Automated SPME–GC–MS monitoring of headspace metabolomic responses of E. coli to biologically active components extracted by the coating

Monitoring extracellular metabolites of bacteria is very useful for not only metabolomics research but also for assessment of the effects of various chemicals, including antimicrobial agents and drugs. Herein, we describe the automated headspace solid-phase microextraction (HS-SPME) method coupled with gas chromatography–mass spectrometry (GC–MS) for the qualitative as well as semi-quantitative determination of metabolic responses of Escherichia coli to an antimicrobial agent, cinnamaldehyde. The minimum inhibitory concentration of cinnamaldehyde was calculated to be 2 g L⁻¹. We found that cinnamaldehyde was an important factor influencing the metabolic profile and growth process. A higher number of metabolites were observed during the mid-logarithmic growth phase. The metabolite variations (types and concentrations) induced by cinnamaldehyde were dependent on both cell density and the dose of cinnamaldehyde. Simultaneously, 25 different metabolites were separated and detected (e.g., indole, alkane, alcohol, organic acids, esters, etc.) in headspace of complex biological samples due to intermittent addition of high dose of cinnamaldehyde. The study was done using an automated system, thereby minimizing manual workup and indicating the potential of the method for high-throughput analysis. These findings enhanced the understanding of the metabolic responses of E. coli to cinnamaldehyde shock effect and demonstrated the effectiveness of the SPME–GC–MS based metabolomics approach to study such a complex biological system.     

Ultrasonication extraction and gel permeation chromatography clean-up for the determination of polycyclic aromatic hydrocarbons in edible oil by an isotope dilution gas chromatography–mass spectrometry

An analytical method for the determination of US EPA priority pollutant 16 polycyclic aromatic hydrocarbons (PAHs) in edible oil was developed by an isotope dilution gas chromatography–mass spectrometry (GC–MS). Extraction was performed with ultrasonication mode using acetonitrile as solvent, and subsequent clean-up was applied using narrow gel permeation chromatographic column. Three deuterated PAHs surrogate standards were used as internal standards for quantification and analytical quality control. The limits of quantification (LOQs) were globally below 0.5 ng/g, the recoveries were in the range of 81–96%, and the relative standard deviations (RSDs) were lower than 20%. Further trueness assessment of the method was also verified through participation in international cocoa butter proficiency test (T0638) organised by the FAPAS with excellent results in 2008. The results obtained with the described method were satisfying (z ≤ 2). The method has been applied to determine PAH in real edible oil samples.     

Sonochemical reactions of dissolved organic matter

Property changes of Aldrich and Pahokee peat dissolved organic matter (DOM) at different ultrasonic frequencies and energy densities were systematically investigated. Exposure of DOM to ultrasound resulted in decreases in TOC, Color₄₆₅, specific UV absorbance (SUVA), aromaticity and molecular weight, while DOM acidity increased. Compared to 20 kHz ultrasound, greater sonochemical transformation of DOM occurred at 354 kHz and at higher energy density, due to greater ^·OH radical production. The changes to DOM properties suggest that ultrasound may significantly affect DOM-pollutant interactions (e.g.facilitate desorption of hydrophobic organics from DOM or promote complexation between metallic cations and DOM).     

Characterization of aquatic dissolved organic matter by asymmetrical flow field-flow fractionation coupled to UV-Visible diode array and excitation emission matrix fluorescence

Flow field-flow fractionation (FlFFF) with on-line UV/Visible diode array detector (DAD) and excitation emission matrix (EEM) fluorescence detector has been developed for the characterization of optical properties of aquatic dissolved organic matter (DOM) collected in the Otonabee River (Ontario, Canada) and Athabasca River (Alberta, Canada). The molecular weight (MW) distribution of DOM was estimated using a series of organic macromolecules ranging from 479 to 66,000 Da. Both the number-average (M_n) and weight-average (M_w) molecular weights of Suwannee River fulvic acid (SRFA) and Suwannee River humic acid (SRHA) determined using these macromolecular standards were comparable to those obtained using polystyrenesulfonate (PSS) standards, suggesting that organic macromolecules can be used to estimate MW of natural organic colloids. The MW of eight river DOM samples determined by this method was found to have an M_n range of 0.8–1.1 kDa, which agrees with available literature estimates. The FlFFF-DAD-EEM system provided insight into the MW components of river DOM including the optical properties by on-line absorbance and fluorescence measurement. A red-shift in emission and excitation wavelength maxima associated with lower spectral slope ratios (S_R = S_275–295:S_350–400) was related to higher MW DOM. However, DOM of different origins at similar MW also showed significant difference in optical properties. A difference of 47 and 40 nm in excitation and emission peak C maxima was found. This supports the hypothesis that river DOM is not uniform in size and optical composition.     

