Chromatography-mass Spectrometry-selected Ion Monitoring Research Articles

Gas chromatography-mass spectrometry selected ion monitoring and gas chromatography-tandem mass spectrometry selected reaction monitoring analyses of mono-, di- and tri-unsaturated C25 highly branched isoprenoid alkene biomarkers in sea ice and sediment samples: A comparative study.

The efficiency of selected ion monitoring (SIM) and selected reaction monitoring (SRM) analyses for the quantification of three mono-, di- and tri-unsaturated highly branched isoprenoid (HBI) alkenes (IP25 , IPSO25 and HBI III, respectively), often used as proxies for the occurrence of Arctic and Antarctic sea ice or the adjacent open waters, was compared. Gas chromatography (GC)-mass spectrometry (MS)/SIM and GC/MS/MS/SRM analyses were carried out on dilute solutions made from purified standards of these three HBIs, and then on hydrocarbon fractions of several sediment and sea ice sample extracts. More efficient and specific SRM transitions were selected after collision-induced dissociation of each precursor ion at different collision energies. SRM analysis avoided any overestimation of IP25 resulting from the contribution of the coeluting 13 C mass isotopomer of IPSO25 (M+ ˙ + 2) to the SIM target ion. In contrast, SRM analysis is less reliable for IPSO25 quantification in cases where several regio-isomers are present, likely due to intense double bond migrations following electron impact. In the case of HBI III, SRM analysis constitutes a potentially suitable alternative to SIM analysis, especially in terms of improving limit of detection. Despite the intense migrations of HBI double bonds under electron ionization, the selected SRM transitions should be more suitable than SIM target ions for IP25 and HBI III quantification in complex hydrocarbon fractions of natural samples. However, the advantage is less evident for IPSO25 due to the presence of numerous regio-isomers.

Exposure assessment of polycyclic aromatic hydrocarbons in refined coal tar sealant applications.

Refined coal tar sealant (RCTS) emulsions are used to seal the surface of asphalt pavement. Nine of the 22 polycyclic aromatic hydrocarbons (PAHs) evaluated in this study are classified as known, probable, or possible human carcinogens. Exposure assessment research for RCTS workers has not been published previously. The overall objective of this study was to develop a representative occupational exposure assessment of PAH exposure for RCTS workers based on worksite surveys. The specific aims were to: 1) quantify full-shift airborne occupational exposures to PAHs among RCTS workers; 2) quantify workers' dermal exposures to PAHs; 3) quantify biomarkers of PAH exposure in workers' urine; 4) identify specific job titles associated with RCTS exposure; and 5) apply these results to a biological exposure index to assess risk of potential genotoxicity from occupational exposures. A total of twenty-one RCTS workers were recruited from three companies. Personal and area air samples were collected using a modification of NIOSH Method 5515. Dermal exposure was assessed by hand and neck wipes before and after shifts. Twenty-two PAHs were quantified via gas chromatography-mass spectrometry selected ion monitoring. Internal dose was estimated by quantifying select PAH metabolites in pre- and post-shift urine samples using on-line solid phase extraction-high performance liquid chromatography-tandem mass spectrometry. PAH levels in the worker breathing zones were highest for naphthalene, acenaphthene, and phenanthrene, with geometric means of 52.1, 11.4, and 9.8μg/m3, respectively. Hand wipe levels of phenanthrene, fluoranthene and pyrene were the highest among the 22 PAHs with geometric means of 7.9, 7.7, and 5.5μg/cm2, respectively. Urinary PAH biomarkers for naphthalene, fluorene, phenanthrene, and pyrene were detected in all workers and were higher for post-shift samples than those collected pre-shift. Urinary concentrations of the metabolite 1-hydroxypyrene were greater than the American Conference of Governmental Industrial Hygienists (ACGIH) Biological Exposure Index (BEI) for this metabolite in 89 percent of post-shift samples collected on the final day of the work week or field survey. Statistically significances were found between concentrations of fluorene, naphthalene, and phenanthrene in the breathing zone of workers and their corresponding urinary PAH biomarkers. Workers were placed in two work place exposure groups: applicators and non-applicators. Applicators had higher total PAH concentrations in personal breathing zone (PBZ) air samples than non-applicators and were more likely to have post-shift hand wipe concentrations significantly higher than pre-shift concentrations. Concentrations of post-shift urinary biomarkers were higher, albeit not significantly, for applicators than non-applicators. The exposure results from RCTS worker samples cannot be explained by proximal factors such as nearby restaurants or construction. Air and skin concentration levels were substantially higher for RCTS workers than previously published levels among asphalt workers for all PAHs. PAH profiles on skin wipes were more consistent with RCTS sealant product than air samples. Last day post-shift urinary concentrations of 1-hydroxypyrene greatly exceeded the ACGIH BEI benchmark of 2.5μg/L in 25 of 26 samples, which suggests occupational exposure and risk of genotoxicity. When pyrene and benzo[a]pyrene were both detected, concentration ratios from personal exposure samples were used to calculate the adjusted BEI. Concentrations of 1-hydroxypyrene exceeded the adjusted BEIs for air, hand wipes, and neck wipes in most cases. These results indicate the need to increase safety controls and exposure mitigation for RCTS workers.

