Quantitative Gas Chromatography Research Articles

A-316 A quantitative gas chromatography tandem mass spectrometry method for metabolic profiling of urine organic acids and acylglycines

Abstract Background The analysis of urine organic acids (UOA) and acylglycines (UAG) are an essential investigation for the diagnosis inborn errors of metabolism (IEMs). The vast majority of methods for these analytes use gas chromatography mass spectrometry (GC-MS) and are only qualitative. Gas chromatography tandem mass spectrometry (GC-MS/MS) offers greater sensitivity and selectivity and enables the combination of qualitative screening with quantitative analysis. We describe the development and validation results of a quantitative GC-MS/MS method. Methods Urine creatinine concentrations were measured (CobasPro, Roche Diagnostics) and normalized to 1.25 mmol/L. After internal standard addition, each urine specimen, calibration standard and QC were incubated with a hydroxylamine solution followed by a liquid-liquid extraction. Further sample concentration permitted the isolated UOA and UAG to be derivatized using N,O-bis(trimethylsilyl)trifluoroacetamine (BSTFA) with 1% chlorotrimethylsilane (TMCS) prior to injection. GC-MS/MS analysis was performed using a GC-MS-TQ8050 triple quadrupole system (Shimadzu) with a 30 m DB-5MS (Agilent J&W) capillary column (0.25 mm, ID of 0.25 µm). Split injection mode was deployed with an initial column temperature of 70°C. The mass spectrometer was operated in electron ionization (EI) and multiple reaction monitoring (MRM) modes. Results The optimal temperature gradient for high-resolution UOA and UAG separations was derived and their respective retention times determined. MS/MS detection parameters were optimized to permit the identification of precursor and production ions for all (N=36) derivatized analytes. Total analysis time was 33.8 min per injection. This method permits the measurement of 11 samples in one batch and is highly linear for all analytes (R^2 >0.99 and average slope 1.04). The limit of quantification (LOQ) was established and ranged from <0.05 to 2.1 µmol/mmol creatinine. Intra- and inter-day imprecision (CV%) was determined using matrix-matched abnormal and normal QC. Intra-day imprecision (N=10) ranged from 1.3% (2-ketoglutaric acid) to 9.7% (3-hydroxybutyric acid). Inter-day imprecision (N=30 over 15 days) ranged from 2.2% (ethylmalonic acid and methylmalonic acid) to 20.5% (3-hydroxy-3-methylglutaric acid). The method had an average accuracy of 113±14% for analytes included in the ERNDiM quantitative organic acids (urine) external quality assurance program. For analytes not included in this proficiency testing, the method showed good agreement when compared to a national reference laboratory, with average Deming correlation coefficients and regression slopes being 0.9944 and 1.1, respectively. No interferences were detected when monitoring the analyte quantifier and qualifier ion ratios in patient urine. Conclusions The devised GC-MS/MS protocol permits the unbiased identification and quantification of UOA and UAG used to investigate IEMs. The method has acceptable sensitivity, specificity, precision and accuracy for clinical routine clinical testing.

Water-Insoluble Copper Complexes As Electrocatalysts for CO2 Reduction

The electrochemical reduction of carbon dioxide (CO2R) constitutes a promising CO2 utilization technology, allowing the production of valuable fuels and chemicals by using electrical energy from renewable sources. Nevertheless, poor selectivity and high required overpotentials hamper practical applications of the CO2R, which highlights the need for the development of more efficient electrocatalysts. Different copper-based materials have been extensively studied as electrocatalysts for CO2R. Particularly, copper complexes exhibited interesting properties under operational conditions: many of them undergo structural modifications (e.g. changes in oxidation state and/or coordination number), which potentially affect activity, selectivity and stability. Interestingly, some complexes exhibit reversible restructuring under specific conditions, which can be an interesting property to reach higher stability. In this presentation, we will show the results obtained for the electrocatalysis of water-insoluble copper complexes for the CO2R. The activity, selectivity and stability of both [Cu(bzimpy)Cl2] and [Cu(pyrben)2(NO3)]NO3 were investigated in K2SO4 0.1 mol L-1 (CO2 sat.) under different electrochemical conditions. Here, bzimpy and pyrben stand for 2,6-bis(2-benzimidazolyl)pyridine and 2-(2-pyridyl)benzimidazole, respectively. Qualitative EC-MS (Electrochemistry Coupled to Mass Spectrometry) analysis evidenced that both complexes are active for CO2 reduction and that product distribution changes according to the applied potential. Quantitative gas chromatography measurements revealed that, at -1.24 V vs. RHE, [Cu(bzimpy)Cl2] promotes the formation of H2, CO, CH4 and C2H4 with faradaic efficiencies of 26, 3.4, 9.2 and 31%, respectively. On the other hand, [Cu(pyrben)2(NO3)]NO3 shows FE values of 24, 8.8, 15 and 24% for H2, CO, CH4 and C2H4, respectively. After 1.6 h of electrolysis, the former exhibited stable current and FE of ~42% towards ethylene formation.

