Efficacy of head space solid-phase microextraction coupled to gas chromatography-mass spectrometry method for determination of the trace extracellular hydrocarbons of cyanobacteria.

Hydrocarbons are widespread in cyanobacteria, and the biochemical synthetic pathways were recently identified. Intracellular fatty alka(e)nes of cyanobacteria have been detected by liquid-liquid extraction (LLE) coupled to gas chromatography-mass spectrometry (GC/MS). However, whether fatty alka(e)nes can be released to cyanobacterial culture media remains to be clarified. This work develops a sensitive method for analyzing the trace level of extracellular hydrocarbons in cyanobacterial culture media by head space solid-phase microextraction (HS-SPME) coupled to GC/MS. Headspace (HS) extraction mode using polydimethylsiloxane fiber to extract for 30min at 50°C was employed as the optimal extraction conditions. Five cyanobacterial fatty alka(e)nes analogs including pentadecene (C15:1), pentadecane (C15:0), heptadecene (C17:1), heptadecane (C17:0), nonadecane (C19:0) were analyzed, and the data obtained from HS-SPME-GC/MS method were quantified using internal standard peak area comparisons. Limits of detection (LOD), limits of quantitation (LOQ), linear dynamic range, precisions (RSD) and recovery for the analysis of extracellular fatty alka(e)nes of cyanobacteria by HS-SPME-GC/MS were evaluated. The LODs limits of detection (S/N = 3) varied from 10 to 21 ng L-1. The correlation coefficients (r) of the calibration curves ranged from 0.9873 to 0.9977 with a linearity from 0.1 to 50 µg L-1. The RSD values were ranging from 7.8 to 14.0% and from 4.0 to 8.8% at 1.0 µg L-1 and 10.0 µg L-1 standard solutions, respectively. Comparative analysis of extracellular fatty alka(e)nes in the culture media of model cyanobacteria Synechocystis sp. PCC 6803 demonstrated that sensitivity of HS-SPME-GC/MS method was significantly higher than LLE method. Finally, we found that heptadecane can be released into the culture media of Synechocystis sp. PCC 6803 at the later growth period.

GC-MS Analysis and Volatile Profile Comparison for the Characteristic Smell from Liang-wai Gan Cao (Glycyrrhiza uralensis) and Honey-Roasting Products.

The characteristic smell of Liang-wai Gan Cao (Glycyrrhiza uralensis) and honey-roasting products was comprehensively analyzed using gas chromatography-mass spectrometry (GC-MS). Steam distillation and headspace solid-phase microextraction (HS-SPME) were used to extract volatile organic compounds (VOCs). Multiple fibers of SPME may reflect the samples' comprehensive information to the greatest extent, depending on their chemical characters. After chemometric resolution and spectra interpretation, many aroma compounds could be identified from GC-MS data. As a result, principal component analysis was set for the differentiation of several G. uralensis samples in different regions, and some important peaks could be found. Next, VOCs' profiles of honey-roasting products suggested that the flavors could be influenced by honey and pharmaceutical technologies.

Hydrogen peroxide-independent production of α-alkenes by OleTJE P450 fatty acid decarboxylase.

BACKGROUND: Cytochrome P450 OleTJE from Jeotgalicoccus sp. ATCC 8456, a new member of the CYP152 peroxygenase family, was recently found to catalyze the unusual decarboxylation of long-chain fatty acids to form α-alkenes using H2O2 as the sole electron and oxygen donor. Because aliphatic α-alkenes are important chemicals that can be used as biofuels to replace fossil fuels, or for making lubricants, polymers and detergents, studies on OleTJE fatty acid decarboxylase are significant and may lead to commercial production of biogenic α-alkenes in the future, which are renewable and more environmentally friendly than petroleum-derived equivalents. RESULTS: We report the H2O2-independent activity of OleTJE for the first time. In the presence of NADPH and O2, this P450 enzyme efficiently decarboxylates long-chain fatty acids (C12 to C20) in vitro when partnering with either the fused P450 reductase domain RhFRED from Rhodococcus sp. or the separate flavodoxin/flavodoxin reductase from Escherichia coli. In vivo, expression of OleTJE or OleTJE-RhFRED in different E. coli strains overproducing free fatty acids resulted in production of variant levels of multiple α-alkenes, with a highest total hydrocarbon titer of 97.6 mg·l-1. CONCLUSIONS: The discovery of the H2O2-independent activity of OleTJE not only raises a number of fundamental questions on the monooxygenase-like mechanism of this peroxygenase, but also will direct the future metabolic engineering work toward improvement of O2/redox partner(s)/NADPH for overproduction of α-alkenes by OleTJE.

Characterization of the bafilomycin biosynthetic gene cluster from Streptomyces lohii.

New hope for old bones: The plecomacrolide bafilomycin has been explored for decades as an anti-osteoporotic. However, its structural complexity has limited the synthesis of analogues. The cloning of the bafilomycin biosynthetic gene cluster from the environmental isolate Streptomyces lohii opens the door to the production of new analogues through bioengineering.