Multi-Residual Determination of Multi-Class Pesticides in Groundwater by Direct Immersion Solid-Phase Microextraction with Gas Chromatography-Selected Ion Monitoring Mass Spectrometry (GC–MS/SIM) Detection

Multi-Residual Determination of Multi-Class Pesticides in Groundwater by Direct Immersion Solid-Phase Microextraction with Gas Chromatography-Selected Ion Monitoring Mass Spectrometry (GC–MS/SIM) Detection

Determination of 24 Pesticides Residues in Leather Products by Solid-Phase Microextraction Coupled with Gas Chromatography–Mass Spectrometry

Considering the high content of oil and complex residual additives in leather samples, a new analytical method based on the solid-phase extraction technique and gas chromatography-selected ion monitoring mass spectrometry (GC-SIM-MS) was developed to determine 24 organic compounds involving the organochlorine pesticides (OCPs), organophosphorous pesticides (OPPs) and pyrethroids pesticides residues in leather. The extraction conditions (such as the extraction solution, purification procedure and solid-phase extraction column) were optimized using the positive leather samples based on the recovery rates of the pesticides. The best extraction solution, solid-phase extraction column and chromatography column were n-hexane and ethyl acetate (1+1, volume) mixed solution, Carb-PSA (1.0 g, 6mL) and DB-1701 (length: 30 m, inside diameter: 0.25 mm, film thickness: 0.25 μm). The optimized extraction time and temperature were 20 min and 25°C, respectively. The detection limits of 24 pesticide residues range from 0.05 to 0.10 mg/kg, and the recoveries range from 74% to 116%. The relative standard deviations (RSD, n=6) range from 5.42% to 12.00%. The developed method presented a simple, rapid, sensitive, and inexpensive method to detect 24 pesticides in skin and leather and was successfully applied to the detect them in leather products (cowhide, sheep leather and pig leather).

A Selective Gas Chromatography-Selected Ion Monitoring Mass Spectrometry (GC‑MS/SIM) Method to Determine Polychlorinated Biphenyls in Fuller's Earth

Polychlorinated biphenyls (PCBs) are carcinogenic to humans and can be found in Fuller’s earth used for the regeneration of used transformer oil. This work describes a highly selective gas chromatographic-selected ion monitoring mass spectrometric method (GC-MS/SIM) for the simultaneous determination of three Aroclor PCB congener mixtures (1242, 1254, and 1260). The separation of the PCBs could be systematically varied by changing the temperature conditions of the chromatographic column. Methods were first selected from the literature, and the resolution of the PCBs was then optimized by adjusting k* (effective retention), a (selectivity), and N (column efficiency), enabling the detection of 83 PCBs in 35 min. The extract of PCBs from Fuller’s earth was obtained by ultrasonication (employing hexane and acetone as solvents) and cleanup with Florisil and was concentrated to about 500 µL. The ions analyzed in SIM mode were divided into groups according to the similarity (m/z) of the PCB fragmentation profiles. The limits of detection (LOD) and quantification (LOQ) were 0.19 and 0.64 µg kg-1, respectively. The method developed was used to determine the PCBs present in Fuller’s earth provided by an electric power company. The mean total PCBs concentration was found 1.9 µg kg-1 in Fuller’s earth.