Development and validation of a quantitative method for the analysis of delta-9-tetrahydrocannabinol (delta-9-THC), delta-8-tetrahydrocannabinol (delta-8-THC), delta-9-tetrahydrocannabinolic acid (THCA), and cannabidiol (CBD) in botanicals, edibles, liquids, oils, waxes, and bath products by gas chromatography mass spectrometry (GC/MS).

A quantitative gas chromatography mass spectrometry (GC/MS) method was developed for delta-9-tetrahydrocannabinol (delta-9-THC), delta-8-tetrahydrocannabinol (delta-8-THC), tetrahydrocannabinolic acid (THCA), and cannabidiol (CBD) in matrices including plant material, liquids and oils, waxes, edibles, and bath and body products. Samples were prepared by homogenization, extraction of the cannabinoids into solvent, liquid/liquid extraction, and derivatization. The GC/MS method was validated from 0.15% to 5.00% (weight basis) to encompass the 0.3% legal distinction between hemp and marijuana. Validation was performed assessing imprecision/bias, calibration model, recovery, interferences, limit of detection, matrix matching, carryover, accuracy, and an assessment of CBD conversion to delta-9-THC. The calibration curves were quadratic weighted 1/x with r2 > 0.990. The method had a detection limit of 0.075% in plant material for each analyte. Analyte recovery was greater than 70% in plant material. Carryover was not observed up to concentrations equivalent to 100% analyte, and no forensically significant conversion of CBD to delta-9-THC was observed. One cannabinoid isomer, 9(R)-delta-7-tetrahydrocannabinol (9(R)-delta-7-THC), was determined to interfere with the quantitation of delta-9-THC, but could be differentiated based on mass spectrum. The method was determined to be suitable for quantitation of delta-9-THC, delta-8-THC, delta-9-THCA, and CBD and was able to differentiate hemp samples from marijuana samples.

A systematic derivatization technique for characterization of ethoxylates by GC and GCMS

AbstractEthoxylation is one of the common industrial process in which ethylene oxide (EO) is added to compounds containing active hydrogen atom (OH, COOH, SH, NH, etc.) to form surfactants. Alcohol ethoxylates are a major class of non‐ionic surfactants composed of an alkyl chain combined with few EO units. They are widely used in homecare products, personal care products, textiles, oilfield, construction, lubricants, food, paper, leather. The analysis of these ethoxylates possess several challenges since they contain ‘n’ number of EO units (n > 1). With increase in “n,” the product becomes more and more viscous and the boiling point increases making it difficult to analyze by normal gas chromatography (GC) technique. This paper describes a method of derivatizing these ethoxylates so that they can be eluted on a high temperature GC. BSTFA (N, O‐bis (trimethylsilyl) trifluoroacetamide) is used as the derivatizing agent (silylating agent). The ethoxylate combines with BSTFA to form the corresponding trimethylsilylated (TMS) product, which have comparatively lower boiling point. Thus, we detect trimethyl silyl product, which is the resemblance for the ethoxylated product. In this paper, we present a quantitative GC method for the ethoxylates of a few simple alcohols (2‐ethyl hexanol, glycerin), and complex vegetable oils (castor oil). The products formed during the reaction have also been identified on a gas chromatograph coupled with mass spectrometer (GCMS). GCMS shows the mass corresponding to the TMS derivatized product. The derivatization technique not only helps in increasing the volatility of these compounds but also improves the chromatographic efficiency, selectivity, and enhances the detectability.

B-201 Development of a Quantitative Gas Chromatography Tandem Mass Spectrometry Method for Metabolic Profiling of Urine Organic Acids

