Gas Chromatography Pyrolysis Isotope Ratio Mass Spectrometry Research Articles

The Effect of Fava Bean (Vicia faba L.) Protein Ingestion on Myofibrillar Protein Synthesis at Rest and after Resistance Exercise in Healthy, Young Men and Women: A Randomised Control Trial.

The aim of the present study was to evaluate the effect of feeding fava bean (Vicia faba L.) protein (FBP) on resting and post-exercise myofibrillar fractional synthetic rate (myoFSR). In a parallel, double-blind, randomised control trial, sixteen young, healthy recreationally active adults (age = 25 (5) years, body mass = 70 (15) kg, stature = 1.72 (0.11) m, mean (SD)) ingested 0.33 g·kg−1 FBP (n = 8) or a negative control (CON, i.e., EAA-free mixture) (n = 8), immediately after a bout of unilateral knee-extensor resistance exercise. Plasma, saliva, and m. vastus lateralis muscle samples were obtained pre-ingestion and 3 h post-ingestion. MyoFSR was calculated via deuterium labelling of myofibrillar-bound alanine, measured by gas chromatography–pyrolysis–isotope ratio mass spectrometry (GC-Pyr-IRMS). Resistance exercise increased myoFSR (p = 0.012). However, ingestion of FBP did not evoke an increase in resting (FBP 29 [−5, 63] vs. CON 12 [−25, 49]%, p = 0.409, mean % change [95% CI]) or post-exercise (FBP 78 [33, 123]% vs. CON 58 [9, 107]%, p = 0.732) myoFSR. Ingestion of 0.33 g·kg−1 of FBP does not appear to enhance resting or post-exercise myoFSR in young, healthy, recreationally active adults.

Accessing the position-specific 18O/16O ratios of lignin monomeric units from higher plants with highly selective hydrogenolysis followed by GC/Py/IRMS analysis

The 18O/16O of lignin at bulk, molecular and positional levels can be used to extract valuable information about climate, plant growth environment, plant physiology, and plant metabolism. Access to the individual oxygen isotope compositions (δ18O) in the lignin monomeric units is, however, challenging as depolymerization of lignin to release the monomeric units may cause isotope fractionation. We have developed a novel method to measure the δ18O of the three oxygens (O-3, O-4 and O-5) attached to the aromatic ring of the monomeric units (bearing no oxygen in their side chains) releasable by highly selective W2C/AC (tungsten carbide supported by activated carbon)-catalyzed hydrogenolysis of lignin. O-4 is obtained by measuring the δ18O of H-type monomeric unit, while O-3 and O-5 can be calculated following isotope mass balance between H, G and S-type monomeric units measurable simultaneously with GC/Py/IRMS (gas chromatography-pyrolysis-isotope ratio mass spectrometry). The measurement precisions are better than 1.15 mUr and 4.15 mUr at molecular and positional levels, respectively. It was shown that there were a δ18OH > δ18OG > δ18OS isotopic order in the herbaceous plant lignin and an (inclusive) opposite order in woody plant lignin. Such differences in isotopic order is likely to be caused by the fact that both L-tyrosine, which carries an 18O-enriched leaf water signal, and L-phenylalanine, which carries mainly a molecular O2 isotopic signal, serve as the precursors for lignin biosynthesis in herbaceous plants while only the latter serves as precursor for lignin biosynthesis in woody plants. We have highlighted the potential application of such molecular and positional levels isotopic signals in plant physiological, metabolic, lignin biosynthetic and climate studies.

The Effect of Whey Protein Supplementation on Myofibrillar Protein Synthesis and Performance Recovery in Resistance-Trained Men.

Background: The aim of this study was to investigate the effect of whey protein supplementation on myofibrillar protein synthesis (myoPS) and muscle recovery over a 7-d period of intensified resistance training (RT). Methods: In a double-blind randomised parallel group design, 16 resistance-trained men aged 18 to 35 years completed a 7-d RT protocol, consisting of three lower-body RT sessions on non-consecutive days. Participants consumed a controlled diet (146 kJ·kg−1·d−1, 1.7 g·kg−1·d−1 protein) with either a whey protein supplement or an isonitrogenous control (0.33 g·kg−1·d−1 protein). To measure myoPS, 400 ml of deuterium oxide (D2O) (70 atom %) was ingested the day prior to starting the study and m. vastus lateralis biopsies were taken before and after RT-intervention. Myofibrillar fractional synthetic rate (myoFSR) was calculated via deuterium labelling of myofibrillar-bound alanine, measured by gas chromatography-pyrolysis-isotope ratio mass spectrometry (GC-Pyr-IRMS). Muscle recovery parameters (i.e., countermovement jump height, isometric-squat force, muscle soreness and serum creatine kinase) were assessed daily. Results: MyoFSR PRE was 1.6 (0.2) %∙d−1 (mean (SD)). Whey protein supplementation had no effect on myoFSR (p = 0.771) or any recovery parameter (p = 0.390–0.989). Conclusions: Over an intense 7-d RT protocol, 0.33 g·kg−1·d−1 of supplemental whey protein does not enhance day-to-day measures of myoPS or postexercise recovery in resistance-trained men.

