Application of in vivo solid phase microextraction (SPME) in capturing metabolome of apple (Malus ×domestica Borkh.) fruit.

An in vivo direct-immersion SPME sampling coupled to comprehensive two-dimensional gas chromatography - time-of-flight mass spectrometry (GCxGC-ToFMS) was employed to capture real-time changes in the metabolome of 'Honeycrisp' apples during ripening on the tree. This novel sampling approach was successful in acquiring a broad metabolic fingerprint, capturing unique metabolites and detecting changes in metabolic profiles associated with fruit maturation. Several metabolites and chemical classes, including volatile esters, phenylpropanoid metabolites, 1-octen-3-ol, hexanal, and (2E,4E)-2,4-hexadienal were found to be up-regulated in response to fruit maturation. For the first time, Amaryllidaceae alkaloids, metabolites with important biological activities, including anti-cancer, anti-viral, anti-parasitic, and acetylcholinesterase (AChE) inhibitory activity, were detected in apples. Considering the elimination of oxidative degradation mechanisms that adversely impact the representativeness of metabolome obtained ex vivo, and further evidence that lipoxygenase (LOX) pathway contributes to volatile production in intact fruit, in vivo DI-SPME represents an attractive approach for global plant metabolite studies.

Quantitation and identification of ethanol and inhalant compounds in whole blood using static headspace gas chromatography vacuum ultraviolet spectroscopy.

Gas chromatography (GC) and vacuum ultraviolet spectroscopy (VUV) are powerful and complementary techniques for the analysis of small molecules in forensics. Most notably, flame ionization detection (FID) is commonly used with GC to identify and quantify volatile compounds. An FID's price point and ease of use makes it an attractive approach for routine laboratories that are in high demand for forensics analysis, but with the contingency that an FID relies on retention time for identification and quantification. A new and innovative method using static headspace gas chromatography coupled with vacuum ultraviolet (VUV) spectroscopy was developed for the quantitative determination of ethanol in blood and identification of inhalants. This study investigates the possibility of using VUV as an alternative technique to traditional methods that use FID and mass spectrometry (MS) in toxicology and forensic analysis. VUV brings both identification and quantitation based on Beer-Lambert's Law while using a simple single-column solution. This paper investigates 25 compounds, including ethanol, methanol, acetone, benzene, and toluene using a 130-240 nm wavelength range for identification and quantification using GC-VUV, even when coelutions occur. Ethanol was examined under a concentration range of 9 to 495 mg/dl, and the method was found to be linear with an r2 = 0.997 and a LOD of 3.1 mg/dl. Ethanol was fully separated from other volatile organic compounds (VOCs) as well as endogenous materials present in blood. Nonaromatic VOCs were analyzed at concentration ranges of 2.4 to 99 mg/dl with LODs around 0.2 mg/dl; aromatic VOCs were analyzed from 0.5 to 24 mg/dl with LODs ∼ 0.1 mg/dl.

Fast gas chromatography-atmospheric pressure (photo)ionization mass spectrometry of polybrominated diphenylether flame retardants.

Gas chromatography (GC) and mass spectrometry (MS) are powerful, complementary techniques for the analysis of environmental toxicants. Currently, most GC-MS instruments employ electron ionization under vacuum, but the concept of coupling GC to atmospheric pressure ionization (API) is attracting revitalized interest. API conditions are inherently compatible with a wide range of ionization techniques as well high carrier gas flows that enable fast GC separations. This study reports on the application of atmospheric pressure chemical ionization (APCI) and a custom-built photoionization (APPI) source for the GC-MS analysis of polybrominated diphenyl ethers (PBDEs), a ubiquitous class of flame retardants. Photoionization of PBDEs resulted in the abundant formation of molecular ions M•+ with very little fragmentation. Some photo-oxidation was observed, which differentiated critical BDE isomers. Formation of protonated molecules [M+H]+ did not occur in GC-APPI because the ionization energy of H2O (clusters) exceeds the energy of the ionizing photons. Avoiding mixed-mode ionization is a major advantage of APPI over APCI, which requires careful control of the source conditions. A fast GC-API-MS method was developed using helium and nitrogen carrier gases that provides good separation of critical isomers (BDE-49/71) and elution of BDE 209 in less than 7 min (with He) and 15 min (with N2). It will be shown that the GC-APPI and GC-APCI methods match the sensitivity and improve upon the selectivity and throughput of established methods for the analysis of PBDEs using standard reference materials (NIST SRM 1944 and SRM 2585) and selected environmental samples.

Vacuum Ultraviolet Spectroscopy as a New Tool for GC Analysis of Terpenes in Flavors and Fragrances.