Abstract Background Urine organic acids (UOA) analysis is an essential investigation for inborn errors of metabolism (IEMs). The vast majority of methods use gas chromatography mass spectrometry (GC-MS) and are only qualitative. Gas chromatography tandem mass spectrometry (GC-MS/MS) offers greater sensitivity and selectivity, and enables the combination of qualitative screening with quantitative analysis. We describe the development and preliminary validation results of a quantitative GC-MS/MS method. Methods Urine creatinine concentrations were measured (CobasPro, Roche Diagnostics) and normalized to 1.25 mmol/L. After internal standard addition, each urine specimen, calibration standard and QC were incubated with a hydroxylamine solution followed by a liquid-liquid extraction. Further sample concentration permitted the isolated UOA to be derivatized using N,O-bis(trimethylsilyl)trifluoroacetamine (BSTFA) with 1% chlorotrimethylsilane (TMCS) prior to injection. GC-MS/MS analysis was performed using a GC-MS-TQ8050 triple quadrupole system (Shimadzu) with a 30 m DB-5MS (Agilent J&W) capillary column (0.25 mm, ID of 0.25 µm). Split injection mode was deployed with an initial column temperature of 70°C. The mass spectrometer was operated in electron ionization (EI) and multiple reaction monitoring (MRM) modes. The linear concentration ranges of the targeted metabolites within the devised GC-MS/MS protocol were determined. Results The optimal temperature gradient for high-resolution UOA separations was derived and their respective retention times determined. MS/MS detection parameters were optimized to permit the identification of precursor and production ions for all (N = 38) derivatized UOA. Total analysis time was 33.8 min per injection. The achieved linear concentration ranges for each metabolite was respectively determined. Linear concentration ranges and correlation coefficients of representative UOA: 3-hydroxyisovaleric acid (range = 9 to 708 µmol/L, R2 = 0.9874); lactic acid (range = 15 to 751 µmol/L, R2 = 0.9936); 3-hydroxybutyric acid (range = 15 to 751 µmol/L, R2 = 0.9991); and 2-methyl-3-hydroxybutyric acid (range = 10 to 501 µmol/L, R2 = 0.9996). The correlation coefficients for all remaining UOA ranged from R2 = 0.9458 (4-hydroxyphenylpyruvic acid) to R2 = 0.9998 (3-methylglutanoic acid and ethylmalonic acid). Conclusion The devised GC-MS/MS protocol permits the unbiased identification and quantification of UOA used to investigate IEMs. The linear concentration ranges for all analytes were determined and are appropriate for monitoring these markers of inherited enzyme deficiencies. The analytical performance of this UOA GC-MS/MS method must be further validated and its diagnostic performance for selected IEMs characterized.

RNAi-mediated silencing of SlitPer disrupts sex pheromone communication behavior in Spodoptera litura.

The 24-hour circadian rhythm is considered crucial for insect sexual communication. However, its molecular mechanisms and signalling pathways, especially the roles of the clock gene period (Per), remain largely unclear. The sex pheromone communication behavior of Spodoptera litura displays typical circadian rhythm characteristics. Thus, it represents an excellent model for functional analyses of the clock gene Per. In this study, we investigated the potential roles of SlitPer in regulating sex pheromone communication in S. litura using RNAi, qRT-PCR, GC, and behavioral assays. The qRT-PCR results showed that the expression levels of SlitPer and two desaturase genes (SlitDes5 and SlitDes11) in the siPer group significantly differed in most time points from those in the siNC group. The dynamic variation in the three major sex pheromone titers and calling behavior of S. litura females in the siPer group were disordered. In addition, the mating rates of siPer S. litura females significantly decreased by 33.33%. The oviposition of mated siPer females was substantially reduced by 84.84%. These findings provide a fundamental basis for elucidating the molecular mechanism by which Per regulates sex pheromone communication behavior in lepidopteran species. This article is protected by copyright. All rights reserved.

Antifungal Activities of Sida acuta Burm. f. Leaf Extracts on the Fruit Rot Pathogen of Annona muricata L.

This study investigated the antifungal activities of Sida acuta Burm. f. leaf extracts against fruit rot fungi of Annona muricata (sour sop) and the phytochemical constituent of the plant. Fruits of sour sop (A. muricata) and leaves of Sida acuta used for the study were collected from different locations within Abraka community, Delta State. Qualitative and quantitative phytochemical screening and Gas Chromatography – Mass Spectrometry analysis were carried out on the extract. Fungi causing rot of A. muricata was isolated and antifungal activities of S. acuta on isolated fungi was carried out using the poisoned food method. The result showed that both methanol and chloroform extracts of the plant contained flavonoid, tannin, saponin, steroid, glycoside, alkaloid, anthraquinone, terpenoid, and reducing sugar. The GC-MS result revealed the presence of 20 compounds with 3-Trifluoromethylbenzylamine, N,N-diundecyl and L-Proline, N-(2-trif¬luoromethylbenzoyl)-, isohexyl ester as the most abundant in both methanolic and chloroform extracts. The study identified Rhizopus stolonifer as the fungi associated with the rot of A. muricata and showed that as the concentration of the extract increased, its antimicrobial activity was significantly higher in the organ¬isms. The phytochemical composition and antifungal effects make S. acuta leaf extract a potential agent for use against fungal infections in the future.

