Search results for: Gas chromatography–mass spectrometry (GC-MS)

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: M. Hakami, A. Jammaly, I. Attafi, M. Oraiby, M. Jeraiby

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We reviewed an unusual case of a 65-year-old male taking an herbal mixture containing compounds with anticholinesterase activity for a long period of time, presented with acute my myocardial infarction and multiple organ dysfunction syndrome followed by death. Clinically, there are findings correlated with anticholinesterase activity, such as bilateral miosis, diaphoresis, vomiting and fasciculation without a history of any toxic ingestion or exposure. Gas chromatography–mass spectrometry screening studies identified the presence of thymol, anethole in the herbal extract and butylated hydroxytoluene in the blood sample. Hence, with this case report, we intend to highlight the necessity of evaluating the long-term use of the herbal mixture.

Keywords: cholinesterase inhibitors, thymole, anethole, butylatedhydroxytoluene, cardiac toxicity, myocardial infarction

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Authors: Wanthani Paengsri, Thanyarat Chuesaard, Napapha Promsawan

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Spilanthes acmella Murr. was extracted with methanol, yielding methanol crude extract 5.86 %w/w. This study aimed to examine the chemical composition and antioxidant activity of methanolic crude extract. The chemical composition of methanolic crude extract was analyzed by gas chromatography-mass spectrometry (GC-MS). The predominant components were found to be palmitic acid (40.08%), 2-hexadecanoyl glycerol (6.96%) and octadecanoic acid (4.06%). Antioxidant activity was determined using 2,2-diphenyl-1-picryl hydrazyl (DPPH) free radical, for evaluating free radicle scavenging activity. The methanolic extract at 150 µg/mL showed an antioxidant activity with high of radical scavenging activity (75.23%).

Keywords: antioxidant activity, GC-MS analysis, Spilanthes, Phak-Kratt Hauwaen

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Authors: Thanida Sritangthong, Suksun Amornraksa

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By-product upgrading is crucial in hydrocarbon industries as it can increase overall profit margin of the business. Microwave-assisted pyrolysis is relatively new technique which induces heat directly to raw materials. This results in a more energy saving and more energy-efficient process. It is also a promising method to enhance and accelerate chemical reactions, thus reducing the pyrolysis reaction time and increasing the quality of value-added products from different kinds of feedstocks. In this study, upgrading opportunity of fuel oil by-product from an olefins plant is investigated by means of microwave pyrolysis. The experiment was conducted in a lab-scale quartz reactor placed inside a 1,100 watts household microwave oven. Operating temperature was varied from 500 to 900C to observe the consequence on the quality of pyrolysis products. Several microwave receptors i.e. activated carbon, silicon carbide (SiC) and copper oxide (CuO) were used as a material to enhance the heating and reaction in the reactor. The effect of residence time was determined by adjusting flow rate of N2 carrier gas. The chemical composition and product yield were analyzed by using gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS). The results showed that hydrogen, methane, ethylene, and ethane were obtained as the main gaseous products from all operating temperatures while the main liquid products were alkane, cycloalkane and polycyclic aromatic groups. The results indicated that microwave pyrolysis has a potential to upgrade low value hydrocarbons to high value products.

Keywords: fuel oil, heavy hydrocarbons, microwave pyrolysis, pyrolysis

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Authors: Louise H. Gracioso, Marcela P.G. Baltazar, Ingrid R. Avanzi, Bruno Karolski, Luciana J. Gimenes, Claudio O. Nascimento, Elen A. Perpetuo

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Introduction: Higher copper concentrations promote a selection pressure on organisms such as plants, fungi and bacteria, which allows surviving only the resistant organisms to the contaminated site. This selective pressure keeps only the organisms most resistant to a specific condition and subsequently increases their bioremediation potential. Despite the bacteria importance for biosphere maintenance, it is estimated that only a small fraction living microbial species has been described and characterized. Due to the molecular biology development, tools based on analysis 16S ribosomal RNA or another specific gene are making a new scenario for the characterization studies and identification of microorganisms in the environment. News identification of microorganisms methods have also emerged like Biotyper (MALDI / TOF), this method mass spectrometry is subject to the recognition of spectroscopic patterns of conserved and features proteins for different microbial species. In view of this, this study aimed to isolate bacteria resistant to copper present in a Copper Processing Area (Sossego Mine, Canaan, PA) and identifies them in two different methods: Recent (spectrometry mass) and conventional. This work aimed to use them for a future bioremediation of this Mining. Material and Methods: Samples were collected at fifteen different sites of five periods of times. Microorganisms were isolated from mining wastes by culture enrichment technique; this procedure was repeated 4 times. The isolates were inoculated into MJS medium containing different concentrations of chloride copper (1mM, 2.5mM, 5mM, 7.5mM and 10 mM) and incubated in plates for 72 h at 28 ºC. These isolates were subjected to mass spectrometry identification methods (Biotyper – MALDI/TOF) and 16S gene sequencing. Results: A total of 105 strains were isolated in this area, bacterial identification by mass spectrometry method (MALDI/TOF) achieved 74% agreement with the conventional identification method (16S), 31% have been unsuccessful in MALDI-TOF and 2% did not obtain identification sequence the 16S. These results show that Biotyper can be a very useful tool in the identification of bacteria isolated from environmental samples, since it has a better value for money (cheap and simple sample preparation and MALDI plates are reusable). Furthermore, this technique is more rentable because it saves time and has a high performance (the mass spectra are compared to the database and it takes less than 2 minutes per sample).