Background: Traditional detectors such as flame ionization detection and MS have issues with coeluting isomers like terpenes; however, unique vacuum UV (VUV) absorbance spectra can be used to deliberately compress chromatography. Objective: Deconvolution capabilities under various run conditions of GC-MS and GC-VUV are compared. Methods: A standard terpenes mix and tea tree essential oil were run on both GC-MS (63 and 14 min run times) and GC-VUV (22, 11, and 7 min run times). Results: The three GC-VUV methods showed good precision for 10 terpenes, as well as with the 63 min GC-MS method. The 14 min GC-MS method struggled precisely quantifying some terpenes. Highlights: GC-VUV allows for faster run times while providing the same level of quantitative accuracy.

Lab-simulated downhole leaching of formaldehyde from proppants by high performance liquid chromatography (HPLC), headspace gas chromatography-vacuum ultraviolet (HS-GC-VUV) spectroscopy, and headspace gas chromatography-mass spectrometry (HS-GC-MS).

The ability of different methods to analyze formaldehyde and other leachates from proppants was investigated under lab-simulated downhole conditions. These methods include high performance liquid chromatography (HPLC), headspace gas chromatography-vacuum ultraviolet spectroscopy (HS-GC-VUV), and headspace gas chromatography-mass spectrometry (HS-GC-MS). Two different types of resin-coated proppants, phenol-formaldehyde- and polyurethane-based, were examined. Each proppant was tested at different time intervals (1, 4, 15, 20, or 25 hours) to determine the timeframe for chemical dissolution. Analyses were performed at room temperature and heated (93 °C) to examine how temperature affected the concentration of leachates. Multiple matrices were examined to mimic conditions in subsurface environment including deionized water, a solution surrogate to mimic the ionic concentration of produced water, and recovered produced water. The complexity of these samples was further enhanced to simulate downhole conditions by the addition of shale core. The influence of matrix components on the analysis of formaldehyde was greatly correlated to the quantity of formaldehyde measured. Of the three techniques surveyed, HS-GC-MS was found to be better suited for the analysis of formaldehyde leachates in complex samples. It was found that phenol-formaldehyde resin coated proppants leached higher concentrations of formaldehyde than the polyurethane resin coated proppants.

Gas chromatography-vacuum ultraviolet detection for classification and speciation of polychlorinated biphenyls in industrial mixtures.

Polychlorinated biphenyls (PCBs) are a group of synthetic chlorinated compounds that have been widely used as dielectric fluids in capacitors and transformers. Due to their toxicity, persistence, and bioaccumulation in the food chain, PCBs are an environmental concern and among the most analyzed compounds in environmental analysis. The most common analytical methods for analysis of PCBs are based on gas chromatography-electron capture detection (GC-ECD) and gas chromatography-mass spectrometry (GC-MS). However, the number of possible congeners (209), similarities of physical and chemical properties, and complexity of sample matrices make it difficult to distinguish and accurately speciate PCB congeners using existing methods. This study presents a new method using gas chromatography with vacuum ultraviolet detection (GC-VUV), which offers absorption detection in the range of 120-240nm, where all chemical species have absorption. The VUV absorption spectra for all 209 PCB congeners were collected and shown to be differentiable. The capability of VUV data analysis software for deconvolution of co-eluting signals was also demonstrated. An automated time interval deconvolution (TID) procedure was applied to rapidly speciate individual PCBs, as well as classify commercial Aroclor mixtures based on their degree of chlorination. The data showed excellent agreement between the stated nominal and determined degrees of chlorination (less than 1% deviation for highly chlorinated mixtures). GC-VUV was verified to provide excellent specificity, high sensitivity (100-150pg limit of detection), and fast data acquisition for this application.

Quantitative evaluation of the occupant kinematic response of the THUMS 50th-percentile male model relative to PMHS laboratory rollover tests.