Preparation of C18 composite solid-phase microextraction fiber and its application to the determination of organochlorine pesticides in water samples

In this work, a C18 composite solid-phase microextraction (SPME) fiber was prepared with a new method and applied to the analysis of organochlorine pesticides (OCPs) in water sample. A stainless steel wire (o.d. 127 μm) was used as the substrate, and a mixture of the C18 particle (3.5 μm) and the 184 silicone was used as the coating material. During the process of fiber preparation, a section of capillary column was used to fix the mixture onto the stainless steel wire and to ensure the constant of coating thickness. The prepared fiber showed excellent thermal stability and solvent resistance. By coupling with gas chromatography–mass spectrometry (GC–MS), the fiber exhibited wide linearity (2–500 ng L⁻¹) and good sensitivity for the determination of six OCPs in water samples, the OCPs tested included hexachlorobezene, trans-chlordane, cis-chlordane, o,p-DDT, p,p-DDT and mirex. Not only the extraction performance of the newly prepared fiber was more than seven times higher than those of commercial fibers, the limits of detections (LODs) (0.059–0.151 ng L⁻¹) for OCPs achieved under optimized conditions were also lower than those of reported SPME methods. The fiber was successfully applied to the determination of OCPs in real water samples by using developed SPME–GC–MS method.     

Application of hollow fiber liquid-phase microextraction in identification of oil spill sources

In this study, hollow fiber based liquid-phase microextraction (HF-LPME), coupled with GC, GC–MS and GC–IRMS detections, was employed to determine petroleum hydrocarbons in spilled oils. According to the results, the HF-LPME method collected more low-molecular weight components, such as C₇–C₁₁n-alkanes, naphthalene, and phenanthrene, than those collected in conventional liquid–liquid extraction (LLE). The results also showed that this method had no remarkable effect on the distributions of high-molecular weight compounds such as >C₁₈n-alkanes, C₁–C₃ phenanthrene, and hopanes. Also, the carbon isotopic compositions of individual n-alkanes in the two preparation processes were identical. Accordingly, HF-LPME, as a simple, fast, and inexpensive sample preparation technique, could become a promising method for the identification of oil spill sources.     

Pyrolysis-gas chromatography-mass spectrometry for studying N-vinyl-2-pyrrolidone-co-vinyl acetate copolymers and their dissolution behaviour

Knowledge on the solubility behaviour and dissolution rate of speciality and commodity polymers is very important for the use of such materials in high-tech applications. We have developed methods for the quantification and characterization of dissolved copolymers of N-vinyl-2-pyrrolidone (VP) and vinyl acetate (VA) during dissolution in water. The methods are based on pyrolysis (Py) performed in a programmed-temperature vaporization injector with subsequent identification and quantification of the components in the pyrolysate using capillary gas chromatography–mass spectrometry (GC–MS). By injecting large volumes and applying cryo-focussing at the top of the column, low detection limits could be achieved.The monomer ratio was found to have the greatest effect on the dissolution rate of the PVP-co-VA copolymers. The material with the highest amount of VA (50%) dissolves significantly slower than the other grades. Size-exclusion chromatography (SEC) and Py–GC–MS were used to measure molecular weights and average chemical compositions, respectively. Combined off-line SEC//Py–GC–MS was used to determine the copolymer composition (VP/VA ratio), as a function of the molecular weight for the pure polymers. In the dissolution experiments, a constant VP/VA ratio across the dissolution curve was observed for all copolymers analysed. This suggests a random distribution of the two monomers over the molecules.     