Keywords: copper mining area, bioremediation, microorganisms, identification, MALDI/TOF, RNA 16S

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Authors: M. S. M. Yusof, R. Ramli, S. K. C. Soh, N. Ismail, N. Ngah

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This study presents the synthesis of a series of methoxybenzoylthiourea amino acid derivatives. The compounds were obtained from the reactions between 2/3/4-methoxybenzoyl isothiocyanate with threonine. All of the compounds were characterized via mass spectrometry, ¹H and ¹³C NMR spectrometry, UV-Vis spectrophotometer and FT-IR spectroscopy. Mass spectra for all of the compounds showed the presence of molecular ion [M]⁺ peaks at m/z 312, which are in agreement to the calculated molecular weight. For ¹H NMR spectra, the presence of OCH₃, C=S-NH and C=O-NH protons were observed within range of δ_H 3.8-4.0 ppm, 11.1-11.5 ppm and 10.0-11.5 ppm, respectively. ¹³C NMR spectra in all compounds displayed the presence of OCH₃, C=O-NH, C=O-OH and C=S carbon resonances within range of δ_C 55.0-57.0 ppm, 165.0-168.0 ppm, 170.0-171.0 ppm and 180.0-182.0 ppm, respectively. In UV spectra, two absorption bands have been observed and both were assigned to the n-π* and π-π* transitions. Six vibrational modes of v(N-H), v(O-H), v(C=O-OH), v(C=O-NH), v(C=C) aromatic and v(C=S) appeared in the FT-IR spectra within the range of 3241-3467 cm^-1, 2976-3302 cm^-1, 1720-1768 cm^-1, 1655-1672 cm^-1, 1519-1525 cm^-1 and 754-763 cm^-1, respectively. The antibacterial activity for all of the compounds was screened against Staphylococcus aureus, Staphylococcus epidermidis, Salmonella typhimurium and Escherichia coli. However, no activity was observed.

Keywords: methoxybenzoyl isothiocyanate, amino acid, threonine, antibacterial

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Authors: Odunayo Christy Atewolara-Odule, Olapeju O. Aiyelaagbe

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The search for new natural anti-oxidants has grown tremendously over the years because reactive oxygen species (ROS) production and oxidative stress have been linked to a large number of human degenerative diseases, such as cancer, cardiovascular diseases, inflammation, and diabetes. Tapinanthus bangwensis, a parasitic plant commonly known as mistletoe belonging to the Loranthaceae family, is mostly employed traditionally to treat inflammation, cancer, diabetes, and hypertension to mention a few. In this study, air-dried pulverized leaves and stem of Tapinanthus bangwensis were successively extracted with n-hexane, ethyl acetate, and methanol to give the corresponding crude extracts. The extracts were purified by column chromatography and high-performance liquid chromatography to give the isolated compounds. Structural elucidation was done using mass spectrometry, Fourier transform infra-red, 1D and 2D NMR spectroscopy. The antioxidant activity of the compounds was evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ascorbic acid as standard. Three compounds; Friedelin, Eudesmic acid (3,4,5-trimethoxybenzoic) and Methyl-3,4,5-trimethoxybenzoate were isolated from the extracts of Tapinanthus bangwensis. Friedelin was isolated from the ethyl acetate extract of the stem while the two other compounds were isolated from the methanol extract of the leaves. The percentages of free radical scavenging activities of the compounds are as follows: Friedelin, 73.69%, methyl-3,4,5-trimethoxybenzoate, 79.33% and eudesmic, 87.68% anti-oxidant activity which were quite comparable to 93.96% given by ascorbic acid. We are reporting, to our best knowledge, for the first time the occurrence of friedelin and eudesmic acid in Tapinanthus bangwensis. The high anti-oxidant activity of these compounds supports the use of this plant in the management of diabetes and hypertension as they will be useful in combating complications arising from the disease.

Keywords: column chromatography, eudesmic acid, friedelin, Tapinanthus bangwensis

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Authors: Muhammad A. Ado, Intan S. Ismail, Hasanah M. Ghazali, Faridah Abas

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In this study, we have investigated the phytochemical constituents of soluble fractions of dichloromethane (DCM) of methanolic leaves extract of the Callicarpa maingayi. The phytochemicals investigation has resulted in the isolation of three triterpenoids (euscaphic acid (1), arjunic acid (2), and ursolic acid (3)) together with two flavones apigenin (4) and acacetin (5)), two phytosterols (stigmasterol 3-O-β-glycopyranoside (6) and sitosterol 3-O-β-glycopyranoside (7)), and one fatty acid (n-hexacosanoic acid (8)). Six (6) compounds isolated from this species were isolated for the first time (1, 2, 3, 4, 5, and 8). Their structures were elucidated and identified by spectral methods of one and two-dimensional NMR techniques, gas chromatography-mass spectrometry, and comparison with the previously reported literature. The biological activity of three compounds (1-3) was carried out on acetylcholinesterase inhibition activity. Compound (3) was found to displayed good inhibition against AChE with an IC₅₀ value of 21.5 ± 0.022 μM.