OBJECTIVE: The objective of the current study was to evaluate the whole-body kinematic response of the Total Human Model for Safety (THUMS) occupant model in controlled laboratory rollover tests by comparing the model response to postmortem human surrogate (PMHS) kinematic response targets published in 2014. METHODS: A computational model of the parametric vehicle buck environment was developed and the AM50 THUMS occupant model (Ver 4.01) was subjected to a pure dynamic roll at 360°/s in trailing-side front-row seating position. A baseline configuration was defined by a baseline posture representing the average of all PMHS postures, with a friction coefficient of 0.4 for the belt and 0.6 for the seat. To encompass challenges in controlling boundary conditions from the PMHS tests and ensure the robustness of the model evaluation, a total of 12 simulations were performed to investigate the following: 1. The effect of initial posture by adding 3 additional postures representing PMHS extremes. 2. The effect of belt tension by varying tension from the nominal vehicle retractor belt tension of 5 N to the 35 N belt tension used in the PMHS tests. 3. The effect of friction between the environment (belt, seat) and THUMS. Trajectories (head, T1, T4, T10, L1, and sacrum), spinal segment rotations (head-to-T1, T1-to-T4, T4-to-T10, T10-to-L1, and L1-to-sacrum) relative to the rollover buck and spinal segment elongation/compression calculated from the simulations were compared to PMHS corridors using a correlation method (CORA). RESULTS: THUMS baseline response showed lower correlation (overall CORA score = 0.63) with the PMHS response in rollover compared to other crash modes. THUMS and PMHS demonstrated similar kinematic responses in the longitudinal axis and vertical axis but significantly different lateral excursion relative to the seat. In addition, no spinal elongation was observed for THUMS compared to the PMHS. The posture, pretension, and belt frictions were found to alter model kinematics, especially on THUMS lateral axis motion. The posture was judged to be the most sensitive parameter evaluated because a change of 30 mm in the lateral axis results in up to an 80 mm of change in observed displacement. CONCLUSIONS: Though the model response in the lateral axis is significantly different than that of the PMHS, it is unclear whether this difference is the result of extrinsic factors (posture, pretension, and friction), where exact values in experiment are unknown or by model intrinsic factors (e.g., spine stiffness). These differences in occupant kinematics could potentially subject the PMHS and THUMS to very different loading conditions under roof impact in rollover crashes: different occupant posture and different roof impact location. Therefore, different injury mechanisms and severity might be predicted by the current model relative to the PMHS. Consequently, though the information provided in the current study could be useful for improving model biofidelity for rollover crashes, additional studies are required to properly solve this issue.

Capturing Plant Metabolome with Direct-Immersion in Vivo Solid Phase Microextraction of Plant Tissues.

For the first time, an in vivo sampling mode of direct immersion-solid phase microextraction (DI-SPME) was employed to capture the metabolome of living plant specimens, using apple (Malus × domestica Borkh.) as a model system. Metabolites were extracted from apple tissues and introduced by thermal desorption into a comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry instrument. The feasibility of this sampling approach, based on exploitation of microextraction principles, including negligible depletion of free analyte concentrations, solventless sampling and sample preparation, and on-site compatibility, was determined in global metabolite analysis. Rather than adopting an approach of traditional sample preparation, requiring metabolism quenching and laborious sample preparation, the objective of the study was to capture the metabolome in vivo, evaluate the feasibility of the approach to provide unbiased extraction coverage, and compare analytical precision when different SPME sampling modes are employed. The potential of in vivo DI-SPME in quantitative plant metabolomics was assessed by evaluating changes in metabolic fingerprints in response to fruit maturation. The in vivo SPME sampling approach has been demonstrated as capable of sampling living systems with high reproducibility, considering that nearly 50% of hundreds of evaluated compounds included in the determination of analytical performance met the 15% RSD FDA criterion. Esters were extracted with high repeatability (% RSD for hexyl butanoate and butyl butanoate of 16.5 and 5.9, respectively, from 9 determinations in 3 apples) and found to be upregulated in response to apple fruit maturation.

Determination of emerging contaminants in wastewater utilizing comprehensive two-dimensional gas-chromatography coupled with time-of-flight mass spectrometry.

An analytical method for identification of emerging contaminants of concern, such as pesticides and organohalogens has been developed and utilized for true discovery-based analysis. In order to achieve the level of sensitivity and selectivity necessary for detecting compounds in complex samples, comprehensive gas chromatography coupled with time-of-flight mass spectrometry (GC×GC-TOFMS) was utilized to analyze wastewater samples obtained from the Pennsylvania State University wastewater treatment facility (WWTF). Determination of emerging contaminants through a process of combining samples which represent "normal background" and comparing this to new samples was developed. Results show the presence of halogenated benzotriazoles in wastewater samples as well as soil samples from Pennsylvania State University agricultural fields. The trace levels of chlorinated benzotriazoles observed in the monitoring wells present on the property indicate likely environmental degradation of the chlorinated benzotriazoles. Preliminary investigation of environmental fate of the substituted benzotriazoles indicates their likely degradation into phenol; an Environmental Protection Agency (USEPA) priority pollutant.

Comparison of Atmospheric Pressure Ionization Gas Chromatography-Triple Quadrupole Mass Spectrometry to Traditional High-Resolution Mass Spectrometry for the Identification and Quantification of Halogenated Dioxins and Furans.