A thermophilic L-lactic acid producer of high optical purity: isolation and identification

Biodegradable polymers, specifically polylactide, are an important part of food packaging and medical devices. Microbiological synthesis uses cheap renewable raw materials and industrial waste to produce a high yield of lactic acid, the monomer of polylactide. This method needs new effective lactic acid producing strains, e.g., thermophilic bacteria.
 The research involved thermophilic bacterial strains isolated from soil and compost samples. Their ability to produce organic acids and extracellular enzymes was tested using the method of high-performance liquid chromatography (HPLC) and microbiological tests respectively. The real-time polymerase chain reaction method (PCR) detected L-lactate dehydrogenase structural genes of L-lactate dehydrogenase of Bacillaceae. Strain T7.1 was fermented using glucose and yeast extract as carbon and nitrogen sources, respectively. The optical purity of lactic acid was evaluated using quantitative gas chromatography on a chiral column to separate lactate isomers. The molecular genetic analysis of the 16S rRNA gene sequence was applied to identify strain T7.1.
 The chromatographic analysis proved that 10 out of 13 isolated thermophilic strains were effective lactic acid producers. They demonstrated proteolytic, amylolytic, or cellulase activities. During the fermentation, strain T7.1 produced 81 g/L of lactic acid with a peak productivity at 1.58 g/(L·h). The optical purity of the product exceeded 99.9% L-lactate. The genetic analysis identified strain T7.1 as Weizmannia coagulans (Bacillus coagulans).
 The research revealed a promising thermophilic producer of optically pure L-lactic acid. Further research is needed to optimize the cultivation conditions, design an effective and cheap nutrient medium, and develop engineering and technological solutions to increase the yield.

Study of Essential Oil Isolated from Achiote (Bixa orellana) Leaves: Chemical Composition, Enantiomeric Distribution and Antimicrobial, Antioxidant and Anticholinesterase Activities.

Bixa orellana is a native and cultivated species of Ecuador commonly known as achiote (annatto), this species is very versatile with a wide variety of uses and applications of its leaves, fruits and seeds. In this study, the chemical composition, enantiomeric distribution and biological activity of essential oil isolated from the leaves of Bixa orellana were determined. Hydrodistillation was used to isolate the essential oil. Gas chromatography coupled with mass spectrometry was used to determine the qualitative composition, a gas chromatograph equipped with a flame ionization detector was used to determine quantitative composition and gas chromatography on an enantioselective column was used to determine enantiomeric distribution. Antibacterial activity was determined using the broth microdilution method, for which we used three Gram-positive cocci bacteria, a Gram-positive bacilli bacterium and three Gram-negative bacilli bacteria. 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic (ABTS) acid radical cation and 2,2-diphenyl-1-picrylhydryl (DPPH) free radical were used as reagents for determining the antioxidant activity of the essential oil. The spectrophotometric method was used to analyze acetylcholinesterase inhibitory effect of the essential oil. The yield of leaves in essential oil was 0.13 ± 0.01% (v/w). A total of 56 chemical compounds, which represent 99.25% of the total composition, were identified in the essential oil. Sesquiterpene hydrocarbons were the most representative group in number of compounds and relative abundance with 31 compounds and 69.06%, respectively. The principal constituents were found to germacrene D (17.87 ± 1.20%), bicyclogermacrene (14.27 ± 0.97%), caryophyllene < (E)- > (8.56 ± 1.24%) and pinene <α-> (6.34 ± 0.13%). Six pairs of enantiomers were identified in the essential oil of Bixa orellana. The essential oil presented strong activity against the Enterococcus faecium (ATCC 27270) with an MIC of 250 μg/mL and weak activity against Enterococcus faecalis (ATCC 19433) and Staphylococcus aureus (ATCC 25923) with an MIC of 1000 μg/mL. The antioxidant activity of the essential oil was strong according to ABTS methods with a SC50 of 61.49 ± 0.04 µg/mL and was moderate in DPPH with a SC50 of 224.24 ± 6,4 µg/mL. Additionally, the essential oil reported moderate anticholinesterase activity with an IC50 of 39.45 ± 1.06 µg/mL.

Switchgrass Metabolomics Reveals Striking Genotypic and Developmental Differences in Specialized Metabolic Phenotypes.