Keywords: acetylcholinesterase, Callicarpa maingayi, euscaphic acid, ursolic acid

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Authors: George Madalin Danila, Mihaiella Cretu, Cristian Puscasu

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The recycling of used antifreeze/coolant is a widely discussed and intricate issue. Complying with government regulations for the proper disposal of hazardous waste poses a significant challenge for today's automotive and industrial industries. In recent years, global focus has shifted toward Earth's fragile ecology, emphasizing the need to restore and preserve the natural environment. The business and industrial sectors have undergone substantial changes to adapt and offer products tailored to these evolving markets. The global antifreeze market size was evaluated at US 5.4 billion in 2020 to reach USD 5,9 billion by 2025 due to the increased number of vehicles worldwide, but also to the growth of HVAC systems. This study presents the evaluation of an ion exchange resin-based installation designed for the recycling of engine coolants, specifically ethylene glycol (EG) and propylene glycol (PG). The recycling process aims to restore the coolant to meet the stringent ASTM standards for both new and recycled coolants. A combination of physical-chemical methods, gas chromatography-mass spectrometry (GC-MS), and inductively coupled plasma mass spectrometry (ICP-MS) was employed to analyze and validate the purity and performance of the recycled product. The experimental setup included performance tests, namely corrosion to glassware and the tendency to foaming of coolant, to assess the efficacy of the recycled coolants in comparison to new coolant standards. The results demonstrate that the recycled EG coolants exhibit comparable quality to new coolants, with all critical parameters falling within the acceptable ASTM limits. This indicates that the ion exchange resin method is a viable and efficient solution for the recycling of engine coolants, offering an environmentally friendly alternative to the disposal of used coolants while ensuring compliance with industry standards.

Keywords: engine coolant, glycols, recycling, ion exchange resin, circular economy

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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: Shima Behkami, Nor Shahirul Umirah Idris, Sharifuddin Md. Zain, Kah Hin Low, Mehrdad Gholami, Nima A. Behkami, Ahmad Firdaus Kamaruddin

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In this work, Inductively Coupled Plasma Mass Spectrometry (ICP-MS), Isotopic Ratio Mass Spectrometry (IRMS) and Ultrasound Milko Tester were used to study milk samples obtained from various geographical locations in Malaysia. ICP-MS was used to determine the concentration of trace elements in milk, water and soil samples obtained from seven dairy farms at different geographical locations in peninsular Malaysia. IRMS was used to analyze the milk samples for isotopic ratios of δ13C, 15N and 18O. Nutritional parameters in the milk samples were determined using an ultrasound milko tester. Data obtained from these measurements were evaluated by Principal Component Analysis (PCA) and Hierarchical Analysis (HA) as a preliminary step in determining geographical origin of these milk samples. It is observed that the isotopic ratios and a number of the nutritional parameters are responsible for the discrimination of the samples. It was also observed that it is possible to determine the geographical origin of these milk samples solely by the isotopic ratios of δ13C, 15N and 18O. The accuracy of the geographical discrimination is demonstrated when several milk samples from a milk factory taken from one of the regions under study were appropriately assigned to the correct PCA cluster.

Keywords: inductively coupled plasma mass spectroscopy ICP-MS, isotope ratio mass spectroscopy IRMS, ultrasound, principal component analysis, hierarchical analysis, geographical origin, milk

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Authors: Zlatina Peteva, Stanislava Georgieva, Mona Stancheva, Lubomir Makedonsky

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Yessotoxins (YTX) and pectenotoxins (PTX) are lipophilic polyether marine biotoxins that accumulate in shellfish and are regulated in the European Union. Aim of this study is investigation of the presence of YTXs and PTXs in Black Sea mussel (Mytilus galloprovincialis) on the North Black Sea cast of Bulgaria. YTX and PTX-2 have been identified in samples of the digestive glands of wild and cultivated samples. Mussel samples are harvested from two locations in important farming area on the Bulgarian North Black Sea coast in the period September 2016 – May 2017. Samples are analyzed by liquid chromatography tandem mass spectrometry (LC–MS/MS). In about 50 % of the samples analyzed the obtained concentration of YTX ranged from 10 – 5000 pg/g hepatopancreas and PTX-2 ranged from 300-7000 pg/g hepatopancreas. This is to our knowledge the first report of occurrence of lipophilic toxins in mussels from Bulgaria.

Keywords: yessotoxins, pectenotoxins, Black sea, mussels

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Authors: Yonghwa Lee, Yoon Suk Kim, Yongsub Yi

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This study was to confirm the effects of anti-melanogenesis and anti-inflammatory effects from Opuntia humifusa fruit and stem extracts. A potent anti-oxidant activity was shown from the leaf extract at IC50 value of 38.33±1.07 μg/mL and fruit extract at IC50 value of 40.23±2.21 μg/mL by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. Also, phenolic contents were confirmed total phenolic assay by high performance liquid chromatography (HPLC). Fraction of taxifolin from leaf extract was identified using HPLC and gas chromatography/mass spectrometry. The extracts of Opuntia humifusa fruit and stem were confirmed about toxicity effect in B16 F1 by cell viability. Melanin contents were decreased. Opuntia humifusa fruit and stem extracts had a positive effect of melanin synthesis inhibition for skin whitening. In investigating the anti-inflammatory activities of Opuntia humifusa, the results of cell viability indicated that taxifolin did not show cytotoxicity on RAW264.7 cells at 500 μM of concentration. The results show that taxifolin inhibited lipopolysaccharide (LPS)-induced production of Nitrite oxide (NO). In addition, taxifolin indicated the inhibition of lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF) -α and interleukin (IL) -6 productions by cytokine assay and cyclooxygenase (COX)-2 expression by western blot analysis, meaning that taxifolin had a significant anti-inflammatory effect. Our results suggested that taxifolin from Opuntia humifusa has anti-melanogenesis and anti-inflammatory activities.

