide content and also the most commonly consumed product. Saudi adults had a mean exposure of acrylamide for coffee, potato, biscuit, and cereal (0.07439, 0.04794, 0.01125, 0.003371 µg/kg-b.w/day), respectively. On the other hand, exposure to acrylamide in Saudi infants and children to the same types of food was (0.1701, 0.1096, 0.02572, 0.00771 µg/kg-b.w/day), respectively. Most groups have a percentile that exceeds the tolerable daily intake (TDI) cancer value (2.6 µg/kg-b.w/day). Overall, the MOE results show that the Saudi population is at high risk of acrylamide-related disease in all food types, and there is a chance of cancer risk in all age groups (all values ˂10,000). Furthermore, it was found that in non-cancer risks, the acrylamide in all tested foods was within the safe limit (˃125), except for potato chips, in which there is a risk for diseases in the population. With potato and coffee as raw materials, additional studies were conducted to assess different factors, including temperature, cocking time, and additives affecting the acrylamide formation in fried potato and roasted coffee, by systematically varying processing temperatures and time values, a mitigation of acrylamide content was achieved when lowering the temperature and decreasing the cooking time. Furthermore, it was shown that the combination of the addition of chitosan and NaCl had a large impact on the formation.

Keywords: risk assessment, dietary exposure, MOA, acrylamide, hazard

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Authors: Simran Baweja, Bhavika Kalal, Surajit Maity

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Photoinduced tautomerization reactions have been the centre of attention among scientific community over past several decades because of their significance in various biological systems. 7-azaindole (7AI) is considered as a model system for DNA base pairing and to understand the role of such tautomerization reactions in mutations. To the best of our knowledge, extensive studies have been carried on 7-azaindole and its solvent clusters exhibiting proton/ hydrogen transfer in both solution as well as gas phase. Derivatives of above molecule, like 2,7- and 2,6-diazaindoles are proposed to have even better photophysical properties due to the presence of -aza group on the 2nd position. However, there are a few studies in the solution phase which suggest the relevance of these molecules, but there are no experimental studies reported in the gas phase yet. In our current investigation, we present the first gas phase spectroscopic data of 2,7-diazaindole (2,7-DAI) and its solvent cluster (2,7-DAI-H2O). In this, we have employed state-of-the-art laser spectroscopic methods such as fluorescence excitation (LIF), dispersed fluorescence (DF), resonant two-photon ionization time of flight mass spectrometry (2C-R2PI), photoionization efficiency spectroscopy (PIE), IR-UV double resonance spectroscopy i.e. fluorescence-dip infrared spectroscopy (FDIR) and resonant ion-dip infrared spectroscopy (IDIR) to understand the electronic structure of the molecule. The origin band corresponding to S1 ← S0 transition of the bare 2,7-DAI is found to be positioned at 33910 cm-1 whereas the origin band corresponding to S1 ← S0 transition of the 2,7-DAI-H2O is positioned at 33074 cm-1. The red shifted transition in case of solvent cluster suggests the enhanced feasibility of excited state hydrogen/ proton transfer. The ionization potential for the 2,7-DAI molecule is found to be 8.92 eV, which is significantly higher that the previously reported 7AI (8.11 eV) molecule, making it a comparatively complex molecule to study. The ionization potential is reduced by 0.14 eV in case of 2,7-DAI-H2O (8.78 eV) cluster compared to that of 2,7-DAI. Moreover, on comparison with the available literature values of 7AI, we found the origin band of 2,7-DAI and 2,7-DAI-H2O to be red shifted by -729 and -280 cm-1 respectively. The ground and excited state N-H stretching frequencies of the 27DAI molecule were determined using fluorescence-dip infrared spectra (FDIR) and resonant ion dip infrared spectroscopy (IDIR), obtained at 3523 and 3467 cm-1, respectively. The lower value of vNH in the electronic excited state of 27DAI implies the higher acidity of the group compared to the ground state. Moreover, we have done extensive computational analysis, which suggests that the energy barrier in excited state reduces significantly as we increase the number of catalytic solvent molecules (S= H2O, NH3) as well as the polarity of solvent molecules. We found that the ammonia molecule is a better candidate for hydrogen transfer compared to water because of its higher gas-phase basicity. Further studies are underway to understand the excited state dynamics and photochemistry of such N-rich chromophores.