Quantitative analysis of fatty-acid-based biofuels produced by wild-type and genetically engineered cyanobacteria by gas chromatography-mass spectrometry.

Simple and rapid quantitative determination of fatty-acid-based biofuels is greatly important for the study of genetic engineering progress for biofuels production by microalgae. Ideal biofuels produced from biological systems should be chemically similar to petroleum, like fatty-acid-based molecules including free fatty acids, fatty acid methyl esters, fatty acid ethyl esters, fatty alcohols and fatty alkanes. This study founded a gas chromatography-mass spectrometry (GC-MS) method for simultaneous quantification of seven free fatty acids, nine fatty acid methyl esters, five fatty acid ethyl esters, five fatty alcohols and three fatty alkanes produced by wild-type Synechocystis PCC 6803 and its genetically engineered strain. Data obtained from GC-MS analyses were quantified using internal standard peak area comparisons. The linearity, limit of detection (LOD) and precision (RSD) of the method were evaluated. The results demonstrated that fatty-acid-based biofuels can be directly determined by GC-MS without derivation. Therefore, rapid and reliable quantitative analysis of fatty-acid-based biofuels produced by wild-type and genetically engineered cyanobacteria can be achieved using the GC-MS method founded in this work.

[Analysis of organochlorine pesticides in buckwheat using pressurized liquid extraction and gas chromatography].

Seven organochlorine pesticides (OCPs) in buckwheat were extracted by a homemade 24 cells auto-pressurized liquid extraction instrument (APLE) and then analyzed by gas chromatography with electronic capture detection. The optimal extraction condition was found to be 100 degrees C and 10 MPa for 5 min with acetone/n-hexane (1:1, v/v) as the extraction solvent. The extraction was performed twice for each sample to obtain complete extraction. The buckwheat was mixed with florisil and active carbon before filling into extraction cells. Concentrated sulfuric acid was used in clean up step of the extractant. The standard mixture of OCPs including alpha-benzene hexachloride (BHC), gamma-BHC, delta-BHC, p, p'-dichloro-diphenyl-dichloroethylene (p,p'-DDE), p,p'-dichloro-diphenyl-dichloroethane (p, p'-DDD), o, p-dichloro-diphenyl trichloro-ethane (o,p-DDT), p,p'-DDT was used for the identification and quantification in gas chromatographic analysis. The absolute recoveries of the 7 organochlorine pesticides were between 68%-126% with the relative standard deviations lower 14.7%. The detection limits were between 0.051 - 0.18 ng/g. The relative recoveries were as high as 116%-148% compared with Soxhlet extraction. It shows that the recoveries of APLE are much better than Soxhlet extraction, and the time of extraction for APLE is only about 15 min which is much less than conventional extraction methods.

[Analysis of phenolic compounds in aqueous samples by gas chromatography coupled with headspace solid-phase microextraction using poly (phthalazine ether sulfone ketone) coated fiber].

The direct trace analysis of phenolic compounds in aqueous samples was performed by headspace solid-phase microextraction/gas chromatography (HS-SPME/GC). A laboratory made poly (phthalazine ether sulfone ketone) (PPESK, 30 microm) coated fiber was used to extract the phenols from aqueous samples. The parameters affecting the extraction efficiency, such as extraction temperature and time, pH value, and salt concentration, were optimized. The low pH value and high salt concentration can increase the extraction efficiency of phenols. The limits of detection (LODs) were from 0.003 to 0.041 microg/L, which were within the range of EPA Method 604. The relative standard deviations (RSDs) were less than 16%. Compared with commercial polyacrylate (PA) fiber (85 microm), the PPESK fiber shows high affinity toward phenolic compounds, and therefore, high absolute recoveries. The phenols were detected with the recoveries of 100.5%-111.8% for a tap water sample and 94.8%-117.3% for a seawater sample at the spiked level of 20 microg/L.

Poly(phthalazine ether sulfone ketone) as novel stationary phase for stir bar sorptive extraction of organochlorine compounds and organophosphorus pesticides.

A novel poly(phthalazine ether sulfone ketone) (PPESK) film prepared by immersion precipitation technique was coated on stir bars for sorptive extraction. Scanning electron micrographs showed that the coating has a denser porous surface (about 1 microm in thickness) with a sponge-like sublayer, and the thickness of the coating was 250 microm. The PPESK coated stir bar has high thermostability (290 degrees C) and long lifetime (50 times). The extraction properties of this stir bar were evaluated for the extraction of both polar and semi-polar analytes, including organochlorine compounds and organophosphorus pesticides. The PPESK stir bar was proved to show higher affinity towards polar compounds than that of PDMS coated stir bar and higher sample load compared with corresponding PPESK fiber. It was applied to the determination of organochlorine compounds in seawater samples and organophosphorus pesticides in juices by gas chromatographic analysis. The effect of sample matrix was evaluated at optimized condition of extraction temperature, extraction time and salt concentration. Limits of detection were in the range of 0.05-2.53 ng L(-1) for organochlorine compounds in seawater samples using electron capture detector (ECD), with precisions of less than 11% RSD. Limits of detection for organophosphorus pesticides were in the range of 0.17-2.25 ng L(-1) and 2.47-10.3 ng L(-1) in grape and peach juice, respectively, using thermionic specified detector (TSD), with precisions of less than 12% RSD and 20% RSD, respectively.