Keywords: anti-melanogenesis, anti-inflammatory, Opuntia humifusa, taxifolin

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Authors: Y. T. Boon, M. N. Naim, R. Zakaria, N. F. Abu Bakar, N. Ahmad, I. W. Lenggoro

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In this study, encapsulation of agarwood oil with non-ionic surfactant, Tween 80 was prepared at critical micelle concentration of 0.0167 % v/v to produce the most stable nano-emulsion in aqueous. The encapsulation has minimized the bioactive compounds degradation in various pH conditions thus prolong their shelf life and maintained its initial oil grade. The oil grading of the prepared samples were conducted using the gel electrophoresis instead of using common analytical industrial grading such as gas chromatography- mass spectrometry (GC- MS). The grading method was chosen due to their unique zeta potential value after the encapsulation process. This paper demonstrates the feasibility of applying the electrophoresis principles to separate the encapsulated agarwood oil or grading of the emulsified agarwood oil. The results indicated that the grading process are potential to be further investigate based on their droplet size and zeta potential value at various pH condition when the droplet were migrate through polyacrylamide gel.

Keywords: electrophoretic mobility, essential oil, nanoemulsion, polyacrylamide gel electrophoresis, tween 80, zeta potential

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Authors: M. A. Ferhat, M. N. Boukhatem, F. Chemat

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Eucalyptus essential oil is extracted from Eucalyptus globulus of the Myrtaceae family and is also known as Tasmanian blue gum or blue gum. Despite the reputation earned by aromatic and medicinal plants of Algeria. The objectives of this study were: (i) the extraction of the essential oil from the leaves of Eucalyptus globulus Labill., Myrtaceae grown in Algeria, and the quantification of the yield thereof, (ii) the identification and quantification of the compounds in the essential oil obtained, and (iii) the determination of physical and chemical properties of EGEO. The chemical constituents of Eucalyptus globulus essential oil (EGEO) of Blida origin has not previously been investigated. Thus, the present study has been conducted for the determination of chemical constituents and different physico-chemical properties of the EGEO. Chemical composition of the EGEO, grown in Algeria, was analysed by Gas Chromatography-Mass Spectrometry. The chemical components were identified on the basis of Retention Time and comparing with mass spectral database of standard compounds. Relative amounts of detected compounds were calculated on the basis of GC peak areas. Fresh leaves of E. globulus on steam distillation yielded 0.96% (v/w) of essential oil whereas the analysis resulted in the identification of a total of 11 constituents, 1.8 cineole (85.8%), α-pinene (7.2%), and β-myrcene (1.5%) being the main components. Other notable compounds identified in the oil were β-pinene, limonene, α-phellandrene, γ-terpinene, linalool, pinocarveol, terpinen-4-ol, and α-terpineol. The physical properties such as specific gravity, refractive index and optical rotation and the chemical properties such as saponification value, acid number and iodine number of the EGEO were examined. The oil extracted has been analyzed to have 1.4602-1.4623 refractive index value, 0.918-0.919 specific gravity (sp.gr.), +9 - +10 optical rotation that satisfy the standards stipulated by European Pharmacopeia. All the physical and chemical parameters were in the range indicated by the ISO standards. Our findings will help to access the quality of the Eucalyptus oil which is important in the production of high value essential oils that will help to improve the economic condition of the community as well as the nation.

Keywords: chemical composition, essential oil, eucalyptol, gas chromatography

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Authors: Saurabh B. Ganorkar, Atul A. Shirkhedkar

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The need for investigations protecting against toxic impurities though seems to be a primary requirement; the impurities which may prove non - toxic can be explored for their therapeutic potential if any to assist advanced drug discovery. The essential role of pharmaceutical analysis can thus be extended effectively to achieve it. The present study successfully achieved these objectives with characterization of major degradation products as impurities for Zileuton which has been used for to treat asthma since years. The forced degradation studies were performed to identify the potential degradation products using Ultra-fine Liquid-chromatography. Liquid-chromatography-Mass spectrometry (Time of Flight) and Proton Nuclear Magnetic Resonance Studies were utilized effectively to characterize the drug along with five major oxidative and hydrolytic degradation products (DP’s). The mass fragments were identified for Zileuton and path for the degradation was investigated. The characterized DP’s were subjected to In-Silico studies as XP Molecular Docking to compare the gain or loss in binding affinity with 5-Lipooxygenase enzyme. One of the impurity of was found to have the binding affinity more than the drug itself indicating for its potential to be more bioactive as better Antiasthmatic. The close structural resemblance has the ability to potentiate or reduce bioactivity and or toxicity. The chances of being active biologically at other sites cannot be denied and the same is achieved to some extent by predictions for probability of being active with Prediction of Activity Spectrum for Substances (PASS) The impurities found to be bio-active as Antineoplastic, Antiallergic, and inhibitors of Complement Factor D. The toxicological abilities as Ames-Mutagenicity, Carcinogenicity, Developmental Toxicity and Skin Irritancy were evaluated using Toxicity Prediction by Komputer Assisted Technology (TOPKAT). Two of the impurities were found to be non-toxic as compared to original drug Zileuton. As the drugs are purposed and repurposed effectively the impurities can also be; as they can have more binding affinity; less toxicity and better ability to be bio-active at other biological targets.