Extraction of climatic signals from fossil organic compounds in marine sediments up to 11.7 Ma old (IODP-U1318)

This study focuses on the extraction of climate signals and processes using a combined approach which includes the analysis of a high number of lipid molecules in marine sediments, and the chemometric analysis of the acquired data. Neutral and acidic fractions of marine sediments from site IODP-U1318 (south-west of the UK, Porcupine Seabight) were quantified by GC–MS. The alkenone unsaturation index, U^k′₃₇, was estimated from the composition of C₃₇ alkenones and it was then used for the estimation of sea surface temperatures (SST) for reference. Principal component analysis (PCA), explained 77.45% of the total data variance, and differentiated neutral fraction GC–MS total ion current (TIC) profiles according to SST values of the different sediment sections. GC–MS TIC chromatograms were correlated to sea surface temperatures (SST) by partial least squares regression (PLSR). The compounds more robustly in line with SST values at each sediment section explained 93% of the SST variance and they were identified using the variable importance in projection (VIP) scores method. The proposed approach enables an objective identification of organic compounds sensitive to SST variability throughout complete chromatographic profiles. As a result of this multivariate unbiased approach, lipid composition of sediments was differentiated between compounds of marine (long chain n-alkanes, long chain n-alkan-1-ols) and terrestrial (short chain n-alkan1-ols, alkenols, cholesterol, squalene) origin, whose concentrations were directly and inversely correlated to SST, respectively.     

Preparation,characterization, and applications of a novel solid-phase microextraction fiber by sol-gel technology on the surface of stainless steel wire for determination of poly cyclic aromatic hydrocarbons in aquatic environmental samples

A novel solid-phase microextraction(SPME) fiber was prepared using sol–gel technology with ethoxylated nonylphenol as a fiber coating material. The fiber was employed to develop a headspace SPME–GC–MS method suitable for quantification of 13 polycyclic aromatic hydrocarbons (PAHs) in water samples. Surface characteristics of the fibers were inspected by energy dispersive X-ray (EDX) spectroscopy as well as by scanning electron microscopy (SEM). The SEM measurements showed the presence of highly porous nano-sized particles in the coating. Important parameters affecting the extraction efficiency such as extraction temperature and time, desorption conditions as well as ionic strength have been evaluated and optimized. In the next step, the validation of the new method have been performed, finding it to be specific in the trace analysis of PAHs, with the limit of detection (LOD) ranging from 0.01 to 0.5 μg L⁻¹ and the linear range from the respective LOD to 200 μg L⁻¹with RSD amounting to less than 8%. The thermal stability of the fibers was investigated as well and they were found to be durable at 280 °C for 345 min. Furthermore, the proposed method was successfully applied for quantification of PAHs in real water samples.     

Direct monitoring of ochratoxin A in cheese with solid-phase microextraction coupled to liquid chromatography-tandem mass spectrometry

An in situ application of solid-phase microextraction (SPME) as a sampling and sample preparation method coupled to HPLC-MS/MS for direct monitoring of ochratoxin A (OTA) distribution at different locations in a single cheese piece is proposed. To be suited to the acidic analyte, the extraction phase (carbon-tape SPME fiber) was acidified with aqueous solution of HCl at pH 2, instead of the traditional sample pre-treatment with acids before SPME sampling. For calibration, kinetic on-fiber-standardization was used, which allowed the use of short sampling time (20 min) and accurate quantification of the OTA in the semi-solid cheese sample. In addition, the traditional kinetic calibration that used deuterated compounds as standards was extended to use a non-deuterated analogue ochratoxin B (OTB) as the standard of the analyte OTA, which was supported by both theoretical discussion and experimental verification. Finally, the miniaturized SPME fiber was adopted so that the concentration distribution of OTA in a small-sized cheese piece could be directly probed. The detection limit of the resulting SPME method in semi-solid gel was 1.5 ng/mL and the linear range was 3.5–500 ng/mL. The SPME–LC-MS/MS method showed good precision (RSD: ∼10%) and accuracy (relative recovery: 93%) in the gel model. The direct cheese analysis showed comparable accuracy and precision to the established liquid extraction. As a result, the developed in situ SPME–LC-MS/MS method was sensitive, simple, accurate and applicable for the analysis of complicated lipid-rich samples such as cheese.     