Fast temperature programming on a stainless-steel narrow-bore capillary column by direct resistive heating for fast gas chromatography.

A direct resistive-heating fast temperature programming device for fast gas chromatography was designed and evaluated. A stainless-steel (SS) capillary column acted both as a separation column and as a heating element. A fast temperature controller with the deviation derivative proportional-integral-derivative (DDPID) control algorithm, which was suitable for ramp control using ramp-to-setpoint function, was used to facilitate the fast pulse heating. The SS resistive-heating column can generate linear temperature ramps up to 10 degrees C/s and can re-equilibrium from 250 degrees C down to 50 degrees C within 30s. With n-alkanes as the test analytes, the relative standard deviations (RSDs) of retention time were between 0.19 and 0.59% and the RSDs of their peak areas were less than 4% for all but one. The results indicated that this technique could be used for both qualitative and quantitative analysis. Phenolic and nitroaromatic compounds were also analyzed by using the SS resistive-heated system. The combination of a short narrow-bore SS column and rapid heating rates provides sufficient separation efficiency for relatively simple mixtures at drastically reduced analysis time. The total analysis time including equilibration time was less than 2 min for all test mixtures in this study.

[Preparation of poly (trifluoropropyl) methylsiloxane coating for solid-phase microextraction and its application for the analysis of phenolic compounds in sea water].

The poly (trifluoropropyl) methylsiloxane (PTFPMS) coating was prepared as solid-phase microextraction (SPME) coating by sol-gel technique. The coating has a porous microstructure and the thickness was 10 microm. Since the PTFPMS phase contains the functional group of -C2H4CF3, it exhibits high affinity to compounds containing electron-attracting groups. The PTFPMS phase can withstand desorption temperature up to 280 degrees C. The lifetime of the fiber SPME is more than 150 times. This coating was used to analyse the phenolic compounds in water samples by head-space solid-phase microextraction (HS-SPME) method. The parameters affecting the extraction efficiency, such as extraction mode, extraction temperature, extraction time, ion strength and pH, were optimized. The limits of detection (LODs) of the method were 0.02 - 1.2 microg/L. The relative standard deviations (RSDs) of single fiber and fiber-to-fiber (n = 5 ) were less than 4.4%, and 15.7%, respectively. The results indicated that the sol-gel method has good repeatability for the preparation of the PTFPMS coating.

A new poly(phthalazine ether sulfone ketone)-coated fiber for solid-phase microextraction to determine nitroaromatic explosives in aqueous samples.

A novel polar solid-phase microextraction (SPME) fiber coated with poly(phthalazine ether sulfone ketone) (PPESK) was prepared by immersion precipitation technique. The microstructure of the coating exhibits a sponge-like sublayer supporting a dense cracking shaped top layer (about 1 microm in thickness). This coating shows long lifetime (up to 100 times) and is stable at desorption temperature up to 290 degrees C due to the rigid aromatic rings in chemical structure. We evaluated the extraction-desorption properties of the PPESK fiber for nitroaromatic explosives in aqueous samples. The parameters affecting the extraction were optimized, including extraction temperature and time, salt addition, desorption temperature and time. Limits of detection (LOD), precisions and linear dynamic range for the analysis of explosives by SPME-GC/TSD or ECD were evaluated. Limits of detection of the new fiber was three orders of magnitude lower than those with carbowax/divinylbenzene (CW/DVB), and the relative standard deviation (RSD) of single fiber and fiber-to-fiber were less than 9.3 and 10.4%, respectively. The results demonstrated that the PPESK coating exhibited high extraction efficiency for nitroaromatic compounds due to the pi-pi interaction, dipole-dipole interactions and interactions by polar functional groups. The method was applied to the analysis of nitroaromatic explosives in real aqueous samples including seawater and groundwater samples, with relative recoveries better than 90.7%.

Study of conical columns with 10 degrees opening angle for preparative liquid chromatography.

The efficiency and sample capacity of conical liquid chromatographic columns with 10 degrees opening angle were studied at different ratio of cross section areas of inlet to outlet (A(in)/A(out)) and column dimension. As the A(in)/A(out) ratio changed from 4 to 2.25, the reduced plate height (h) was reduced and the h value decreased 12% when the column dimension was scaled up proportionally because of relatively smaller dead volume on both end of the column. Compared to cylindrical columns having corresponding lengths and volumes, the conical columns with 10 degrees opening angle were superior in column efficiency, resolution and the maximum peak concentration at column outlet; the loadability of conical column was improved 30-40% on injection volume and 50-60% on sample mass, respectively, over cylindrical columns.