Keywords: photoinduced tautomerization reactions, gas phse spectroscopy, ), IR-UV double resonance spectroscopy, resonant two-photon ionization time of flight mass spectrometry (2C-R2PI)

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Authors: Guo-Hui Lu, Jing-Yi Cai, Ke-Yan Tan, Xiao-Cai Yin, Yu Zheng, Peng-Wei Shao, Yong-Liang Yang

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Soil erosion around abandoned mines is one of the important geological agents for pollutant diffuses to the lower reaches of the local river basin system. River loading of pollutants is an important parameter for remediation of abandoned mines. In order to obtain information on pollutant transport and diffusion downstream in mining area, the small tributary system of the Xiaxi River in Wanshan District of Guizhou Province was selected as the research area. Sediment and suspended matter samples were collected and determined for Pb, As, Hg, Zn, Co, Cd, Cu, Ni, Cr, and Mn by inductively coupled plasma mass spectrometry (ICP-MS) and atomic fluorescence spectrometry (AFS) with the pretreatment of wet digestion. Discussions are made for pollution status and spatial distribution characteristics. The total Hg content in the sediments ranged from 0.45 to 16.0 g/g (dry weight) with an average of 5.79 g/g, which was ten times higher than the limit of Class II soil for mercury by the National Soil Environmental Quality Standard. The maximum occurred at the intersection of the Jin River and the Xiaxi River. The potential ecological hazard index (RI) was used to evaluate the ecological risk of heavy metals in the sediments. The average RI value for the whole study area suggests the high potential ecological risk level. High Cd potential ecological risk was found at individual sites.

Keywords: heavy metal, risk assessment, sediment, suspended matter, Wanshan mercury mine, small tributary system

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Authors: Patricia O. Carvalho, Marcia C. F. Messias, Laura Credidio, Carlos A. R. Martinez

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Lipidomic strategy can provide important information regarding cancer pathogenesis mechanisms and could reveal new biomarkers to enable early diagnosis of rectal adenocarcinoma (RAC). This study set out to evaluate lipoperoxidation biomarkers, and lipidomic signature by gas chromatography (GC) and electrospray ionization-qToF-mass spectrometry (ESI-qToF-MS) combined with multivariate data analysis in plasma from 23 RAC patients (early- or advanced-stages cancer) and 18 healthy controls. The most abundant ions identified in the RAC patients were those of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) while those of lisophosphatidylcholine (LPC), identified as LPC (16:1), LPC (18:1) and LPC (18:2), were down-regulated. LPC plasmalogen containing palmitoleic acid (LPC (P-16:1)), with highest VIP score, showed a low tendency in the cancer patients. Malondialdehyde plasma levels were higher in patients with advanced cancer (III/IV stages) than in the early stages groups and the healthy group (p<0.05). No differences in F2-isoprostane levels were observed between these groups. This study shows that the reduction in plasma levels of LPC plasmalogens associated to an increase in MDA levels may indicate increased oxidative stress in these patients and identify the metabolite LPC (P-16:1) as new biomarkers for RAC.

Keywords: biomarkers, lipidomic, plasmalogen, rectal adenocarcinoma

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Authors: Nataliya Venelinova

Abstract:

The paper proposes a new interdisciplinary model of reconsidering the role of mass communication effects by coverage of terrorism. The idea of 4P model is based on the synergy, created by the information strategy of threat, predominantly used by terrorist groups, the effects of mediating the symbolic action of the terrorist attacks or the taking of responsibility of any attacks, and the reshaped public perception for security after the attacks being mass communicated. The paper defines the mass communication cycle of terrorism, which leads not only to re-agenda setting of the societies, but also spirally amplifying the effect of propagating fears by over-informing on terrorism attacks. This finally results in the outlining of the so called 4P-model of information terrorism: mass propaganda, panic, paranoia and pandemic.