High-speed counter-current chromatographic isolation of ricinine,an insecticide from Ricinus communis

The alkaloid ricinine, an insecticide for leaf-cutting ant (Atta sexdens rubropilosa), was obtained from Ricinus communis. A two-phase solvent system composed of CH₂Cl₂/EtOH/H₂O (93:35:72, v/v/v) was used for high-speed counter-current chromatographic (HSCCC) isolation of ricinine in high yield and with over 96% purity, as determined by liquid and gas chromatography–mass spectrometry (LC–MS and GC–MS). Identification of ricinine was performed by comparison of ¹H NMR, ¹³C NMR and LC–MS/MS data.     

Optimisation of a headspace-solid-phase micro-extraction method for simultaneous determination of organometallic compounds of mercury,lead and tin in water by gas chromatography–tandem mass spectrometry

In this work, a headspace-solid-phase micro-extraction (HS-SPME) combined with gas chromatography–mass spectrometry (GC–MS) method for multielemental speciation of organometallic compounds of mercury, lead and tin in water samples was upgraded by the introduction of tandem mass spectrometry (MS/MS) as detection technique. The analytical method is based on the ethylation with NaBEt₄ and simultaneous headspace-solid-phase micro-extraction of the derivative compounds followed by GC–MS/MS analysis. The main experimental parameters influencing the extraction efficiency such as derivatisation time, extraction time and extraction temperature were optimized. The overall optimum extraction conditions were the following: a 50 μm/30 μm divinyl-benzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) SPME fibre, 150 min derivatisation time, 15 min extraction time, sample agitation at 250 rpm and 40 °C extraction temperature. The analytical characteristics of the HS-SPME method combined with GC–MS and GC–MS/MS were evaluated. The combination of both techniques HS-SPME and GC–MS/MS allowed to attain lower limits of detection (4–33 ng l⁻¹) than those obtained by HS-SPME–GC–MS (17–45 ng l⁻¹). The proposed method presented good linear regression coefficients (r² > 0.9970) and repeatability (4.8–21.0%) for all the compounds under study. The accuracy of the method measured as the average percentage recovery of the compounds in spiked river water and seawater samples was higher than 80% for all the compounds studied, except for monobutyltin in the river water sample. A study of the uncertainty associated with the analytical results was also carried out.     

Analytical performance of a triple quadrupole mass spectrometer compared to a high resolution mass spectrometer for the analysis of polybrominated diphenyl ethers in fish

Polybrominated diphenyl ethers (PBDEs) are a class of flame retardants used globally in many consumer products and industrial applications. Traditionally, gas chromatography–high resolution mass spectrometry (GC–HR-MS) is the method of choice for analysis of PBDEs in environmental samples because it offers high sensitivity and selectivity, resulting in less interferences. However, the specificity offered by gas chromatography-triple quadrupole tandem mass spectrometry (GC–QQQ-MS/MS), operated in selected reaction monitoring mode, provides a more affordable alternative to GC–HR-MS for the analysis of PBDEs in complex environmental samples. In this study, an analytical method was developed for the analysis of 41 PBDE congeners in fish using GC–QQQ-MS/MS. Results from the analysis of three fish species [lake trout (Salvelinus namaycush), yellow perch (Perca flavescens), and round goby (Neogobius melanostomus)] using GC–QQQ-MS/MS were compared with those obtained by GC–HR-MS. These species were selected because they represent varying levels of lipid-rich matrix and contaminant loads. Instrumental limits of detection for the GC–QQQ-MS/MS ranged from 0.04 pg to 41 pg, whereas those for the GC–HR-MS ranged from 5 pg to 85 pg. The PBDE values obtained from these two methods were highly correlated, R² values >0.7, for all three fish species, supporting the suitability of GC–QQQ-MS/MS for analysis of PBDEs in fish with varying fat content.     