The goal of this study was to qualify gas chromatography coupled to atmospheric pressure ionization tandem mass spectrometry (APGC-MS/MS) as a reliable and valid technique for analysis of halogenated dioxins and furans that could be used in place of more traditional gas chromatography coupled to high-resolution mass spectrometry (GC-HRMS) analysis. A direct comparison of the two instrumental techniques was performed. APGC-MS/MS system sensitivity was demonstrated to be on the single femtogram level. The APGC-MS/MS analysis also demonstrated method detection limits (MDLs) in both sediment and fish that were 2-18 times lower than those determined for the GC-HRMS. Inlet conditions were established to prevent issues with sample carry-over, due largely to the enhanced sensitivity of this technique. Additionally, this work utilized direct injection for sample introduction through the split/splittless inlet. Finally, quantification of both sediment and fish certified reference materials were directly compared between the APGC-MS/MS and GC-HRMS. The APGC-MS/MS performed similarly to, if not better than, the GC-HRMS instrument in the analysis of these samples. This data is intended to substantiate APGC-MS/MS as a comparable technique to GC-HRMS for the analysis of dioxins and furans.

Evaluation of conditions of comprehensive two-dimensional gas chromatography that yield a near-theoretical maximum in peak capacity gain.

The peak capacity gain (Gn) of a GC×GC system is the ratio of the system peak capacity to that of an optimized one-dimensional GC analysis lasting the same time and providing the same detection limit. A near-theoretical maximum in Gn has been experimentally demonstrated in GC×GC-TOF based on a 60m×0.25mm primary column. It was found that Gn was close to 9 compared to the theoretical maximum of about 11 for this system. A six-sigma peak capacity of 4500 was obtained during an 80min heating ramp from 50°C to 320°C. Using peak deconvolution, 2242 individual peaks were determined in a Las Vegas runoff water sample. This is the first definitive experimental demonstration known to us of an order-of-magnitude Gn. The key factors enabling this gain were: relatively sharp (about 20ms at half height) reinjection pulses into the secondary column, relatively long (60m) primary column, the same diameters in primary and secondary columns, relatively low retention factor at the end of the secondary analysis (k≅5 instead of 15, optimal for ideal conditions), optimum flow rate in both columns, and helium (rather than hydrogen) used as the carrier gas. The latter, while making the analysis 65% longer than if using H2, was a better match to the reinjection bandwidth and cycle time.

Comprehensive characterization of the halogenated dibenzo-p-dioxin and dibenzofuran contents of residential fire debris using comprehensive two-dimensional gas chromatography coupled to time of flight mass spectrometry.

A comprehensive approach was taken to characterize the polyhalogenated dibenzo-p-dioxin and dibenzofuran contents of fire debris. Household and electronics fire simulations were performed to create samples representative of those firefighters most typically come in contact with. Sample analysis was performed using GC×GC-TOFMS to provide a comprehensive profile of the halogenated dioxins and furans present among the two types of fire debris. Both the household fire and electronics fire simulations produced a significant amount of polybrominated dibenzofurans. Only the electronics rich fire simulation produced mixed halogenated (Br/Cl) dibenzofurans in amounts above the limit of detection of the analytical method. Of the mixed halogenated dibenzofurans identified, a majority were those having no commercially available standard to allow for specific congener identification. GC×GC-TOFMS was extremely beneficial for the identification of compound classes due to the manner in which compounds classify in the two-dimensional chromatographic plane, thus aiding data reduction for these materials.

Physician leadership in changing times.

Today, hospitals and physicians are reorganizing themselves in novel ways to take advantage of payment incentives that reward shared accountability for the total health care experience. These delivery system changes will take place with our without physician leadership. To optimize change on behalf of patients, physicians must play a conscious role in shaping future health care delivery organizations. As physician leaders of three of the nation׳s largest integrated health care delivery systems - Kaiser Permanente, Virginia Mason Medical Center, and the Mayo Clinic Health System - we call on physicians to view leadership and the development of leaders as key aspects of their role as patient advocates.

Comprehensive two-dimensional gas chromatography with a multi-capillary second dimension: a new column-set format for simultaneous optimum linear velocity operation.