Keywords: information terrorism, mass communication cycle, public perception, security

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Authors: Takeyuki Suzuki, Ismiyarto, Da-Yang Zhou, Kaori Asano, Hiroaki Sasai

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The development of catalytic asymmetric reaction is important for the synthesis of natural products. To construct the multiple stereogenic centers, the desymmetrization of meso compounds is powerful strategy for the synthesis of chiral molecules. Oxidative desymmetrization of meso diols using chiral iridium catalyst provides a chiral hydroxyl ketone. The reaction is practical and an environmentally benign method which does not require the use of stoichiometric amount of heavy metals. This time we report here catalytic asymmetric synthesis of catalponol based on tandem coupling of meso-diols and an aldehyde. The tandem reaction includes oxidative desymmetrization of meso-diols, aldol condensation with an aldehyde. The reaction of meso-diol, benzaldehyde in the presence of a catalytic amount of chiral Ir complex and CsOH in tetrahydrofuran afforded the desired benzylidene ketone in 82% yield with 96% ee (enantiomeric excess). Next, we applied this benzylidene ketone derivative to the synthesis of catalponol. The corresponding benzylidene ketone was obtained in 87% yield with 99% ee. Finally, catalponol was synthesized by the regio- and stereo-selective reduction of dienone moiety in good yield.

Keywords: catalponol, desymmetrization, iridium, oxidation

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Authors: E. D. Paul, F. G. Okibe, C. E. Gimba, S. Yakubu

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Some physicochemical characteristics and heavy metal concentrations of water samples collected from ten boreholes in Samaru, Zaria, Kaduna state, Nigeria were analysed in order to assess the drinking water quality. Physicochemical parameters were determined using classical methods while the heavy metals were determined using Atomic Absorption Spectrometry. Results of the analysis obtained were as follows: Temperature 29 – 310C, pH 5.74 – 6.19, Electrical conductivity 3.21 – 7.54 µs, DO 0.51 – 1.00 mg/L, BOD 0.0001 – 0.006 mg/L, COD 160 – 260 mg/L, TDS 2.08 – 4.55 mg/L, Total Hardness 97.44 – 401.36 mg/L CaCO3, and Chloride 0.97 – 59.12 mg/L. Concentrations of heavy metals were in the range; Zinc 0.000 – 0.7568 mg/L, Lead 0.000 – 0.070 mg/L and Cadmium 0.000 – 0.009 mg/L. The implications of these findings are discussed.

Keywords: ground water, water quality, heavy metals, Atomic Absorption Spectrometry (AAS)

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Authors: R. Tudugala, B. M. A. I. Balasooriya, W. M. Ediri Arachchi, R. W. M. W. K. Rathnayake, T. D. Premaratna

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Brachytherapy involves placing a source of radiation near the cancer site which gives promising prognosis for cervical cancer treatments. The purpose of this study was to evaluate the effect of random variation of treated volumes in between fractions in the 2D image based fractionated high dose rate brachytherapy for cervical cancer at National Cancer Institute Maharagama, Sri Lanka. Dose plans were analyzed for 150 cervical cancer patients with orthogonal radiographs (2D) based brachytherapy. ICRU treated volumes was modeled by translating the applicators with the help of “Multisource HDR plus software”. The difference of treated volumes with respect to the applicator geometry was analyzed by using SPSS 18 software; to derived patient population based estimates of delivered treated volumes relative to ideally treated volumes. Packing was evaluated according to bladder dose, rectum dose and geometry of the dose distribution by three consultant radiation oncologist. The difference of treated volumes depends on types of the applicators, which was used in fractionated brachytherapy. The means of the “Difference of Treated Volume” (DTV) for “Evenly activated tandem (ET)” length” group was ((X_1)) -0.48 cm3 and ((X_2)) 11.85 cm3 for “Unevenly activated tandem length (UET) group. The range of the DTV for ET group was 35.80 cm3 whereas UET group 104.80 cm3. One sample T test was performed to compare the DTV with “Ideal treatment volume difference (0.00cm3)”. It is evident that P value was 0.732 for ET group and for UET it was 0.00 moreover independent two sample T test was performed to compare ET and UET groups and calculated P value was 0.005. Packing was evaluated under three categories 59.38% used “Convenient Packing Technique”, 33.33% used “Fairly Packing Technique” and 7.29% used “Not Convenient Packing” in their fractionated brachytherapy treatments. Random variation of treated volume in ET group is much lower than UET group and there is a significant difference (p<0.05) in between ET and UET groups which affects the dose distribution of the treatment. Furthermore, it can be concluded nearly 92.71% patient’s packing were used acceptable packing technique at NCIM, Sri Lanka.

