Quantitative Analysis of Metabolites at the Single-Cell Level by Hydrogen Flame Desorption Ionization Mass Spectrometry.

To date, direct quantitation of cellular metabolites at the picoliter level or in a single cell is still a challenge due to tiny sampling materials, the accuracy of the sampling volume, and the ubiquitous matrix effect. Herein, picoliter magnitude quantitative analysis was performed using a pressure-assisted microsampling probe coupled to the hydrogen flame desorption ionization mass spectrometer (HFDI-MS). The sampling was accurately controlled with a picoliter pump, and the analytes were rapidly vaporized and quantitatively transferred to the gas phase by adequate heat. The vapor-phase analytes reacted with protonated water cluster ions by the proton-transfer reaction (PTR). The accurate sampling, flash thermal desorption, and proton-transfer ionization processes were conducted spatiotemporally, which could greatly reduce matrix effects to facilitate the quantitation of analytes without the internal standard. Furthermore, this workflow enabled the quantitation of cellular metabolites at the picoliter/single-cell level.

Increasing Signal-to-Noise Ratio in Gas Chromatography - Electroantennography Using a Deans Switch Effluent Chopper.

Gas-chromatography-electroantennographic detection (GC-EAD) is a technique used in the identification of volatile organic compounds (VOCs), such as pheromones and plant host odors, which are physiologically relevant to insects. Although pheromones often elicit large EAD responses, other behaviorally relevant odors may elicit responses that are difficult to discern from noise. Lock-in amplification has long been used to reduce noise in a wide range of applications. Its utility when incorporated with GC-EAD was demonstrated previosuly by chopping (or pulsing) effluent-laden air that flowed over an insect antenna. This method had the disadvantage that it stimulated noise-inducing mechanoreceptors and, in some cases, disturbed the electrochemical interfaces in a preparation, limiting its performance. Here, the chopping function necessary for lock-in amplification was implemented directly on the GC effluent using a simple Deans switch. The technique was applied to excised antennae from female Heliothis virescens responding to phenethyl alcohol, a common VOC emitted by plants. Phenethyl alcohol was always visible and quantifiable on the flame ionization detector (FID) chromatogram, allowing the timing and amount of stimulus delivered to the antennal preparation to be measured. In our new chopper EAG configuration, the antennal preparation was shielded from air currents in the room, further reducing noise. A dose-response model in combination with a Markov-chain monte-carlo (MCMC) method for Bayesian inference was used to estimate and compare performance in terms of error rates involved in the detection of insect responses to GC peaks visible on an FID detector. Our experiments showed that the predicted single-trial phenethyl alcohol detection limit on female H. virescens antennae (at a 5.0% expected error rate) was 140,330 pg using traditional EAG recording methods, compared to 2.6-6.3 pg (5th to the 95th percentile) using Deans switch-enabled lock-in amplification, corresponding to a 10.4-12.7 dB increase in signal-to-noise ratio.

Comparison and validation of 2 analytical methods for the determination of free fatty acids in dairy products by gas chromatography with flame ionization detection.

Accurate quantification of free fatty acids (FFA) in dairy products is important for quality control, nutritional, antimicrobial, authenticity, legislative, and flavor purposes. In this study, the performance of 2 widely used gas chromatographic flame ionization detection methods for determination of FFA in dairy products differing in lipid content and degree of lipolysis were evaluated. We used a direct on-column approach where the isolated FFA extract was injected directly and a derivatization approach where the FFA were esterified in the injector to methyl esters using tetramethylammonium hydroxide as a catalyst. A comprehensive validation was undertaken to establish method linearity, limits of detection, limits of quantification, accuracy, and precision. Linear calibrations of 3 to 700mg/L (R(2)>0.999) and 20 to 700mg/L (R(2)>0.997), and limits of detection and limits of quantification of 0.7 and 3mg/L and 5 and 20mg/L were obtained for the direct injection on-column and the derivatization method, respectively. Intraday precision of 1.5 to 7.2% was obtained for both methods. The direct injection on-column method had the lower levels of limits of detection and quantification, because FFA are directly injected onto the GC as opposed to the split injection used in the derivatization method. However, the direct injection on-column method experienced accumulative column phase deterioration and irreversible FFA absorption because of the acidic nature of the injection extract, which adversely affected method robustness and the quantification of some longer chain FFA. The derivatization method experienced issues with quantification of butyric acid at low concentrations because of coelution with the injection solvent peak, loss of polyunsaturated FFA due to degradation by tetramethylammonium hydroxide, and the periodic emergence of by-product peaks of the tetramethylammonium hydroxide reaction that interfered with the quantification of some short-chain FFA. The derivatization method is more robust, and because the derivatization step can be automated, it is more suitable for routine analysis of FFA in dairy products. However, considerable scope exists to develop an alternative gas chromatography with flame ionization detection method to quantify FFA in dairy products without any limitations that is robust and accurate.