Switchgrass (Panicum virgatum L.) is a bioenergy crop that grows productively on lands not suitable for food production and is an excellent target for low-pesticide input biomass production. We hypothesize that resistance to insect pests and microbial pathogens is influenced by low-molecular-weight compounds known as specialized metabolites. We employed untargeted liquid chromatography–mass spectrometry, quantitative gas chromatography–mass spectrometry (GC–MS), and nuclear magnetic resonance spectroscopy to identify differences in switchgrass ecotype metabolomes. This analysis revealed striking differences between upland and lowland switchgrass metabolomes as well as distinct developmental profiles. Terpenoid- and polyphenol-derived specialized metabolites were identified, including steroidal saponins, di- and sesqui-terpenoids, and flavonoids. The saponins are particularly abundant in switchgrass extracts and have diverse aglycone cores and sugar moieties. We report seven structurally distinct steroidal saponin classes with unique steroidal cores and glycosylated at one or two positions. Quantitative GC–MS revealed differences in total saponin concentrations in the leaf blade, leaf sheath, stem, rhizome, and root (2.3 ± 0.10, 0.5 ± 0.01, 2.5 ± 0.5, 3.0 ± 0.7, and 0.3 ± 0.01 μg/mg of dw, respectively). The quantitative data also demonstrated that saponin concentrations are higher in roots of lowland (ranging from 3.0 to 6.6 μg/mg of dw) than in upland (from 0.9 to 1.9 μg/mg of dw) ecotype plants, suggesting ecotypic-specific biosynthesis and/or biological functions. These results enable future testing of these specialized metabolites on biotic and abiotic stress tolerance and can provide information on the development of low-input bioenergy crops.

Characterization of key odorants in fried red and green huajiao (Zanthoxylum bungeanum maxim. and Zanthoxylum schinifolium sieb. et Zucc.) oils

Fried huajiao oil (FHO) samples prepared with red or green huajiao are widely applied in different Chinese cuisines due to their own aroma characteristics. To investigate their different aroma profiles, 2 red and 3 green FHOs were analyzed by quantitative descriptive sensory analysis (QDA) and gas chromatography–olfactometry/aroma intensity (GC–O/AI). QDA results showed a distinct difference among FHOs in terms of all sensory attributes. Thirty odorants with high OAVs and AIs were screened from 5 FHOs, among which β-myrcene, (E)-2-heptenal, limonene, α-terpineol and p-cymene were the major characteristic compounds of FHOs. In addition, through orthogonal partial least square discriminate analysis (OPLS-DA), linalool, linalyl acetate, and 1,8-cineole were considered as the volatile markers for classification of FHOs with red and green huajiao. Thereafter, aroma recombination and omission tests were performed to characterize the key aroma compounds of red and green FHOs.

Chemical recycling of end-of-life tires by intermediate pyrolysis using a twin-auger reactor: Validation in a laboratory environment

This work deals with the chemical recycling of end-of-life tires (ELT) by means of intermediate pyrolysis to produce tire pyrolysis oil (TPO), raw recovered carbon black (rCB), and tire pyrolysis gas (TPG). Here, the operational features of a twin-auger pyrolyzer, as well as the characteristics of the resulting products were satisfactorily validated in a laboratory environment. Hence, the twin-auger pyrolyzer used in this work aligns with the characteristics associated with the fourth technology readiness level (TRL-4). In order to assure this statement, both the statistical repeatability of yields and, the consistency of the product’s properties were assessed. As such, 27 experiments divided into 4 replicates were conducted at the conditions which maximized the TPO yield, while minimizing that of raw rCB: ELT mass flow rate of 1.16 kg/h, reactor temperature of 475 °C, solid residence time of 3.5 min, and N2 flow rate of 300 mL/min. Thereafter, we explored the main physicochemical properties of different pyrolysis product samples, randomly selected throughout the experimental campaign. The TPO was characterized in terms of density, viscosity, total acid number (TAN), flash point, elemental composition, heating value, distillation characteristics, and contents of both water and carbon Conradson. The contents of saturates, aromatics, resins, and asphaltenes (SARA) were also determined. The raw rCB was characterized by proximate, elemental, and heating value analyses. Textural properties such as surface area (SBET), total pore volume (VT), micropore volume (VMin), and mesopores volume (VMeso), were identified as well, including SEM images analysis. Finally, the chemical composition of the TPG was measured by quantitative gas chromatography (H2, CH4, CO, CO2, C2H6, C2H4, C3H8, H2S). The analysis of variance (ANOVA) suggested that the twin-auger pyrolyzer is repeatable in terms of TPO, raw rCB, and TPG yields, with a reliability of 95 %. Likewise, the characterization of the products revealed that their properties remained quite stable across the experimental runs. These results demonstrate the stability and suitability of the experimental facility, which leads to the conclusion that the twin-auger reactor is a reliable and promising technology for the chemical recycling of ELT by means of intermediate pyrolysis.