Keywords: brachytherapy, cervical cancer, high dose rate, tandem, treated volumes

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Authors: Siraj Beyan Mohamed, Woldia University

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The study is accompanied in northern Ethiopian basement rocks, Finarwa area, and its surrounding areas, south eastern Tigray. From the field observations, the geology of the area haven been described and mapped based on mineral composition, texture, structure, and colour of both fresh and weather rocks. Inductively coupled plasma mass spectrometry (ICP-MS) and atomic absorption spectrometry (AAS) have conducted to analysis gold and base metal mineralization. The ore mineral under microscope are commonly base metal sulphides pyrrhotite, Chalcopyrite, pentilanditeoccurring in variable proportions. Galena, chalcopyrite, pyrite, and gold mineral are hosted in quartz vein. Pyrite occurs both in quartz vein and enclosing rocks as a primary mineral. The base metal sulfides occur as disseminated, vein filling, and replacement. Geochemical analyses result determination of the threshold of geochemical anomalies is directly related to the identification of mineralization information. From samples, stream sediment samples and the soil samples indicated that the most promising mineralization occur in the prospect area are gold(Au), copper (Cu), and zinc (Zn). This is also supported by the abundance of chalcopyrite and sphalerite in some highly altered samples. The stream sediment geochemical survey data shows relatively higher values for zinc compared to Pb and Cu. The moderate concentration of the base metals in some of the samples indicates availability base metal mineralization in the study area requiring further investigation. The rock and soil geochemistry shows the significant concentration of gold with maximum value of 0.33ppm and 0.97 ppm in the south western part of the study area. In Finarwa, artisanal gold mining has become an increasingly widespread economic activity of the local people undertaken by socially differentiated groups with a wide range of education levels and economic backgrounds incorporating a wide variety of ‘labour intensive activities without mechanisation.

Keywords: gold, base metal, anomaly, threshold

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Authors: Rowan Moore, Kai Scheffler, Eugen Damoc, Jennifer Sutton, Aaron Bailey, Stephane Houel, Simon Cubbon, Jonathan Josephs