Keywords: UFLC, LC-MS-TOF, NMR, Zileuton, impurities, toxicity, bio-activity

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Authors: B. G. Muhammad, S. M. Jafar

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Neutron Activation Analysis (NAA) and Inductively Coupled Plasma Mass Spectrometry (ICP-MS) were employed to analyze the level of trace elements concentrations in sediment samples and their bioaccumulation in some aquatic species selected randomly from surface water resources in the Northern peninsula of Malaysia. The NAA results of the sediment samples indicated a wide range in concentration of different elements were observed. Fe, K, and Na were found to have major concentration values that ranges between 61,000 ± 1400 to 4,500 ± 100 ppm, 20100±1000 to 3100±600 and 3,100±600 and 200±10 ppm, respectively. Traces of heavy metals with much more contamination health concern, such as Cr and As, were also identified in many of the samples analyzed. The average specific activities of 40K, 232Th and 226Ra in soil and the corresponding radium equivalent activity and the external hazard index were all found to be lower than the maximum permissible limits (370 Bq kg-1 and 1).

Keywords: external hazard index, Neutron Activation Analysis, radium equivalent, trace elements concentrations

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Authors: Boris Nemzer, Nebiyu Abshiru, Z. B. Pietrzkowski

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Coffee cherry is one of the most ubiquitous agricultural commodities which possess nutritional and human health beneficial properties. Between the two most widely used coffee cherries Coffea arabica (Arabica) and Coffea canephora (Robusta), Coffea arabica remains superior due to its sensory properties and, therefore, remains in great demand in the global coffee market. In this study, the phytochemical contents and pharmacokinetics of Coffeeberry® Energy (CBE), a commercially available Arabica whole coffee fruit caffeine extract, are investigated. For phytochemical screening, 20 mg of CBE was dissolved in an aqueous methanol solution for analysis by mass spectrometry (MS). Quantification of caffeine and chlorogenic acids (CGAs) contents of CBE was performed using HPLC. For the bioavailability study, serum samples were collected from human subjects before and after 1, 2 and 3 h post-ingestion of 150mg CBE extract. Protein precipitation and extraction were carried out using methanol. Identification of compounds was performed using an untargeted metabolomic approach on Q-Exactive Orbitrap MS coupled to reversed-phase chromatography. Data processing was performed using Thermo Scientific Compound Discover 3.3 software. Phytochemical screening identified a total of 170 compounds, including organic acids, phenolic acids, CGAs, diterpenoids and hydroxytryptamine. Caffeine & CGAs make up more than, respectively, 70% & 9% of the total CBE composition. For serum samples, a total of 82 metabolites representing 32 caffeine- and 50 phenolic-derived metabolites were identified. Volcano plot analysis revealed 32 differential metabolites (24 caffeine- and 8 phenolic-derived) that showed an increase in serum level post-CBE dosing. Caffeine, uric acid, and trimethyluric acid isomers exhibited 4- to 10-fold increase in serum abundance post-dosing. 7-Methyluric acid, 1,7-dimethyluric acid, paraxanthine and theophylline exhibited a minimum of 1.5-fold increase in serum level. Among the phenolic-derived metabolites, iso-feruloyl quinic acid isomers (3-, 4- and 5-iFQA) showed the highest increase in serum level. These compounds were essentially absent in serum collected before dosage. More interestingly, the iFQA isomers were not originally present in the CBE extract, as our phytochemical screen did not identify these compounds. This suggests the potential formation of the isomers during the digestion and absorption processes. Pharmacokinetics parameters (Cmax, Tmax and AUC0-3h) of caffeine- and phenolic-derived metabolites were also investigated. Caffeine was rapidly absorbed, reaching a maximum concentration (Cmax) of 10.95 µg/ml in just 1 hour. Thereafter, caffeine level steadily dropped from the peak level, although it did not return to baseline within the 3-hour dosing period. The disappearance of caffeine from circulation was mirrored by the rise in the concentration of its methylxanthine metabolites. Similarly, serum concentration of iFQA isomers steadily increased, reaching maximum (Cmax: 3-iFQA, 1.54 ng/ml; 4-iFQA, 2.47 ng/ml; 5-iFQA, 2.91 ng/ml) at tmax of 1.5 hours. The isomers remained well above the baseline during the 3-hour dosing period, allowing them to remain in circulation long enough for absorption into the body. Overall, the current study provides evidence of the potential health benefits of a uniquely formulated whole coffee fruit product. Consumption of this product resulted in a distinct serum profile of bioactive compounds, as demonstrated by the more than 32 metabolites that exhibited a significant change in systemic exposure.

Keywords: phytochemicals, mass spectrometry, pharmacokinetics, differential metabolites, chlorogenic acids

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Authors: Toan Vu Duc, Son Ha Viet

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This research presents the first comprehensive survey of congener profiles (7 indicator congeners) of polybrominated diphenyl ethers (PBDEs) in sediment samples covering ten sites in CauBay River, Vietnam. Chemical analyses were carried out in gas chromatography–mass spectrometry (GC–MS) for tri- to hepta- brominated congeners. Results pointed out a non-homogenous contamination of the sediment with ∑7 PBDE values ranging from 8.93 to 25.64ng g−1, reflecting moderate to low contamination closely in conformity to other Asian aquatic environments. The general order of decreasing congener contribution to the total load was: BDE 47 > 99 > 100 > 154, similar to the distribution pattern worldwide. PBDEs had rare risks in the sediment of studied area. However, due to the propensity of PBDEs to accumulate in various compartments of wildlife and human food webs, evaluation of biological tissues should be undertaken as a high priority.