Evaluation of low-cost disposable polymeric materials for sorptive extraction of organic pollutants in water samples

The capabilities of four commercially available and low cost polymeric materials for the extraction of polar and non-polar contaminants (log K_ow = −0.07–6.88, from caffeine to octocrylene, respectively) from water samples was compared. Tested sorbents were polyethersulphone, polypropylene and Kevlar, compared to polydimethylsiloxane as reference material. Parameters that affect the extraction process such as pH and ionic strength of the sample, extraction time and desorption conditions were thoroughly investigated. A set of experimental partition coefficients (K_pw), at two different experimental conditions, was estimated for the best suited materials and compared with the theoretical octanol–water (K_ow) partition coefficients of the analytes. Polyethersulphone displayed the largest extraction yields for both polar and non-polar analytes, with higher K_pw and lower matrix effects than polydimethylsiloxane and polypropylene. Thus, a sorptive microextraction method, followed by large volume injection (LVI) gas chromatography–tandem mass spectrometry (GC–MS/MS), was proposed using the former sorbent (2 mg) for the simultaneous determination of model compounds in water samples. Good linearity (>0.99) was obtained for most of the analytes, except in the case of 4-nonylphenol (0.9466). Precision (n = 4) at 50 and 500 ng L⁻¹ levels was in the 2–24% and limits of detection (LODs) were in the 0.6–25 ng L⁻¹ range for all the analytes studied.     

A fast method for the identification of Mycobacterium tuberculosis in sputum and cultures based on thermally assisted hydrolysis and methylation followed by gas chromatography–mass spectrometry

A fast gas chromatography–mass spectrometry (GC–MS) method with minimum sample preparation is described for early diagnosis of tuberculosis (TB). The automated procedure is based on the injection of sputum samples which are then methylated inside the GC injector using thermally assisted hydrolysis and methylation (THM). The THM–GC–MS procedure was optimized for the injection of sputum samples. For the identification of Mycobacterium tuberculosis the known marker tuberculostearic acid (TBSA) and other potential markers were evaluated. Hexacosanoic acid in combination with TBSA was found to be specific for the presence of M. tuberculosis. For validation of the method several sputum samples with different viscosities spiked with bacterial cultures were analyzed. Finally, 18 stored sputum samples collected in Vietnam from patients suspected to suffer from TB were re-analyzed in Amsterdam by microscopy after decontamination/concentration and using the new THM–GC–MS method. No false positives were found by THM–GC–MS and all patients who were diagnosed with TB were also found positive using our newly developed THM–GC–MS method. These results show that the new fast and sensitive THM–GC–MS method holds great potential for the diagnosis of TB.     

Application of an automatic thermal desorption–gas chromatography–mass spectrometry system for the analysis of polycyclic aromatic hydrocarbons in airborne particulate matter

The application of the thermal desorption (TD) method coupled with gas chromatography–mass spectrometry (GC–MS) to the analysis of aerosol organics has been the focus of many studies in recent years. This technique overcomes the main drawbacks of the solvent extraction approach such as the use of large amounts of toxic organic solvents and long and laborious extraction processes. In this work, the application of an automatic TD–GC–MS instrument for the determination of particle-bound polycyclic aromatic hydrocarbons (PAHs) is evaluated. This device offers the advantage of allowing the analysis of either gaseous or particulate organics without any modification. Once the thermal desorption conditions for PAH extraction were optimised, the method was verified on NIST standard reference material (SRM) 1649a urban dust, showing good linearity, reproducibility and accuracy for all target PAHs. The method has been applied to PM10 and PM2.5 samples collected on quartz fibre filters with low volume samplers, demonstrating its capability to quantify PAHs when only a small amount of sample is available.     