Simultaneous analysis of eight benzodiazepines in blood and urine matrix by gas chromatography–mass spectrometry: Implications for air crash investigation

Introduction: Benzodiazepines are the most commonly prescribed class of drugs in India and are capable of impairing the performance of an aviator in therapeutic to subtherapeutic levels. Detection of benzodiazepines, particularly in blood, is not easy, since the concentrations present, especially following prescribed medical use, can be very low. Several publications have addressed estimation of benzodiazepines in plasma or serum; however, few have attempted their detection in whole blood. Urine, although a better specimen, benzodiazepines due to their extensive metabolism, its metabolites are excreted in urine instead of the parent compounds. Materials and Methods: In our laboratory, a method was developed for simultaneous detection and quantification of eight benzodiazepines in whole blood and urine matrix by gas chromatography–mass spectrometry selective ion monitoring (SIM) method. Chromatographic separation was optimized and achieved for separation of all 8 compounds using Agilent DB-5MS column. Retention time, selectivity and sensitivity were achieved by measuring each analyte in SIM mode. The developed method was tested and validated on actual biological samples for lorazepam, temazepam, diazepam, clonazepam, and nitrazepam. Conclusion: A single method was developed for the detection and quantification of eight benzodiazepines in whole blood and urine matrix by GC–MS SIM method. The method was also tested on limited number of actual biological samples for the lorazepam, temazepam, diazepam, clonazepam, and nitrazepam.

Determination of Acrylamide using Solid-phase Extraction and Gas Chromatography-Mass Spectrometry Selected Ion Monitoring in Roasted Cereal Foods

An analytical method has been developed for the determination of acrylamide in cereal-based foods. The method is based on extraction of acrylamide with water, cleaned up through a solid phase extraction (SPE) cartridge, followed by bromination of the acrylamide double bond. The reaction product (2,3-dibromopropionamide) is extracted with ethyl acetate and analyzed by gas chromatography coupled to mass spectrometry (GC/MS) in selected ion monitoring (SIM), employing acrylamide-2,3,3-d3 (AA-d3) as internal standard. The SPE process for sample preconcentration was performed on a commercially available SupelcleanTM Coconut Charcoal cartridge packed with active carbon. In this method, correlation coefficients (R2) of calibration curves appeared to be 0.9997 in the concentration range of 2.5~100 μg/kg. The method detection limit (MDL) and the limit of quantitation (LOQ) of acrylamide were 0.76 μg/kg and 2.32 μg/kg, respectively. The calibration curve showed good linearity above 0.9997 in the ranges of the 2.5~100 μg/kg. For validation purposes, recovery studies were carried out at one concentration levels (10 μg/kg, n=3). The intraday assay accuracy and precision were in the range 104% and 5.6%, respectively, and the interday assay accuracy and precision were in the range 95.8% and 5.3%, respectively. Key words: acrylamide, bromination, GC/MS, SPE, limit of quantitation (LOQ)

Determination of cembrenediols in tobacco by gas chromatography—mass spectrometry-selected ion monitoring with precolumn derivatization