Keywords: residue, risk assessment, PBDEs, sediment

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Authors: Mikhail S. Zastrozhin, Valery V. Smirnov, Dmitry A. Sychev, Ludmila M. Savchenko, Evgeny A. Bryun, Mark O. Nechaev

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Cytochrome P-450 isoenzyme 3A4 takes part in the biotransformation of medical drugs. The activity of CYP isoenzymes depends on genetic (polymorphisms of genes which encoded it) and phenotypic factors (a kind of food, a concomitant drug therapy). The aim of the study was to evaluate a carbamazepine effect on the CYP3A4 activity in patients with alcohol addiction. The study included 25 men with alcohol dependence, who received haloperidol during the exacerbation of the addiction. CYP3A4 activity was assessed by urinary 6-beta-hydroxycortisol/cortisol ratios measured by high performance liquid chromatography with mass spectrometry. The study modeled a graph and an equation of the logarithmic regression, that reflects the dependence of CYP3A4 activity on a dose of carbamazepine: y = 5,5 * 9,1 * 10-5 * x2. The study statistically significant demonstrates the effect of carbamazepine on CYP2D6 isozyme activity in patients with alcohol addiction.

Keywords: CYP3A4, biotransformation, carbamazepine, alcohol abuse

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Authors: Bashar Al-Sabti, Jehad Harbali

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Background: Pyridine is a reactive base that might be used in preparing sitagliptin. International Agency for Research on Cancer classifies pyridine in group 2B; this classification means that pyridine is possibly carcinogenic to humans. Therefore, pyridine should be monitored at the allowed limit in sitagliptin pharmaceutical ingredients. Objective: The aim of this study was to develop a novel ultra high performance liquid chromatography mass spectrometry (UHPLC-MS) method to estimate the quantity of pyridine impurity in sitagliptin pharmaceutical ingredients. Methods: The separation was performed on C8 shim-pack (150 mm X 4.6 mm, 5 µm) in reversed phase mode using a mobile phase of water-methanol-acetonitrile containing 4 mM ammonium acetate in gradient mode. Pyridine was detected by mass spectrometer using selected ionization monitoring mode at m/z = 80. The flow rate of the method was 0.75 mL/min. Results: The method showed excellent sensitivity with a quantitation limit of 1.5 ppm of pyridine relative to sitagliptin. The linearity of the method was excellent at the range of 1.5-22.5 ppm with a correlation coefficient of 0.9996. Recoveries values were between 93.59-103.55%. Conclusions: The results showed good linearity, precision, accuracy, sensitivity, selectivity, and robustness. The studied method was applied to test three batches of sitagliptin raw materials. Highlights: This method is useful for monitoring pyridine in sitagliptin during its synthesis and testing sitagliptin raw materials before using them in the production of pharmaceutical products.

Keywords: genotoxic impurity, pyridine, sitagliptin, UHPLC -MS

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Authors: M. O. Ezegbogu, H. B. Osadolor

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Lipsticks constitute a significant source of transfer evidence, and can, therefore, provide corroborative or inclusionary evidence in criminal investigation. This study aimed to determine the uniqueness and persistence of different lipstick smears using Thin Layer Chromatography (TLC), and Gas Chromatography with a Flame Ionisation Detector (GC-FID). In this study, we analysed lipstick smears retrieved from tea cups exposed to the environment for up to four weeks. The n-alkane content of each sample was determined using GC-FID, while TLC was used to determine the number of bands, and retention factor of each band per smear. This study shows that TLC gives more consistent results over a 4-week period than GC-FID. It also proposes a maximum exposure time of two weeks for the analysis of lipsticks left in the open using GC-FID. Finally, we conclude that neither TLC nor GC-FID can distinguish lipstick evidence recovered from hypothetical crime scenes.

Keywords: forensic science, chromatography, identification, lipstick

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Authors: Bong Keun Kang, Sung Suk Oh, Jeong-Woo Sohn, Jong-Ryul Choi, Young Ho Kim

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In this paper, we describe two-step micro-pattering by using laser cutting and wax printing. Wax printing is performed only on the bridges for hydrophobic barriers. We prepared 405nm blue-violet laser module and wax pencil module. And, this two modules combine x-y plot. The hollow microstructure formed by laser patterning define the hydrophilic flowing paths. However, bridges are essential to avoid the cutting area being the island. Through the support bridges, microfluidic solution spread out to the unnecessary areas. Chromatography blotting paper was purchased from Whatman. We used 20x20 cm and 46x57 cm of chromatography blotting paper. Axis moving speed of x-y plot was the main parameter of optimization. For aligning between the two patterning, the paper sheet was taped at the bottom. After the two-step patterning, temperature curing step was done at 110-130 °C. The resolution of the fabrication and the potential of the multiplex detection were investigated.