Green chromatography determination of fatty acid methyl esters in biodiesel.

This work proposes a green, simple and rapid chromatographic methodology for separation and determination of a group of 13 fatty acids methyl esters (FAMEs) by using a capillary gas chromatography with a flame ionization detector. The method was successfully applied for the determination of FAMEs in biodiesel samples from commercial and waste cooking oils, synthesized by homogeneous catalysis. Detection and quantification limits were in the µg L(-1) level. Direct injection of sample solution was compared with solid-phase extraction and solid-phase microextraction procedures, giving similar results. The lower analysis time represent considerable improvement compared with other papers. The described methodology is especially suitable for process control applications. The samples analysed showed total contents of FAMEs higher than 96.5%, which verifies the European regulations.

Development of headspace-gas chromatography-flame ionization detection procedure to determine volatile fatty acids in zoo organic waste leachates.

In order to determine volatile fatty acids (short chain monocarboxylic acids with 2 to 6 carbon atoms in a molecule) in leachates formed in organic waste piles a procedure based on static headspace (HS) coupled with gas chromatography (GC) have been proposed and optimized. The conditions of HS extraction and sample introduction into a gas chromatograph as well as of chromatographic separation were considered. Using flame ionization detection LODs were of the order of 0.02-0.37 mg/L. The analyzed leachate collecting on and close to a zoo organic waste pile contained VFAs (Volatile Fatty Acids) at a concentration ranging from 5.5 mg/L to 0.88 g/L and from below LOD to 10.1 mg/L for pool water at some distance from the pile, respectively.

Automated method for volatile fatty acids determination in anaerobic processes using in-syringe magnetic stirring assisted dispersive liquid-liquid microextraction and gas chromatography with flame ionization detector.

Volatile fatty acids (VFAs) are key parameters to monitor anaerobic digestion processes. Thus, a fast, simple and precise determination of these analytes is necessary for a timely characterization of the biological processes present in municipal solid waste and wastewater treatment plants. In this work, an automated method for the extraction and preconcentration of VFAs, based on dispersive liquid-liquid microextraction with magnetic stirring in syringe, and gas chromatography with flame ionization detector for the separation and detection, is described. The effect of parameters such as the type and volume of extraction solvent, pH, salting out effect and stirring time, was studied using a multivariate and univariate experimental design. Extraction and preconcentration were performed simultaneously using tert-butyl methyl ether (TBME) as the extraction solvent, after stirring 100 s at a constant rate. The detection limits were in the range of 0.1 - 1.3 mg L-1 and a good linearity was observed up to 1000 mg L-1 of the studied VFAs, with a range of R2 between 0.9997 and 0.9999. The intra and interday precision expressed as relative standard deviation (n= 5) varied between 0.7 and 2.4% and between 1.7 and 7.0%, respectively. Subsequently, the developed method was successfully applied to evaluate the presence of VFAs in wastewater samples from anaerobic treatments and an average relative recovery of 102% was obtained.

Determination of trace organophosphorus pesticides in water samples with TiO2 nanotubes cartridge prior to GC-flame photometric detection.