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Purpose: MS analysis of monoclonal antibodies (mAbs) at the protein and peptide levels is critical during development and production of biopharmaceuticals. The compositions of current generation therapeutic proteins are often complex due to various modifications which may affect efficacy. Intact proteins analyzed by MS are detected in higher charge states that also provide more complexity in mass spectra. Protein analysis in native or native-like conditions with zero or minimal organic solvent and neutral or weakly acidic pH decreases charge state value resulting in mAb detection at higher m/z ranges with more spatial resolution. Methods: Three commercially available mAbs were used for all experiments. Intact proteins were desalted online using size exclusion chromatography (SEC) or reversed phase chromatography coupled on-line with a mass spectrometer. For streamlined use of the LC- MS platform we used a single SEC column and alternately selected specific mobile phases to perform separations in either denaturing or native-like conditions: buffer A (20 % ACN, 0.1 % FA) with Buffer B (100 mM ammonium acetate). For peptide analysis mAbs were proteolytically digested with and without prior reduction and alkylation. The mass spectrometer used for all experiments was a commercially available Thermo Scientific™ hybrid Quadrupole-Orbitrap™ mass spectrometer, equipped with the new BioPharma option which includes a new High Mass Range (HMR) mode that allows for improved high mass transmission and mass detection up to 8000 m/z. Results: We have analyzed the profiles of three mAbs under reducing and native conditions by direct infusion with offline desalting and with on-line desalting via size exclusion and reversed phase type columns. The presence of high salt under denaturing conditions was found to influence the observed charge state envelope and impact mass accuracy after spectral deconvolution. The significantly lower charge states observed under native conditions improves the spatial resolution of protein signals and has significant benefits for the analysis of antibody mixtures, e.g. lysine variants, degradants or sequence variants. This type of analysis requires the detection of masses beyond the standard mass range ranging up to 6000 m/z requiring the extended capabilities available in the new HMR mode. We have compared each antibody sample that was analyzed individually with mixtures in various relative concentrations. For this type of analysis, we observed that apparent native structures persist and ESI is benefited by the addition of low amounts of acetonitrile and formic acid in combination with the ammonium acetate-buffered mobile phase. For analyses on the peptide level we analyzed reduced/alkylated, and non-reduced proteolytic digests of the individual antibodies separated via reversed phase chromatography aiming to retrieve as much information as possible regarding sequence coverage, disulfide bridges, post-translational modifications such as various glycans, sequence variants, and their relative quantification. All data acquired were submitted to a single software package for analysis aiming to obtain a complete picture of the molecules analyzed. Here we demonstrate the capabilities of the mass spectrometer to fully characterize homogeneous and heterogeneous therapeutic proteins on one single platform. Conclusion: Full characterization of heterogeneous intact protein mixtures by improved mass separation on a quadrupole-Orbitrap™ mass spectrometer with extended capabilities has been demonstrated.

Keywords: disulfide bond analysis, intact analysis, native analysis, mass spectrometry, monoclonal antibodies, peptide mapping, post-translational modifications, sequence variants, size exclusion chromatography, therapeutic protein analysis, UHPLC

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Authors: Béla Kovács, Éva Bódi, Farzaneh Garousi, Szilvia Várallyay, Dávid Andrási

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For analysis of elements in different food, feed and food raw material samples generally a flame atomic absorption spectrometer (FAAS), a graphite furnace atomic absorption spectrometer (GF-AAS), an inductively coupled plasma optical emission spectrometer (ICP-OES) and an inductively coupled plasma mass spectrometer (ICP-MS) are applied. All the analytical instruments have different physical and chemical interfering effects analysing food and food raw material samples. The smaller the concentration of an analyte and the larger the concentration of the matrix the larger the interfering effects. Nowadays, it is very important to analyse growingly smaller concentrations of elements. From the above analytical instruments generally the inductively coupled plasma mass spectrometer is capable of analysing the smallest concentration of elements. The applied ICP-MS instrument has Collision Cell Technology (CCT) also. Using CCT mode certain elements have better detection limits with 1-3 magnitudes comparing to a normal ICP-MS analytical method. The CCT mode has better detection limits mainly for analysis of selenium (arsenic, germanium, vanadium, and chromium). To elaborate an analytical method for selenium with an inductively coupled plasma mass spectrometer the most important interfering effects (problems) were evaluated: 1) isobaric elemental, 2) isobaric molecular, and 3) physical interferences. Analysing food and food raw material samples an other (new) interfering effect emerged in ICP-MS, namely the effect of various matrixes having different evaporation and nebulization effectiveness, moreover having different quantity of carbon content of food, feed and food raw material samples. In our research work the effect of different water-soluble compounds furthermore the effect of various quantity of carbon content (as sample matrix) were examined on changes of intensity of selenium. So finally we could find “opportunities” to decrease the error of selenium analysis. To analyse selenium in food, feed and food raw material samples, the most appropriate inductively coupled plasma mass spectrometer is a quadrupole instrument applying a collision cell technique (CCT). The extent of interfering effect of carbon content depends on the type of compounds. The carbon content significantly affects the measured concentration (intensities) of Se, which can be corrected using internal standard (arsenic or tellurium).

Keywords: selenium, ICP-MS, food, food raw material

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