An efficient and sensitive analytical method based on precolumn derivatization and gas chromatography—mass spectrometry—selected ion monitoring (GC—MS—SIM) was proposed and validated for analysis of two cembrenediols (CBDs) which are α-cembrenediol and β-cembrenediol in tobacco samples. CBDs in tobacco samples were extracted by sonication with 50 mL dichloromethane for 10 min before derivatized with 2:3 (v/v) bis(trimethylsilyl)trifluoroacetamide (BSTFA)—pyridine at 20 °C for 100 min. CBDs’ level in tobacco samples was analyzed by GC—MS—SIM and quantified by the internal standard method. The linear range for α-CBD and β-CBD was 13.6–554.6 μg mL−1 and 4.11–162.6 μg mL−1, and the correlation coefficients of both were 0.9998. The limit of detection (LOD) and limit of quantification (LOQ) of α-cembrenediol and β-cembrenediol were 0.40 μg g−1 and 1.34 μg g−1, and 0.27 μg g−1 and 0.90 μg g−1, respectively. Average recoveries of α-CBD and β-CBD were 94.4–99.9% and 91.9–98.2% while the relative standard deviations (RSDs, n = 5) were ranged from 2.67 to 5.6% and 2.04 to 4.22%, respectively. This proposed analytical method has been successfully applied to analyze CBDs in tobacco samples.

The significance of petroleum bitumen in ancient Egyptian mummies.

Mummification was practised in ancient Egypt for more than 3000 years, emerging from initial observations of buried bodies preserved by natural desiccation. The use of organic balms (and other funerary practices) was a later introduction necessitated by more humid burial environments, especially tombs. The dark colour of many mummies led to the assumption that petroleum bitumen (or natural asphalt) was ubiquitous in mummification; however, this has been questioned for more than 100 years. We test this by investigating 91 materials comprising balms, tissues and textiles from 39 mummies dating from ca 3200 BC to AD 395. Targeted petroleum bitumen biomarker (steranes and hopanes) analyses by gas chromatography-mass spectrometry selected ion monitoring (GC-MS SIM, m/z 217 and 191) showed no detectable bitumen use before the New Kingdom (ca 1550–1070 BC). However, bitumen was used in 50% of New Kingdom to Late Period mummies, rising to 87% of Ptolemaic/Roman Period mummies. Quantitative determinations using 14C analyses reveal that even at peak use balms were never more than 45% w/w bitumen. Critically, the dark colour of balms can be simulated by heating/ageing mixtures of fats, resins and beeswax known to be used in balms. The application of black/dark brown balms to bodies was deliberate after the New Kingdom reflecting changing funerary beliefs and shifts in religious ideology.This article is part of the themed issue ‘Quantitative mass spectrometry’.

Validation of an isotope dilution gas chromatography–mass spectrometry method for combined analysis of oxysterols and oxyphytosterols in serum samples

We describe the validation of a method for the analysis of oxysterols, i.e. oxycholesterols and oxyphytosterols, in human serum using gas chromatography–mass spectrometry selected ion monitoring (GC–MS-SIM). Concentrations of 7α- and 7β-hydroxy-, and 7oxo-cholesterol, -campesterol, and -sitosterol as well as 4β-hydroxycholesterol and side-chain oxygenated 24S-, 25-, and 27-hydroxycholesterol were determined by isotope dilution methodology.After saponification at room temperature the oxysterols were extracted, separated from their substrates, cholesterol, campesterol, and sitosterol, by solid phase extraction, and subsequently derivatised to their corresponding trimethylsilyl-ethers prior to GC–MS-SIM. In order to prevent artificial autoxidation butylated hydroxytoluene and ethylenediaminetetraacetic acid were added. The validation of the method was performed according to the International Conference on Harmonisation guidance, including limits of detection and quantification, ranges, recovery and precision. Due to improved instrumental settings and work-up procedure, limits of detection and quantification ranged between 8.0–202.0pg/mL and 28.0–674pg/mL, respectively. Recovery data in five calibration points varied between 91.9% and 116.8% and in serum samples between 93.1% and 118.1%. The mean coefficient of variation (CV) for the recovery of all compounds was <10%. Well satisfying CVs for within-day precision (2.1–10.8%) and for between-day precision (2.3–12.1%) were obtained. More than 20 samples could be processed in a single routine day and test series of about 300 samples can be realised without impairment of the validation parameters during a sequence. Comparison of oxysterol and oxyphytosterol content in serum and plasma revealed no difference.A fully validated isotope dilution methodology for the quantification of oxycholesterols and oxyphytosterols from human serum or plasma is presented.