Flow injection solid-phase extraction using multi-walled carbon nanotubes packed micro-column for the determination of polycyclic aromatic hydrocarbons in water by gas chromatography–mass spectrometry

A flow injection solid-phase extraction preconcentration system using a multi-walled carbon nanotubes (MWCNTs) packed micro-column was developed for the determination of 16 polycyclic aromatic hydrocarbons (PAHs) in water by gas chromatography–mass spectrometry (GC–MS). The preconcentration of PAHs on the MWCNTs was carried out based on the adsorption retention of analytes by on-line introducing the sample into the micro-column system. Methanol was introduced to elute the retained analytes for GC–MS analysis using selected ion monitoring (SIM) mode. Important influence factors were studied in detail, such as sample acidity, sample flow rate, eluent flow rate and volume, dimensions of MWCNTs and amounts of packing material. Limits of detection of 16 PAHs for an extraction of 50 mL water sample were in the range of 0.001–0.15 μg L⁻¹, and the precisions (RSD) were in the range of 4–14%. The optimized method was successfully applied to the determination of 16 PAHs in surface waters, with recoveries in the range of 72–93% for real spiked sample.     

High-throughput approach for analysis of multicomponent gas chromatographic–mass spectrometric signals

Hyphenated techniques such as gas chromatography–mass spectrometry (GC–MS) or high-performance liquid chromatography–mass spectrometry (LC–MS) produce a large amount of data in a form of two-way data matrix. It has been a great challenge to furthest extract the useful information from the data. In this work, a chemometric approach based on a modification of adaptive immune algorithm (AIA) was proposed for a high-throughput analysis of the multicomponent overlapping GC–MS signals. With the proposed method, the chromatographic profile of each component in an overlapping signal can be extracted independently and sequentially along the retention time. In order to show the efficiency of the method, a stimulated GC–MS data of six components with background and an experimental GC–MS data of 40 pesticides were investigated. It was found that the multicomponent overlapping GC–MS signals could be fast and accurately resolved. Furthermore, the quantitative property of the extracted information was also investigated. The correlation coefficients (r) between the peak area and the added volumes of the sample are in the range 0.9658–0.9953.     

Measuring the pollutant transport capacity of dissolved organic matter in complex matrixes

Dissolved organic matter (DOM) facilitated transport in contaminated groundwater was investigated through the measurement of the binding capacity of landfill leachate DOM (Vejen, Denmark) towards two model pollutants (pyrene and phenanthrene). Three different methods for measuring binding capacity were used and evaluated, head-space solid-phase micro-extraction (HS-SPME), enhanced solubility (ES) and fluorescence quenching (FQ). It was concluded that for samples with complex matrixes it was possible to measure the net effect of the DOM binding capacity and the salting out effect of the matrix. It was further concluded that DOM facilitated transport should be taken into account for non-ionic PAHs with lg?K?_OW above 5, at DOM concentrations above 250?mg?C/L. The total DOM concentration was found to be more important for the potential of facilitated transport than differences in the DOM binding capacity.     

Selectable one-dimensional or two-dimensional gas chromatography–mass spectrometry with simultaneous olfactometry or element-specific detection

A novel selectable one-dimensional (¹D) or two-dimensional (²D) gas chromatography–mass spectrometry (selectable ¹D/²D GC–MS) system with selective detection was developed by using capillary flow technology and low thermal mass GC (LTM-GC). The main advantages of this system are the simple and fast selection of ¹D GC–MS or ²D GC–MS operation without any instrumental set-up change (e.g.²D GC–MS can be run just after ¹D GC–MS run), and simultaneous mass spectrometric and olfactometry or element-specific detection for both ¹D and ²D separation to assure selection of a heart-cut region and correct identification of compounds of interest. The feasibility and benefit of the proposed system with selective detection, e.g. olfactometry, nitrogen phosphorus detection (NPD), and pulsed flame photometric detection (PFPD), was demonstrated with an identification of trace amounts of aroma components in beverages (beer and coffee). Using stir bar sorptive extraction (SBSE) and selectable ¹D/²D GC–Olfactometry/MS on a beer sample, β-damascenone could be determined at 1.9 ng mL⁻¹ (RSD 3.1%, n = 6) as a potent aroma compound. In a coffee sample, two odor active compounds were clearly resolved from a 4.2 s heart-cut and were assigned probable identifications as 4,5-dimethyl thiazole and dimethyl trisulfide based on a NIST library search, dual linear retention indices (dual LRI) and elemental information obtained by SBSE in combination with selectable ¹D/²D GC–NPD/PFPD/MS.