This article described a new method for the sensitive determination of organophosphorus pesticides in water samples using SPE in combination with GC-flame photometric detection. In the procedure of method development, TiO(2) nanotubes were used as SPE adsorbents for the enrichment of organophosphorus pesticides from water samples. Several factors, such as eluent and its volume, sample pH, sample volume, sample flow rate, and concentration of humic acid, were optimized. Under the optimal conditions, the proposed method had good linear ranges as 0.1-40 microg/L for each of them, LOD of 0.11, 0.014, and 0.0025 microg/L, and LOQs of 0.37, 0.047, and 0.0083 microg/L for chlorpyrifos, phorate, and methyl parathion, respectively. The proposed method was validated with real environmental water samples and the spiked recoveries were over the range of 86.5-115.1%. All these results indicated that TiO(2) nanotubes, as a new SPE adsorbent, would be used widespread for the preconcentration and determination of environmental pollutants in the future.

Synthesis of nano-sized biphasic calcium phosphate ceramics with spherical shape by flame spray pyrolysis.

Nanometer size biphasic calcium phosphate (BCP) powders with various Ca/P molar ratios satisfied with appropriate phase ratios of HA/beta-TCP were prepared by high temperature flame spray pyrolysis process. The BCP powders had spherical shapes and narrow size distributions irrespective of the ratios of Ca/P. The mean size of the BCP powders measured from the TEM image was 38 nm. The composition ratio of Ca/P was controlled from 1.500 to 1.723 in the spray solution, and required phase ratios of HA/TCP are controlled systematically. The calcium dissolution of the pellets obtained from the BCP powders directly prepared by flame spray pyrolysis in buffer solution increased with the decrease of Ca/P ratios except with the Ca/P ratio of 1.713. The pellet surface with Ca/P ratio of 1.500, which consisted of beta-TCP, was eroded dramatically for 7 days. On the other hand, the pellet surface with Ca/P ratio of 1.667 was stable and did not disintegrate after immersion in Tris-HCl buffer solution based on the SEM observation.

Analysis of the chemical composition of the essential oil of Polygonum minus Huds. using two-dimensional gas chromatography-time-of-flight mass spectrometry (GC-TOF MS).

The essential oil in leaves of Polygonum minus Huds., a local aromatic plant, were identified by a pipeline of gas chromatography (GC) techniques coupled with mass-spectrometry (MS), flame ionization detector (FID) and two dimensional gas chromatography time of flight mass spectrometry (GC x GC-TOF MS). A total of 48 compounds with a good match and high probability values were identified using this technique. Meanwhile, 42 compounds were successfully identified in this study using GC-MS, a significantly larger number than in previous studies. GC-FID was used in determining the retention indices of chemical components in P. minus essential oil. The result also showed the efficiency and reliability were greatly improved when chemometric methods and retention indices were used in identification and quantification of chemical components in plant essential oil.

Post-column reaction with a 3D-printed two-stage microreactor and flame ionization detection for carbon compound independent response in fast gas chromatography.

Fast gas chromatography that leverages the high chromatographic efficiency of narrow bore capillary column technology and temperature programming was successfully integrated with a third-generation low void-volume, 3D-printed two-stage microreactor. Effective management of extra-column effect and the capability to perform post-column backflushing were achieved with the incorporation of a recently commercialized, electronically controlled pneumatic switching device and a deactivated metal three-way microdevice. With this configuration, narrow bore capillary columns having internal diameters between 0.10 and 0.15 mm can be employed to produce chromatographic peaks in the domain of fast gas chromatography, with peak widths at half-height ranging from 0.42 s to 0.92 s for probe compounds having k over a range from 1.7 for toluene to 60 with the last analyte (nC44) eluted in less than 12 min. The carbon independent response capability of the 3D-printed microreactor affords unique and advantaged differentiators, for instance, conducting measurement of the target analytes using one single carbon-containing compound for calibration with an acceptable accuracy of ±10%, achieving a higher degree of accuracy by eliminating the need for multi-level and multi-compound calibration, and improving sensitivity for compounds that are not efficiently ionized by flame ionization detection. Using this platform, repeatability of retention times for 14 probe compounds was less than 0.1% RSD (n = 10), and less than 1.0% RSD (n = 10) for area counts. The utility of the analytical approach was illustrated with relevant, challenging applications.

Chemical composition, antioxidant, anti-inflammatory and antiproliferative activities of the essential oil of Cymbopogon nardus, a plant used in traditional medicine.