Effect of Lactobacillus plantarum-fermented mulberry pomace on antioxidant properties and fecal microbial community

Rich in phenolic compounds and dietary fibers, mulberry pomace is a potential material for gut health. This study investigated the effect of the probiotic Lactobacillus plantarum on mulberry pomace. The antioxidant activity as well as the changes of fecal microbiota and short-chain fatty acids (SCFAs) were determined using chemical models, quantitative polymerase chain reaction (qPCR) and gas chromatography, respectively. Furthermore, the phenolic compounds were characterized by UPLC-MS. The results showed that the antioxidant activity was significantly improved in fermented mulberry pomace (FMP) samples. The fecal microbiota community was modulated by FMP through improving beneficial bacteria (Lactobacillus, Bifidobacterium, Ruminococcus, butyrate-producing bacteria and Akkermansia) and inhibiting pathogenic bacteria (Escherichia coli and Enterococcus). Moreover, FMP showed an increase in the production of SCFAs and lactic acid. The study identified that the corresponding glycosides of cyanidin and quercetin were released during Lactobacillus plantarum fermentation. A total of eighteen phenolic compounds were detected in samples and three compounds (p-hydroxybenzoic acid, catechin and gallocatechin) were only detected in FMP samples. The changes in phenolic compounds might explain the gut-improving functions. This study provided a suggestion that Lactobacillus plantarum fermentation was a potential way to utilize mulberry pomaces as a functional food.

Quantitative GC-MS Analysis of Artificially Aged Paints with Variable Pigment and Linseed Oil Ratios.

In this study, quantitative gas chromatography–mass spectrometry (GC–MS) analysis was used to evaluate the influence of pigment concentration on the drying of oil paints. Seven sets of artificially aged self-made paints with different pigments (yellow ochre, red ochre, natural cinnabar, zinc white, Prussian blue, chrome oxide green, hematite + kaolinite) and linseed oil mixtures were analysed. In the pigment + linseed oil mixtures, linseed oil concentration varied in the range of 10 to 95 g/100 g. The results demonstrate that the commonly used palmitic acid to stearic acid ratio (P/S) to distinguish between drying oils varied in a vast range (from especially low 0.6 to a common 1.6) even though the paints contained the same linseed oil. Therefore, the P/S ratio is an unreliable parameter, and other criteria should be included for confirmation. The pigment concentration had a substantial effect on the values used to characterise the degree of drying (azelaic acid to palmitic acid ratio (A/P) and the relative content of dicarboxylic acids (∑D)). The absolute quantification showed that almost all oil paint mock-ups were influenced by pigment concentration. Therefore, pigment concentration needs to be considered as another factor when characterising oil-based paint samples based on the lipid profile.

Isopentenyl Diphosphate Isomerase (IPI) Gene Silencing Negatively Affects Patchouli Alcohol Biosynthesis in Pogostemon cablin

Pogostemon cablin is a medicinally and economically important plant because it contains a high content of patchouli oil. Patchouli oil is composed of more than 24 unique sesquiterpene compounds, with the most abundant being patchouli alcohol (PA). PA is a sesquiterpene and is mainly synthesized through the mevalonate (MVA) pathway in plants in which isopentenyl diphosphate isomerase (IPI) is a key enzyme. Here, a full-length cDNA encoding IPI (designated as PcIPI) was cloned from P. cablin through the rapid amplification of cDNA ends (RACE). Bioinformatics analysis revealed that the full-length PcIPI includes an 879 bp open reading frame (ORF) that encodes a 292 amino acid protein with a theoretical isoelectric point of 5.55 and a molecular mass of 32.8 kDa. Phylogenetic analysis demonstrated that PcIPI shared the most recent genetic relationship with the IPI of Isodon rubescens (86.43%), Salvia miltiorrhiza (85.38%), Plectranthus barbatus (86.54%), and Prunella vulgaris (86.31%). To verify the function of the PcIPI gene in PA biosynthesis, the P. cablin plants were infected with Agrobacterium tumefaciens bearing a pTRV2-PcIPI construct to induce virus-mediated PcIPI silencing. Quantitative real-time polymerase chain reaction (qRT-PCR) and gas chromatography mass spectrometry (GC-MS) analysis were applied to detect changes in the PcIPI catalytic products and transcription levels. The trend of decreasing transcript level was found to be similar to that of decreasing sesquiterpene content. These results revealed that PcIPI silencing had a negative effect on PA biosynthesis.

Composition and energy value research of pyrolise gases

Energy values of gaseous pyrolysis products of typical forest and aquatic plant biomass of Europe are investigated. Theoretical research of main characteristics of samples of typical plant biomass of Europe such as pine, thorn, microcystis, elodea is produced. Methodology and planning of experimental studies of process of pyrolysis of plant biomass are formulated. Experimental researches of composition of gas pyrolysis products of different types of plant biomass for different temperatures were carried according to laboratory conditions. Pyrolysis of plant biomass was carried out using a specially designed and manufactured pyrolysis plant the main part of which is a pyrolysis reactor. Installation made by our department is intended for slow pyrolysis. In research for determine presence and percentage of product yield qualitative and quantitative gas chromatography assays were used. The lower calorific value of gaseous pyrolysis products of samples of typical forest and aquatic plant biomass of Ukraine are calculated. As a result of calculations it was determined that during pyrolysis of aquatic plants and algae mixture of the obtained gases had the highest values of lower calorific value: 17,10-17,15 MJ/kg – for microcystis and 16,45-16,50 MJ/kg – for elodea. Gas obtained from pyrolysis of pine wood had the highest values of lower calorific value in the range of 13,8 to 13,85 MJ/kg. The lower calorific value of pyrogas obtained from thorn samples ranged from 12,6 MJ/kg to12,65 MJ/kg.