Exploring metabolic syndrome serum free fatty acid profiles based on GC–SIM–MS combined with random forests and canonical correlation analysis

Metabolic syndrome (MetS) is a cluster of metabolic abnormalities associated with an increased risk of developing cardiovascular diseases or type II diabetes. Till now, the etiology of MetS is complex and still unknown. Metabolic profiling is a powerful tool for exploring metabolic perturbations and potential biomarkers, thus may shed light on the pathophysiological mechanism of diseases. In this study, fatty acid profiling was employed to exploit the metabolic disturbances and discover potential biomarkers of MetS. Fatty acid profiles of serum samples from metabolic syndrome patients and healthy controls were first analyzed by gas chromatography–selected ion monitoring–mass spectrometry (GC–SIM–MS), a robust method for quantitation of fatty acids. Then, the supervised multivariate statistical method of random forests (RF) was used to establish a classification and prediction model for MetS, which could assist the diagnosis of MetS. Furthermore, canonical correlation analysis (CCA) was employed to investigate the relationships between free fatty acids (FFAs) and clinical parameters. As a result, several FFAs, including C16:1n-9c, C20:1n-9c and C22:4n-6c, were identified as potential biomarkers of MetS. The results also indicated that high density lipoprotein-cholesterol (HDL-C), triglycerides (TG) and fasting blood glucose (FBG) were the most important parameters which were closely correlated with FFAs disturbances of MetS, thus they should be paid more attention in clinical practice for monitoring FFAs disturbances of MetS than waist circumference (WC) and systolic blood pressure/diastolic blood pressure (SBP/DBP). The results have demonstrated that metabolic profiling by GC–SIM–MS combined with RF and CCA may be a useful tool for discovering the perturbations of serum FFAs and possible biomarkers for MetS.

Authentication of pineapple (Ananas comosus [L.] Merr.) fruit maturity stages by quantitative analysis of γ- and δ-lactones using headspace solid-phase microextraction and chirospecific gas chromatography–selected ion monitoring mass spectrometry (HS-SPME–GC–SIM-MS)

Headspace solid phase microextraction and chirospecific gas chromatography–mass spectrometry in selected ion monitoring mode (HS-SPME–GC–SIM-MS) allowed quantitative determination of δ-lactones (δ-C8, δ-C10) and γ-lactones (γ-C6, γ-C8, γ-C10). A stable isotope dilution assay (SIDA) with d7-γ-decalactone as internal standard was used for quantitative analysis of pineapple lactones that was performed at three progressing post-harvest stages of fully ripe air-freighted and green-ripe sea-freighted fruits, covering the relevant shelf-life of the fruits. Fresh pineapples harvested at full maturity were characterised by γ-C6 of high enantiomeric purity remaining stable during the whole post-harvest period. In contrast, the enantiomeric purity of γ-C6 significantly decreased during post-harvest storage of sea-freighted pineapples. The biogenetical background and the potential of chirospecific analysis of lactones for authentication and quality evaluation of fresh pineapple fruits are discussed.

Metal-tag labeling coupled with high performance liquid chromatography-selected ion monitoring mass spectrometry for absolute quantitation of proteins

A novel method has been established based on metal element chelated tags coupled with high performance liquid chromatography-selected ion monitoring mass spectrometry (HPLC-SIM/MS). The labeling efficiency and stability of metal element chelated tags, the chromatographic retention behavior and MS behavior of the labeled peptides, the linear range and accuracy of this method were examined. The results showed that the metal element chelated tag method has high labeling efficiency and high labeling stability, and the labeled peptides with different kinds of metal tags have consistent chromatographic retention behavior. The method of metal tags coupled with HPLC-SIM/MS has high sensitivity with the limit of quantification (LOQ) up to 1 fmol. The linear range for the method was between 1 fmol to 500 fmol with R2 > 0.99, which means the method has a good linearity. Moreover, this method had an average recovery of 117.01%. The method was used in the absolute quantitation of a protein enolase in Thermoanaerobacter tengcongensis (TTE) with a relative standard deviation of 5.74%, which means high precision. All the results showed that this method is accurate and reliable for the absolute quantitation of proteins. This gives us an alternative for the quantitative determination of proteins in relatively simple biological samples.