Objectives Natural products commonly used in traditional medicine, such as essential oils (EOs), are attractive sources for the development of molecules with anti-proliferative activities for future treatment of human cancers, e.g., prostate and cervical cancer. In this study, the chemical composition of the EO from Cymbopogon nardus was characterized, as well as its antioxidativeproperties and anti-inflammatory and antiproliferative activities on LNCaP cells derived from prostate cancer. Methods The chemical composition of the EO was determined by GC/FID and GC/MS analyses. The antioxidative properties were assessed using DPPH radical scavenging assay and ABTS+• radical cation decolorization assay, and the anti-inflammatory capacity was determined by the inhibition of the lipoxygenase activity. Antiproliferative activity was evaluated by MTT assay. Results Collectively, our data show that the major constituents of C. nardus EO are citronellal (33.06 %), geraniol (28.40 %), nerol (10.94 %), elemol (5.25 %) and delta-elemene (4.09 %). C. nardus EO shows modest antioxidant and anti-inflammatory activity compared to the standard galic acid. C. nardus EO exhibits the best antiproliferative activity on the prostate cancer cell line LNCaP with an IC50 of 58.0 ± 7.9 µg/mL, acting through the induction of the cell cycle arrest. Conclusions This study has determined that C. nardus EO efficiently triggers cytotoxicity and pens a new field of investigation regarding the putative use of this EO in vivo.

Dynamic control of split flow in packed column supercritical fluid chromatography using dual resistively heated restrictors.

Remote control of the vent/detector split flow ratio in packed column supercritical fluid chromatography (pSFC) with flame ionization detector (FID) is demonstrated using a dual heated restrictor method. Restrictors stemming from a Tee at the separation column outlet were, respectively, fixed into an FID and a vent port, and their individual temperatures were controlled using resistively heated wires. Subsequently, both system pressure and split flow could be manipulated. For example, for applied restrictor temperatures examined up to 600 degrees C, corresponding vent/FID split flow ratios between 2 and 7 were observed depending on the port heated. As well, column pressures around 16-23 MPa were also achievable over the same range. Conversely, isobaric altering of the split flow ratio was possible when opposing positive and negative temperature gradients were applied at the two restrictors. Under these conditions, the system pressure varied less than 1% RSD over a 10 min period. As an application, the method was used to establish stable detector operation in the analysis of n-alkanes under pSFC-FID conditions that initiated flame instability. Results indicate that this technique could be a relatively simple and inexpensive means of controlling system pressure and detector split flow ratios in pSFC-FID.

Fast GC-FID based metabolic fingerprinting of Japanese green tea leaf for its quality ranking prediction.

There is a need of reliable, rapid, and cost-effective analysis technique to evaluate food and crop compositions, which are important to improve their qualities and quantities. Prior to fast GC-FID development, metabolic fingerprints, and predictive models obtained from a conventional GC-FID were evaluated by comparison to those derived from GC-TOF-MS. A similar chromatographic pattern with higher sensitivity of polyphenol compounds including epicatechin gallate (ECg) and epigallocatechin gallate (EGCg) had been achieved by using conventional GC-FID. Fast gas chromatograph coupled with flame ionization detector (GC-FID) has been carried out with 10 m x 0.18 mm id x 0.20 microm df capillary column. The analysis time per sample was reduced to less than 14 min compared to those of a conventional GC-FID (38 min) and GC-TOF-MS (28 min). The fast GC-FID also offered reliable retention time reproducibility without significant loss of peak resolution. Projections to latent structures by means of partial least squares (PLS) with orthogonal signal correction filtering (OSC) was applied to the fast GC-FID data. The predictive model showed good model fit and predictability with RMSEP of 3.464, suggesting that fast GC-FID based metabolic fingerprinting could be an alternative method for the prediction of Japanese green tea quality.

The EZ:Faast Family of Amino Acid Analysis Kits: Application of the GC-FID Kit for Rapid Determination of Plasma Tryptophan and Other Amino Acids.