Direct Electrochemical Hydrogenation of Naphthalene to Decalin at Pt and Pt-Ru Alloy Electrodes in Liquid Phase

Decalin has been recognized as one of organic chemical hydrides, which can be obtained by reversible hydrogenation reaction of naphthalene in the presence of a catalyst. However, because naphthalene is solid under the ambient conditions, the naphthalene/decalin system has been hardly turned to practical use for the hydrogen storage so far. Recently, we have, for the first time, demonstrated a direct electrochemical hydrogenation of toluene to methylcylcohexane at Pt electrodes in a microemulsion solution at room temperature [1]. In a microemulsion system, a hydrophobic organic compound and water (or hydrated ion, e.g. proton) can be supplied simultaneously to an electrode surface, and can react together with electron transfers. Thus, any solid organic compounds that form a microemulsion solution with water can react electrochemically with proton at lower temperatures than their melting or boiling points. In the present study, direct electrochemical hydrogenation of naphthalene was investigated by employing the microemulsion technique with Pt and Pt-Ru electrocatalysts sputtered on a Ti sheet. Author has, for the first time, achieved a high Faraday efficiency and selectivity for the naphthalene/decalin conversion under a galvanostatic condition at 60°C.Thin Pt-Ru alloy or pure Pt were prepared on a Ti sheet by RF-sputtering a Pt target with or without Ru target. The composition of the alloys was controlling by varying exposed target area ratio, and analyzed by X-ray photoelectron spectroscopy. Microemulsion electrolytes were prepared from naphthalene, a sulfuric acid solution and two surfactants by mixing and sonicating. All the electrochemical measurements were performed with an H type cell at 60°C. A platinum black and a reversible hydrogen electrode were employed as the counter electrode and reference electrode, respectively. The working electrode was immersed into a microemulsion solution while the counter electrode into a sulfuric acid solution. After the electrolysis, products were analyzed and quantified by gas chromatography (GC) with a flame ionization detector for calculating the Faraday efficiency and selectivity. Toluene was employed as internal standard for quantitative GC analysis.Fig. 1 shows cyclic voltammograms (CVs) at a Pt/Ti electrode taken in 1 M sulfuric acid (black) and a naphthalene-containing microemulsion electrolyte (red) at sweep rate of 1 mV/s. In the sulfuric acid solution, the Pt/Ti electrode exhibited a characteristic reversible hydrogen adsorption wave below 0.3 V. In contrast, the hydrogen adsorption feature completely disappeared from the CV in the microemulsion electrolyte presumably due to an adsorption of naphthalene on the Pt surface. However, reduction current appeared below ca. 0.2 V and steeply increased below 0.05 V. This can be explained by a partial reduction and desorption of the adsorbed naphthalene molecules[2] and subsequent bulk hydrogenation reaction on the vacant sites. It should be pointed out that the steep reduction current was observed at more positive potential than the hydrogen evolution reaction, suggesting a Langmuir-Hinshelwood type reaction with underpotential-deposition (UPD) hydrogen on the electrode surface.The GC analysis after a constant-current electrolysis at 2 mA cm-2 for 150 min revealed the formation of cis- and trans- decalin via10-electron transfer as well as tetralin via 4-electron transfer. In comparison between the isomers, cis-decalin was the dominant species presumably due to a flat orientation of the adsorbed naphthalene molecule [2], which reacted with UPD hydrogens via the Langmuir-Hinshelwood type mechanism. Surprisingly, the Fraday efficiency for the naphthalene/decalin conversion and selectivity for dacalin/tetralin was much improved at a Pt-Ru/Ti electrode, up to 48% and 93%, respectively. These improvements might be ascribed to change in the adsorption energy of naphthalene on the Pt site due to alloying with Ru, or change in the adsorption site to Ru.Reference[1] M. Wakisaka and M. Kunitake, Electrochem. Commun. 64 (2016) 5.[2] S.-L. Yau, Y-G. Kim, and K. Itaya, J. Phys. Chem. B 101 (1997) 3547. Figure 1