Cork as a new (green) coating for solid-phase microextraction: Determination of polycyclic aromatic hydrocarbons in water samples by gas chromatography–mass spectrometry

A new fiber for solid-phase microextraction (SPME) was prepared employing cork as a coating. The morphology and composition of the cork fiber was evaluated by scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR), respectively. The proposed fiber was used for the determination of polycyclic aromatic hydrocarbons (PAHs) in river water samples by gas chromatography–selected ion monitoring–mass spectrometry (GC–SIM–MS). A central composite design was used for optimization of the variables involved in the extraction of PAHs from water samples. The optimal extraction conditions were extraction time and temperature of 60min and 80°C, respectively. The detection and quantification limits were 0.03 and 0.1μgL−1, respectively. The recovery values were between 70.2 and 103.2% and the RSD was ≤15.7 (n=3). The linear range was 0.1–10μgL−1 with r≥0.96 and the fiber-to-fiber reproducibility showed RSD≤18.6% (n=5). The efficiency of the cork fiber was compared with commercially available fibers and good results were achieved, demonstrating the applicability and great potential of cork as a coating for SPME.

Morphactin Substantially Induced the Fourth Internode Growth in Decapitated Tulips: Relevance to Endogenous Levels of Indole-3-Acetic Acid

Abstract Important role of auxin polar transport inhibitors such as TIBA (2,3,5- triiodobenzoic acid), NPA (N-(1-naphthyl)phthalamic acid) and morphactin (methyl 2-chloro-9-hydroxyfluorene-9-carboxylate) in stem growth of tulips was intensively studied. After the application of morphactin above the 3rd node of decapitated tulips, the stimulated growth of the 4th internode was clearly observed. On the other hand, NPA and TIBA were slightly effective in stimulating the 4th internode growth of tulips. Endogenous levels of IAA in the 4th internode after the treatment of auxin polar transport inhibitors were determined using gas-liquid chromatography-mass spectrometry (GC-MS) and gas-liquid chromatography-mass spectrometry selected ion monitoring system (GC-SIM) with deuterium labelled IAA (Indole-2,4,5,6,7-d5-3- acetic acid, d5-IAA) as an internal standard, resulted in significant accumulation of IAA in the 4th internode of tulips after the treatment of morphactin. In the treatment of NPA and TIBA, there was almost no such an accumulation of IAA. These results strongly suggest that stimulated growth of the 4th internode of tulips induced by the application of morphactin is the consequence of accumulated endogenous levels of IAA after the treatment. The possible mode of action of auxin polar transport inhibitors in tulip stem growth is also discussed.

Determination of siloxanes in silicone products and potential migration to milk, formula and liquid simulants