Plasma tryptophan (Trp) and other amino acids (AA) can be determined rapidly by gas (GC) or liquid (LC) chromatography using the Phenomenex EZ:Faast™ family of kits. Three kits are available: (1) GC-FID or -NPD, (2) GC-MS, and (3) LC-MS. The two GC kits can determine 32 AA, whereas the LC-MS can determine 5 additional AA. All three kits, however, share the same experimental procedure up to and including the preparation of derivatized AA. The method is based on solid-phase extraction (SPE), thus saving time on prior removal of plasma or other proteins and interfering substances and can be applied to other body fluids and experimental media and to supernatants of extracts of solid material. Briefly, SPE is performed using a proprietary cation-exchange mechanism. The acid solution of the internal standard ensures that the free amino acids are in an anionic form suitable for cationic binding. The alkaline nature of the elution medium ensures that the AA cations are released prior to derivatization. The latter involves production of chloroformate derivatives of both the amino and carboxylic acid groups. With experience, six plasma samples can be so processed within 12 min. The shortest analytical run is <7 min per sample using the GC-FID/NPD kit. Despite its many steps, the procedure becomes second nature and an enjoyable task. I have now used the GC-FID kit with manual injection to process >1600 plasma and other samples. Limit of detection of AA is 1 µM or less. The procedure has been validated and optimized for Trp and its main five brain uptake competitors.

Novel on-column and inverted operating modes of a microcounter-current flame ionization detector.

Improved operating modes of a microcounter-current flame ionization detector (microFID) are demonstrated. By operating the flame inside the end of a capillary gas chromatography (GC) column, the effective cell volume enclosing the flame is considerably reduced and results in significantly lower gas flows being required to produce optimal sensitivity from the stable flame. For instance, in this mode the tiny counter-current flame is situated "upside down" inside the column on the end of a stainless steel capillary delivering 4mL/min of oxygen and is stabilized by a counter flow of only 10mL/min of hydrogen carrier gas. Under these approximately fourfold reduced gas flow conditions, the microFID carbon response is linear over almost 5 orders of magnitude and yields a detection limit of 6x10(-10)gC/s. These figures agree well with those reported for the original microFID, which also similarly operated under hydrogen-rich conditions. To better simulate the oxygen-rich environment of a conventional FID flame, a novel "inverted" counter-current flow mode was also investigated. In this post-column microFID arrangement, a very lean flame is now situated on the end of a stainless steel capillary delivering 10mL/min of hydrogen, which is opposed by a counter-current flow of only 20mL/min of oxygen. The microFID detection limit obtained in this stable, oxygen-rich counter-current flame mode is 7x10(-11)gC/s with a response that is linear over almost 6 orders of magnitude. These findings are more comparable to those of a conventional FID. Overall, the low-flow sensitive microFID operating modes presented demonstrate that this detector may be potentially useful for adaptation to portable devices and related GC applications.

Determination of radix ginseng volatile oils at different ages by comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry.

Comprehensive 2-D GC (GC x GC) coupled with TOF MS or flame ionization detector (FID) was employed to characterize and quantify the chemical composition of volatile oil in the radixes of Panax ginseng C. A. Mey. (ginseng) at different ages. Thirty-six terpenoids were tentatively identified based on the MS library search and retention index in a ginseng sample at the age of 3 years. An obvious group-type separation was obtained in the GC x GC-TOF MS chromatogram. The data collected by GC x GC-FID were processed using a principal component analysis (PCA) method to classify the samples at different ages. The compounds responsible for the significant differentiation among samples were defined. It was found that the relative abundances of alpha-cadinol, alpha-bisabolol, thujopsene, and n-hexadecanoic acid significantly rise with the increase in age.

Measurement of residual solvents in a drug substance by a purge-and-trap method.

The purge-and-trap (P&T) gas extraction method combined with gas chromatography was studied for its suitability for quantitative residual solvents determination in a water-soluble active pharmaceutical ingredient (API). Some analytical method performance characteristics were investigated, namely, the repeatability, the accuracy and the detection limit of determination. The results show that the P&T technique is--as expected--more sensitive than the static headspace, thus it can be used for the determination of residual solvents pertaining to the ICH Class 1 group. It was found that it could be an alternative sample preparation method besides the static headspace (HS) method.