Comprehensive two-dimensional gas chromatography (GC×GC) suffers from the impossibility to operate both dimensions at their optimum carrier gas velocity at the same time due to the different inner diameters of the columns typically employed. The use of multiple parallel capillary columns in the second dimension (GC×multi-GC) is studied as a means to achieve simultaneous optimum-velocity operation. A programme written in Microsoft Excel(®) was developed to calculate the efficiency of the two dimensions in GC×multi-GC for different numbers of columns in the second dimension. With the aid of this programme the appropriate number of columns was selected. Columns with maximum repeatability were specifically manufactured to grand suitable performance, i.e. to avoid band broadening effects caused by inter-column variations. 1D-GC experiments were carried out on the columns separately and combined in parallel. The performance of the parallel column set was consistent with that of the individual columns, with over 9100 plates generated (approximately 10,000 plates/m). A GC×multi-GC set-up was successfully installed. Model experiments proved the possibility to operate both dimensions at their optimum linear velocity simultaneously. The suitability of the novel second dimension column format to perform multidimensional separations was also shown for a number of selected applications.

Comprehensive two-dimensional gas chromatography time of flight mass spectrometry (GC×GC-TOFMS) for environmental forensic investigations in developing countries.

The disposal and dumping of toxic waste is a matter of growing concern in developing countries, including South Africa. Frequently these countries do not possess access to gas chromatography-high resolution mass spectrometry (GC-HRMS) for the determination of persistent organic pollutants (POPs). This publication describes an alternative approach to the investigation of toxic waste using comprehensive gas chromatography coupled to time of flight mass spectrometry (GC×GC-TOFMS). The technology permits both comprehensive screening of toxic samples for numerous classes of organic pollutants and also quantitative analysis for the individual compounds. This paper describes the use of this technique by analysing samples obtained from a hazardous waste treatment facility in South Africa. After sampling and extraction the samples were analysed for polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and four dioxin-like non-ortho substituted polychlorinated biphenyls (PCBs). The quantitative values, as well as detection limits, obtained using the GC×GC-TOFMS methodology compares well with those obtained using GC-HRMS; the accepted benchmark technology for this analysis. Although GC×GC-TOFMS is not a target compound analytical technique (as is GC-HRMS), it is possible to obtain information on numerous other classes of organic pollutants present in the samples in one analytical run. This is not possible with GC-HRMS. Several different column combinations have been investigated for handling very complex waste samples and suggestions are presented for the most suitable combination.

Characterization of military fog oil by comprehensive two-dimensional gas chromatography.

The most commonly used military fog oil is characterized by comprehensive two-dimensional gas chromatography (GC x GC) coupled to either Flame Ionization Detection (FID) or Time-of-Flight Mass Spectrometric Detection (TOFMS) to advance the knowledge regarding the complete chemical makeup of this complex matrix. Two different GC x GC column sets were investigated, one employing a non-polar column combined with a shape selective column and the other an inverse column set (medium-polar/non-polar). The inverse set maximizes the use of the two-dimensional separation space and segregates aliphatic from aromatic fractions. The shape selective column best separates individual polycyclic aromatic hydrocarbons (PAHs) from the bulk oil. The results reveal that fog oil (FO) is composed mainly of aliphatic compounds ranging from C(10) to C(30), where naphthenes comprise the major fraction. Although many different species of aromatics are present, they constitute only a minor fraction in this oil, and no conjugated PAHs are found. The composition of chemically similar aliphatic constituents limits the analytical power of silica gel fractionation and GC-MS analysis to characterize FO. Among the aliphatic compounds identified are alkanes, cyclohexanes, hexahydroindanes, decalins, adamantanes, and bicyclohexane. The aromatic fraction is composed of alkylbenzene compounds, indanes, tetrahydronaphthalenes, partially hydrogenated PAHs, biphenyls, dibenzofurans and dibenzothiophenes. This work represents the best characterization of military fog oil to date. As the characterization process shows, information on such complex samples can only be parsed using a combination of sample preprocessing steps, multiple detection schemes, and an intelligent selection of column chemistries.

Simplifying the setup for vacuum-outlet GC: using a restriction inside the injection port.

The application of vacuum GC has several advantages over pressurized GC. One of the key characteristics is that the optimal gas velocity is very high. Combined with short capillary columns of wide internal diameter, this results in short analysis times using standard GC-MS equipment. To make vacuum GC possible using a GC-MS system, a restriction must be positioned at the injection side of the column. This restriction is usually made of deactivated 0.1 mm i.d. fused-silica tubing which is coupled to the analytical column. Such restrictions will work, but practical challenges are found in coupling, reducing dead volume and robustness. A new way of making restrictions is by incorporating the restriction into the injection port. Using well-defined short pieces of fused silica with internal diameter of 0.025 mm, one can make a restriction using a Press-Tight type connector, and position this inside the injection port. By doing this, the restriction is very short and at high temperature all the time. Activity plays a minimal role, and also leaks will not be an issue as the coupling is in 100% inert gas. Data obtained using this concept is promising as vacuum GC becomes easier and more robust.