Keywords: µPADs, microfluidic, biosensor, mass-fabrication

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Authors: H. M. El-Rafie, A. H. Abou Zeid, R. S. Mohammed, A. A. Sleem

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Acrocarpus is a genus of flowering plants in the legume family Fabaceae which considered as a large and economically important family. This study aimed to investigate the phytoconstituents of the petroleum ether extract (PEE) of Acrocarpus fraxinofolius bark by Gas chromatography coupled with mass spectrometry (GC/MS) analysis of its fractions (fatty acid and unsaponifiable matter). Concerning this, identification of 52 compounds constituting 97.03 % of the total composition of the unsaponifiable matter fraction. Cycloeucalenol was found to be the major compound representing 32.52% followed by 4a, 14a-dimethyl-A8~24(28)-ergostadien (26.50%) and ß-sitosterol(13.74%), furthermore Gas liquid chromatography (GLC) analysis of the sterol fraction revealed the identification of cholesterol (7.22 %), campesterol (13.30 %), stigmasterol (10.00 %) and β - sitosterol (69.48 %). Meanwhile, the identification of 33 fatty acids representing 90.71% of the total fatty acid constituents. Methyl-9,12-octadecadienoate (40.39%) followed by methyl hexadecanoate (23.64%) were found to be the major compounds. On the other hand, column chromatography and Thin layer chromatography (TLC) fractionation of PEE separate the triterpenoid: 21β-hydroxylup-20(29)-en-3-one and β- amyrin which were structurally identified by spectroscopic analysis (NMR, MS and IR). PEE has been biologically evaluated for 1: management of diabetes in alloxan induced diabetic rats 2: cytotoxic activity against four human tumor cell lines (Cervix carcinoma cell line[HELA], Breast carcinoma cell line [MCF7], Liver carcinoma cell line[HEPG2] and Colon carcinoma cell line[HCT-116] 3: hepatoprotective activity against CCl4-induced hepatotoxicity in rats and the activity was studied by assaying the serum marker enzymes like AST, ALT, and ALP. Concerning this, the anti-diabetic activity exhibited by 100mg of PEE extract was 74.38% relative to metformin (100% potency). It also showed a significant anti-proliferative activity against MCF-7 (IC50= 2.35µg), Hela(IC50=3.85µg) and HEPG-2 (IC50= 9.54µg) compared with Doxorubicin as reference drug. The hepatoprotective activity was evidenced by significant decrease in liver function enzymes, i.e. AST, ALT and ALP by (29.18%, 28.26%, and 34.11%, respectively using silymarin as the reference drug, compared to their concentration levels in an untreated group with liver damage induced by CCl₄. This study was performed for the first time on the bark of this species.

Keywords: Acrocarpus fraxinofolius, antidiabetic, cytotoxic, hepatoprotective

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Authors: Bakhtishod Matmuratov, Sakhiba Madraximova, Rakhmat Esanov, Alimjan Matchanov

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Studies have been performed to obtain complexes of glycyrrhizic acid and kinetins in a 2:1 ratio. The complex of glycyrrhizic acid and kinetins in a 2:1 ratio was considered evidence of the formation of a molecular complex by determining the molecular masses using chromato-mass spectroscopy and analyzing the IR spectra.

Keywords: monoammonium salt of glycyrrhizic acid, glycyrrhizic acid, supramolecular complex, isomolar series, IR spectroscopy

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Authors: Hanaa A. S. Oraby, Amal A. M. Hassan, Mahmoud M. Sakr, Atef A. A. Haiba

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Applying the profiling approach for the assessment of proteomic variations due to genetic modification of the Egyptian tomato cultivar "Edkawy", three complementary approaches were used. These methods are amino acids analysis, gel electrophoresis, and Gas chromatography coupled with mass spectrometry (GC/MS). The results of the present study Show evidence of proteomic variations between both modified tomato and its non-modified counterpart. Amino acids concentrations, and the protein patterns separation on the 1D SDS-PAGE were not similar in the case of transformed tomato compared to that of the non-transformed counterpart. These detected differences are most likely derived from the process of transformation. Results also revealed that the efficiency of GC/MS approach to identify a mixture of unknown proteins is limited. GC/MS analysis was only able to identify few number of protein molecules. Therefore, more advanced and specific technologies like MALDI-TOF-MS are recommended to be employed.

Keywords: GMOs, unintended effects, proteomic variations, 1D SDS-PAGE, GC/MS

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Authors: Pierre Moulis, Markus Herderich, Doris Rauhut, Patricia Ballestra

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Nowadays, it is starting to be more frequent to detect wines with mousy off-flavor. The reasons behind this could be the significant decrease in sulphur dioxide, the increase in pH, and the trend for spontaneous fermentation in wine. This off-flavor can be produced by Brettanomyces bruxellensis or some Lactic acid bacteria. So far there is no study working on the influence of the genetic group on the production of these microorganisms. Objectives: The objectives of this research are to increase knowledge and to have a better understanding of the microbiological phenomena related to the production of the mousy off-flavor in the wine. Methodologies: In this research, microorganisms were screened in an N-heterocycle assay medium (this medium contained all known precursors) and the production of mousy compounds was quantified by Stir Bar Sorptive Extraction-Gas Chromatography-Mass Spectrometry (SBSE-GC-MS). Main contributions: Brettanomyces bruxellensis and Oenococcus oeni could produce mousiness at a different amount depending on the strain. But there is no group effect.