A pressurised solvent extraction procedure coupled with a gas chromatography–mass spectrometry–selective ion monitoring (GC–MS–SIM) method was developed to determine three cyclic siloxanes, octamethylcyclotetrasiloxane (D4), decamethylcyclopentasiloxane (D5), dodecamethylcyclohexasiloxane (D6) and three linear siloxanes, octamethyltrisiloxane (L3), decamethyltetrasiloxane (L4), dodecamethylpentasiloxane (L5), in silicone products. Additionally, two different extraction methods were developed to measure these siloxanes migrating into milk, infant formula and liquid simulants (50 and 95% ethanol in water). The limits of quantification (LOQs) of the six siloxanes ranged from 6 ng/g (L3) to 15 ng/g (D6). Silicone nipples and silicone bakewares were extracted using pressurised solvent extraction (PSE) and analysed using the GC–MS–SIM method. No linear siloxanes were detected in the silicone nipple samples analysed. The three cyclic siloxanes (D4, D5 and D6) were detected in all silicone nipple samples with concentrations ranging from 0.5 to 269 µg/g. In the bakeware samples, except for L3, the other five siloxanes were detected with concentrations ranging from 0.2 µg/g (L4) to 7030 µg/g (D6). To investigate the potential migration of the six siloxanes from silicone nipples to milk and infant formula, a liquid extraction and dispersive clean-up procedure was developed for the two matrices. The procedure used a mix of hexane and ethyl acetate (1 : 1, v/v) as extraction solvent and C18 powder as the dispersive clean-up sorbent. For the liquid simulants, extraction of the siloxanes was achieved using hexane without any salting out or clean-up procedures. The recoveries of the six siloxanes from the milk, infant formula and simulants fortified at 50, 100, 200, 500 and 1000 µg/l ranged from 70 to 120% with a relative standard derivation (RSD) of less than 15% (n = 4). Migration tests were performed by exposing milk, infant formula and the liquid simulants to silicone baking sheets with known concentrations of the six siloxanes at 40°C. No siloxanes were detected in milk or infant formula after 6 h of direct contact with the silicone baking sheet plaques, indicating insignificant migration of the siloxanes to milk or infant formula. Migration tests in the two simulants lasted up to 72 h and the three cyclic siloxanes were detected in 50% ethanol after an 8-h exposure and after 2 h in 95% ethanol. The highest detected concentrations of D4, D5 and D6 were 42, 36 and 155 ng/ml, respectively, indicating very limited migration of D4, D5 or D6 into the two simulants.

Metabolite target analysis of isoprenoid pathway in Saccharomyces cerevisiae in response to genetic modification by GC-SIM-MS coupled with chemometrics

A highly selective, sensitive and nonradioactive analytical method for identification and quantification of intracellular metabolites involved in isoprenoid pathway has been developed by means of gas chromatography-selected ion-monitoring mass spectrometry (GC-SIM-MS). These metabolites are classified into two groups: sterols (squalene, ergosterol, lanosterol) and phosphorylated compounds (geranyl diphosphate, farnesyl pyrophosphate, geranylgeranyl pyrophosphate) based on their physicochemical properties. To quantify both groups in a single analytical run, GPP, FPP and GGPP were cleaved to the parent alcohols, geraniol, farnesol, geranylgeraniol by pyrophosphatase followed by alkaline phosphatase before extraction, separation and detection. This study evaluated several extraction procedures and determined the effects of the type of extraction solvent, times used for extraction. Under optimized GC/EI-MS conditions, six compounds were separated with high efficiency in the selected-ion monitoring (SIM) mode. Linearity of the method was good with correlation coefficients (r 2) in the range of 0.9953–0.9999 and detection limits were 1.53–151.88 ng/ml. The intra-day and inter-day precision of the method, as RSD, were less than 5.31 and 6.04%, respectively. The accuracy of six compounds varied between 87.7 and 110.8%. This assay was successfully applied to the determination of six major metabolites in the pathway for isoprenoid biosynthesis in S. cerevisiae and is sensitive to detect changes following genetic modification. By isolating statistically significant differences among metabolite levels from four biological conditions, we observed discriminatory metabolic features that hinted that the role of erg9 and coq1gene was involved in isoprenoid pathway. Integrating this analytical approach with statistical strategies, we can determine the influence of erg9 and coq1gene on isoprenoid levels of S. cerevisiae, thus leading to improved understanding of the pathway in a multitude of biological systems.