Fatty acid profile of milk from Saanen and Swedish Landrace goats.

Recent years have had an increased demand for goat milk and its products. The quality of goat milk is determined, in part, by the fatty acid (FA) profile, but there is little information about breed influence on the FA profile of goat milk. The aim of this study was to describe and compare FA profiles of goat milk produced by Saanen and Swedish Landrace breeds. FA profiles were analysed by gas chromatography with a flame ionisation detector using 100 m capillary column coated with ionic liquids of extreme polarity (SLB-IL111). The amounts of 19 FAs were measured. Analyses indicated that FA profile in the milk of Saanen goats differs from that of Swedish Landrace goats with the first having higher proportions of most SFA-s and the second having lower proportions of C16:0, C16:1 and C18:1. This knowledge enables the improvement of the quality of goat milk and goat milk-derived products.

High-throughput quantification of sodium saccharin in foods by ambient flame ionization mass spectrometry.

Ambient flame ionization (AFI) coupled with triple quadrupole tandem mass spectrometry was developed for quantitative analysis of sodium saccharin (SAC) in various food samples. Typically, the micro-flame by the combustion of n-butane provided a heating zone for fast desorption and ionization of analytes in milliseconds. Then high ion abundance of analyte could be produced in a short time, which made AFI-MS possess a very high sensitivity for SAC detection and was particularly appropriate for the quantification in multiple reaction monitor (MRM) mode. Liquid samples were introduced into outer flame using dip-it tips in order to facilitate a rapid and high-throughput analysis. Saccharin-d4 was used as the internal standard to compensate for the variations of the ion intensities. With a minimal sample preparation, a linear range of 4-100 µg/mL was developed with all linear relationships of different matrices (including coke, juice, liquors, sunflower seeds and sweetmeats) greater than 0.992. Recoveries for coke and apple matrices were ranged from 88.4% to 108.9% at the concentration of 5, 20, 80 µg/mL and the limits of detection (LODs) were in the range of 0.12-0.21 µg/mL. Furthermore, the feasibility of this method was exhibited by the quantification analysis of SAC in seventeen real samples. These results indicated that AFI-MS was a valuable strategy for rapid screening detection and precise quantification analysis of SAC in food.

A comparison study on a sulfonated graphene-polyaniline nanocomposite coated fiber for analysis of nicotine in solid samples through the traditional and vacuum-assisted HS-SPME.

A simple, rapid, and reliable headspace solid-phase microextraction (HS-SPME) procedure, reinforced by applying vacuum in the extraction vial, was developed. It was applied for the extraction of nicotine in solid samples prior to determination by gas chromatography-flame ionization detection (GC-FID). First, the surface of a narrow stainless steel wire was made porous and adhesive by platinization to obtain a durable, higher surface area, and resistant fiber. Then, a thin film of sulfonated graphene/polyaniline (Sulf-G/PANI) nanocomposite was synthesized and simultaneously coated on the platinized fiber using the electrophoretic deposition (EPD) method. It was demonstrated that the extraction efficiency remarkably increased by applying the reduced-pressure condition in the extraction vial. To evaluate the conventional HS-SPME and vacuum-assisted HS-SPME (VA-HS-SPME) platforms, all experimental parameters affecting the extraction efficiency including desorption time and temperature, extraction time and temperature and moisture content of sample matrix were optimized. The highest extraction efficiency was obtained at 60°C, 10min (extraction temperature and time) and 280°C, 2min (desorption condition), for VA-HS-SPME strategy, while for conventional HS-SPME the extraction and desorption conditions found to be 100°C, 30min and 280°C, 2min, respectively. The Sulf-G/PANI coated fiber showed high thermal stability, good chemical/mechanical resistance, and long lifetime. For analysis of nicotine in solid samples using VA-HS-SPME-GC-FID, linear dynamic range (LDR) was 0.01-30µgg-1 (R2=0.996), the relative standard deviation (RSD%, n=6), for analyses of 1µgg-1 nicotine was calculated 3.4% and limit of detection (LOD) found to be 0.002µgg-1. The VA-HS-SPME-GC-FID strategy was successfully carried out for quantitation of nicotine in hair and tobacco real samples.