Keywords: mousy off-flavor, wine, Brettanomyces bruxellensis, Oenococcus oeni

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Authors: Adriana Muñoz-Talavera, Ana Rosa Rincón-Sánchez, Abraham Escobedo-Moratilla, María Cristina Islas-Carbajal, Miguel Ángel Gómez-Lim

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The highest rates of diabetes incidence and prevalence worldwide will increase the number of diabetic patients requiring insulin or insulin analogues. Then, current production systems would not be sufficient to meet the future market demands. Therefore, developing efficient expression systems for insulin and insulin analogues are needed. In addition, insulin analogues with better pharmacokinetics and pharmacodynamics properties and without mitogenic potential will be required. SCI-57 (single chain insulin-57) is an insulin analogue having 10 times greater affinity to the insulin receptor, higher resistance to thermal degradation than insulin, native mitogenicity and biological effect. Plants as expression platforms have been used to produce recombinant proteins because of their advantages such as cost-effectiveness, posttranslational modifications, absence of human pathogens and high quality. Immunoglobulin production with a yield of 50% has been achieved by transient expression in Nicotiana benthamiana (Nb). The aim of this study is to produce SCI-57 by transient expression in Nb. Methodology: DNA sequence encoding SCI-57 was cloned in pICH31070. This construction was introduced into Agrobacterium tumefaciens by electroporation. The resulting strain was used to infiltrate leaves of Nb. In order to isolate SCI-57, leaves from transformed plants were incubated 3 hours with the extraction buffer therefore filtrated to remove solid material. The resultant protein solution was subjected to anion exchange chromatography on an FPLC system and ultrafiltration to purify SCI-57. Detection of SCI-57 was made by electrophoresis pattern (SDS-PAGE). Protein band was digested with trypsin and the peptides were analyzed by Liquid chromatography tandem-mass spectrometry (LC-MS/MS). A purified protein sample (20µM) was analyzed by ESI-Q-TOF-MS to obtain the ionization pattern and the exact molecular weight determination. Chromatography pattern and impurities detection were performed using RP-HPLC using recombinant insulin as standard. The identity of the SCI-57 was confirmed by anti-insulin ELISA. The total soluble protein concentration was quantified by Bradford assay. Results: The expression cassette was verified by restriction mapping (5393 bp fragment). The SDS-PAGE of crude leaf extract (CLE) of transformed plants, revealed a protein of about 6.4 kDa, non-present in CLE of untransformed plants. The LC-MS/MS results displayed one peptide with a high score that matches SCI-57 amino acid sequence in the sample, confirming the identity of SCI-57. From the purified SCI-57 sample (PSCI-57) the most intense charge state was 1069 m/z (+6) on the displayed ionization pattern corresponding to the molecular weight of SCI-57 (6412.6554 Da). The RP-HPLC of the PSCI-57 shows the presence of a peak with similar retention time (rt) and UV spectroscopic profile to the insulin standard (SCI-57 rt=12.96 and insulin rt=12.70 min). The collected SCI-57 peak had ELISA signal. The total protein amount in CLE from transformed plants was higher compared to untransformed plants. Conclusions: Our results suggest the feasibility to produce insulin analogue SCI-57 by transient expression in Nicotiana benthamiana. Further work is being undertaken to evaluate the biological activity by glucose uptake by insulin-sensitive and insulin-resistant murine and human cultured adipocytes.

Keywords: insulin analogue, mass spectrometry, Nicotiana benthamiana, transient expression

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Authors: Madhusmita Dehingia, Supriyo Sen, Bhuwan Bhaskar, Tulsi Joishy, Mojibur R. Khan

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Human gut microbes and their metabolites are important for maintaining homeostasis in the gut and are responsible for many metabolic and immune mediated diseases. In the present study, we determined the profiles of the gut metabolites of five different ethnic groups (Bodo, Tai-Phake, Karbi, Tea tribe and Tai-Aiton) of Assam. Fecal metabolite profiling of the 39 individuals belonging to the ethnic groups was carried out using Gas chromatography – Mass spectrometry (GC-MS), and comparison was performed among the tribes for common and unique metabolites produced within their gut. Partial Least Squares Discriminant Analysis (PLS-DA) of the metabolites suggested that the individuals grouped according to their ethnicity. Among the 66 abundant metabolites, 12 metabolites were found to be common among the five ethnic groups. Additionally, ethnicity wise some unique metabolites were also detected. For example, the tea tribe of Assam contained the tea components, Aniline and Benzoate more in their gut in comparison to others. Metabolites of microbial origin were also correlated with the already published metagenomic data of the same ethnic group and functional analysis were carried out based on human metabolome database.

Keywords: ethnicity, gut microbiota, GC-MS, metabolites

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Authors: Tara Miller, Tariq Ganief, Jonathan Blackburn

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Millions of babies are still born to HIV-infected mothers each year despite the ramped-up HAART use. However, these infants are HIV uninfected but exposed, which is now a growing population that has weakened immune systems and poorer outcomes. Due to HIV exposure and possible ARV exposure during pregnancy and breastfeeding, these infants are believed to have altered immune responses and microbiomes when compared to their healthy counterparts. The gut microbiome roles an important role in infant development, specifically in the immune system. Research has shown these HIV-exposed, uninfected infants have weaker immune responses to their neonate vaccines, and in developing countries, this leaves them vulnerable to opportunistic disease. By gaining a deeper understanding of the gut microbiome and the products of the microbes via metaproteomic analysis, we can hopefully understand and improve the immune system and health of these infants. To investigate the metaproteome of the infants’ guts, mass spectrometry will be used, followed by data analysis using DIA-NN. The hypothesized results are that the HIV-exposed, uninfected infants have an altered microbiome compared to their healthy counterparts. Additionally, the differences found are hypothesized to be involved with inflammation which would contribute to the poor health of the infants.

Keywords: HIV, mass spectrometry, metaproteomics, microbiome

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