The Chardonnay wine olfactory concept revisited: A stable core of volatile compounds, and fuzzy boundaries

An earlier study by the same team showed that Chardonnay wines have common olfactory properties by which wine experts can recognize them. The specific Chardonnay olfactory space was also tentatively linked to the relative concentrations of 29 volatile compounds, regarded as possible aroma-impact compounds. The question now is whether or not those initial results hold independently of the sample under consideration, that is, whether these sensory and chemical spaces are vintage-specific. A series of investigations was conducted on a new set of 46 wines (23 Chardonnay wines and 23 non Chardonnay wines) using the same sensory (wine typicality level) and physico-chemical (Gas Chromatography–mass spectrometry–selected ion monitoring: GC–MS–SIM) approaches as in the earlier study. The sensory space of Chardonnay wines has been identified again in this new study. Compared with white wines of other grape varieties, Chardonnay wines do have distinctive and recognizable olfactory characteristics. LSD tests carried out on the chemical data and a PLS analysis predicting wine typicality level from the chemical data showed that the relative concentrations of 35 volatile compounds affected the wines' typicality scores. Of the 35 compounds, 18 were already among the 29 possible aroma-impact compounds identified by the earlier study. These 18 common volatile compounds seem to be vintage-independent and as such might represent the core of the Chardonnay wine olfactory space. The others seem to be specific to the new wines tested and may contribute to the fuzziness of the boundaries of the Chardonnay wine olfactory space. These results emphasize the complexity of the odor quality of Chardonnay wines, with some volatile compounds remaining stable across vintages, while others are specific to enological practices and to vintages.

A gas chromatography–mass spectrometry method to monitor detergents removal from a membrane protein sample

In membrane protein biochemical and structural studies, detergents are used to mimic membrane environment and maintain functional, stable conformation of membrane proteins in the absence of lipid bilayers. However, detergent concentration, esp. molar ratio of membrane protein to detergent is usually unknown. Here, a gas chromatography–mass spectrometry selected ion monitoring (GC–MS-SIM) method was developed to quantify four detergents which are frequently used in membrane protein structural studies. To remove excessive detergents, a filtered centrifugation using Centricon tubes was applied. A membrane protein Ig-Beta fragment in four different detergent micelles was exemplified. Detergent concentrations in the upper and lower fraction of the Centricon tube were measured after each round of centrifugation. The results were very consistent to basic properties of detergent micelles in aqueous solvents. Therefore, coupling of GC–MS-SIM and detergent removal by Centricon tubes, detergents concentration, esp. molar ratio of membrane protein to detergent could be controlled, which will expedite membrane protein structural and biochemical studies.

Effect of elevated nitrogen levels on endogenous gibberellin and jasmonic acid contents of three rice (Oryza sativa L.) cultivars

AbstractThree rice (Oryza sativa L.) cultivars ( cv. Daesanbyeo, cv. Dongjinbyeo, cv. Junambyeo) were analyzed for endogenous gibberellin (GA) and jasmonic acid (JA) contents and their changes in response to elevated nitrogen (N) levels. The N fertilizer was applied in the form of urea [(NH2)2CO] at three rates (0, 36.8, 73.6 kg N ha–1). Plant growth (height and dry weight) was enhanced by the first N rate but not further enhanced by the highest rate. The endogenous GA contents were analyzed through high‐performance liquid chromatography (HPLC) and gas chromatography–mass spectrometry–selected ion monitoring (GC‐MS‐SIM) while that of JA with GC‐MS‐SIM. They were analyzed one week after N application and were significantly increased with elevated N levels in all rice cultivars. The bioactive GA1 markedly increased, but its concentration differed in different rice cultivars. Similar fluctuations were observed for endogenous GA8, GA12, GA19, GA20, and GA53 in response to elevated N levels, showing that the rates of biosynthesis of GAs were differently affected by elevated N levels within different rice cultivars. The level of GA20, a precursor of GA1 biosynthesis, was not significantly increased, though GA19, a precursor of GA20, was found to be the most abundant GA type in all rice cultivars. Jasmonic acid content in the plants increased with the basic urea application (36.8 kg N ha–1), but significantly decreased with the double urea level (73.6 kg N ha–1). The results demonstrate that GA and JA are differentially affected in response to elevated N application in rice.