Bioaugmentation Treatment of a PAH-Polluted Soil in a Slurry Bioreactor

A bioslurry reactor was designed and used to treat loamy clay soil polluted with polycyclic aromatic hydrocarbons (PAHs). To this end, biostimulation alone, or combined with bioaugmentation with two bacterial strains (Rhodocccus erythropolis and Pseudomonas stuzeri) previously isolated from the polluted site, was applied. The PAH concentrations decreased notably after 15 days in all of the treatments. The concentrations of the two- and three-ring compounds fell by &gt;80%, and, remarkably, the four- to six-ring PAHs also showed a marked decrease (&gt;70%). These results thus indicate the capacity of bioslurry treatments to improve, notably, the degradation yields obtained in a previous real-scale remediation carried out using biopiles. In this sense, the remarkable results for recalcitrant PAHs can be attributed to the increase pollutants’ bioavailability achieves in the slurry bioreactors. Regarding bioaugmentation, although treatment with R. erythropolis led to a somewhat greater reduction of lighter PAHs at 15 days, the most time-effective treatment was achieved using P. stutzeri, which led to an 84% depletion of total PAHs in only three days. The effects of microbial degradation of other organic compounds were also monitored by means of combined qualitative and quantitative gas chromatography mass spectrometry (GC–MS) tools, as was the evolution of microbial populations, which was analyzed by culture and molecular fingerprinting experiments. On the basis of our findings, bioslurry technology emerges as a rapid and operative option for the remediation of polluted sites, especially for fine soil fractions with a high load of recalcitrant pollutants.

Evaluation of date seed (Phoenix dactylifera L.) oil as crop base stock for environment friendly industrial lubricants

Growing environmental concern and rapid consumption of poor biodegradable mineral oils have triggered the need for the development of eco-friendly biodegradable bio-based oils with low toxicity and excellent lubricating performance. The study aims to produce and evaluate the performance of date seed (Phoenix dactylifera L.) oil, which is abundantly available in the Middle East region, as an alternative bio-based lubricant. The optimum date seed oil (DSO) yield of 6% was achieved using solvent extraction of date seeds with particle size of 1 mm, hexane solvent to seed ratio of 6:1, and processing time of 18 hours. The physicochemical properties of the DSO were found such as its clear yellow color, density of 0.907 g/cm3, iodine value of 13.28 gl2/100 g, saponification value of 162.5 mg KOH/g, and acid value of 1.4 mg KOH/g. Moreover, fatty acid composition was analyzed employing quantitative gas chromatography (GC), and the oleic acid (41.1%), linoleic acid (21.4%), and palmitic acid (11.3%) were found to be the major unsaturated fatty acids. The pour and flash points were around -10 °C and 140 °C, respectively. Thermogravimetric analysis (TGA) revealed that the produced DSO is thermally stable at up to 300 °C. The rheological properties such as viscosity (41.62 cSt at 40 °C and 8.13 cSt at 100 °C) and viscosity index 173 were also assessed. These properties along with tribological properties of the DSO were compared with other bio-based oils and commercial SAE20W40 mineral oil. Overall, the characteristics of the DSO showed good potential to be developed into a bio-based lubricant. Furthermore, its properties can be enhanced by adding suitable additives. The findings of this study will help in developing eco-friendly bio-based industrial lubricants.

Metabolic Phenotypes of Hypoxic-Ischemic Encephalopathy with Normal vs. Pathologic Magnetic Resonance Imaging Outcomes.

Hypoxic-Ischemic Encephalopathy (HIE) is one of the most relevant contributors to neurological disability in term infants. We hypothesized that clinical outcomes of newborns with (HIE) can be associated with changes at plasma metabolic level enabling the detection of brain injury. Plasma samples of a cohort of 55 asphyxiated infants who evolved to moderate/severe HIE were collected between birth and completion of therapeutic hypothermia (TH). Samples were analyzed employing a quantitative gas chromatography–mass spectrometry method for the determination of lactate and pyruvate and an untargeted liquid chromatography–time-of-flight mass spectrometry method for metabolic fingerprinting. Brain injury was assessed employing magnetic resonance imaging (MRI). A critical assessment of the usefulness of lactate, pyruvate, and pyruvate/lactate for outcome prediction was carried out. Besides, metabolic fingerprinting identified a dynamic perturbation of eleven metabolic pathways, including amino acid and purine metabolism, and the steroid hormone biosynthesis, in newborns with pathologic MRI outcomes. Although data suggest the usefulness of lactate and pyruvate monitoring during 72 h for discerning outcomes, only the steroid hormone biosynthesis pathway was significantly altered in early plasma samples (i.e., before the initiation of TH). This study highlights pathways that might potentially be targeted for biomarker discovery or adjuvant therapies to be combined with TH.