Differential Stimulation of Post-Exercise Myofibrillar Protein Synthesis in Humans Following Isonitrogenous, Isocaloric Pre-Exercise Feeding.

The aim of this study was to test the effects of two disparate isonitrogenous, isocaloric pre-exercise feeds on deuterium-oxide (D2O) derived measures of myofibrillar protein synthesis (myoPS) in humans. Methods: In a double-blind parallel group design, 22 resistance-trained men aged 18 to 35 years ingested a meal (6 kcal·kg−1, 0.8 g·kg−1 carbohydrate, 0.2 g·kg−1 fat) with 0.33 g·kg−1 nonessential amino acids blend (NEAA) or whey protein (WHEY), prior to resistance exercise (70% 1RM back-squats, 10 reps per set to failure, 25% duty cycle). Biopsies of M. vastus lateralis were obtained pre-ingestion (PRE) and +3 h post-exercise (POST). The myofibrillar fractional synthetic rate (myoFSR) was calculated via deuterium labelling of myofibrillar-bound alanine, measured by gas chromatography–pyrolysis–isotope ratio mass spectrometry (GC-Pyr-IRMS). Data are a mean percentage change (95% CI). Results: There was no discernable change in myoFSR following NEAA (10(−5, 25) %, p = 0.235), whereas an increase in myoFSR was observed after WHEY (28 (13, 43) %, p = 0.003). Conclusions: Measured by a D2O tracer technique, a disparate myoPS response was observed between NEAA and WHEY. Pre-exercise ingestion of whey protein increased post-exercise myoPS, whereas a NEAA blend did not, supporting the use of NEAA as a viable isonitrogenous negative control.

Oxygen isotope analysis of levoglucosan, a tracer of wood burning, in experimental and ambient aerosol samples.

Levoglucosan is formed from cellulose during biomass burning. It is therefore often used as a specific tracer to quantify the contribution of wood burning to the aerosol loading. The stable oxygen isotope composition (δ18 O value) of biomass is determined by the water cycle and varies regionally, and hence the δ18 O value of levoglucosan could help to identify source regions of organic aerosols. After solvent extraction of the organic fraction and concentration steps, a recently developed methylation derivatisation technique was applied on experimental (i.e. controlled wood-burning experiments) and on ambient aerosol samples from Switzerland and Lithuania. The method achieves sufficient compound separation for isotope analysis in atmospheric particulate matter, enabling δ18 O analysis of levoglucosan by gas chromatography/pyrolysis-isotope ratio mass spectrometry (GC/Pyr-IRMS), with a precision better than 1.0 ‰ and an accuracy of 0.3 ‰. The δ18 O value of the levoglucosan released during controlled wood-burning experiments was not significantly different from the cellulose δ18 O values, which implies very little or no isotope fractionation during wood burning under the given conditions. While the δ18 O values of levoglucosan in Swiss samples were as expected for the source region, those in Lithuania were 1-4 ‰ lower than expected. This may be due to differences in vegetation (grass vs wood) or burning conditions (high vs low temperatures). Low oxygen isotope fractionation between cellulose and levoglucosan and clear differences in levoglucosan δ18 O values between the Swiss and Lithuanian ambient samples demonstrate that our new method is useful for source appointment studies on wood-burning-derived aerosols.

A novel methylation derivatization method for δ(18)O analysis of individual carbohydrates by gas chromatography/pyrolysis-isotope ratio mass spectrometry.

The oxygen isotope ratio (δ(18)O) of carbohydrates derived from animals, plants, sediments, and soils provides important information about biochemical and physiological processes, past environmental conditions, and geographical origins, which are otherwise not available. Nowadays, δ(18)O analyses are often performed on carbohydrate bulk material, while compound-specific δ(18)O analyses remain challenging and methods for a wide range of individual carbohydrates are rare. To improve the δ(18)O analysis of individual carbohydrates by gas chromatography/pyrolysis-isotope ratio mass spectrometry (GC/Pyr-IRMS) we developed a new methylation derivatization method. Carbohydrates were fully methylated within 24 h in an easy-to-handle one-pot reaction in acetonitrile, using silver oxide as proton acceptor, methyl iodide as methyl group carrier, and dimethyl sulfide as catalyst. The precision of the method ranged between 0.12 and 1.09‰ for the δ(18)O values of various individual carbohydrates of different classes (mono-, di-, and trisaccharides, alditols), with an accuracy of a similar order of magnitude, despite high variation in peak areas. Based on the δ(18)O values of the main isomers, important monosaccharides such as glucose and fructose could also be precisely analyzed for the first time. We tested the method on standard mixtures, honey samples, and leaf carbohydrates extracted from Pinus sylvestris, showing that the method is also applicable to different carbohydrate mixtures. The new methylation method shows unrivalled accuracy and precision for δ(18)O analysis of various individual carbohydrates; it is fast and easy-to-handle, and may therefore find wide-spread application.

Habitual Myofibrillar Protein Synthesis Is Normal in Patients with Upper GI Cancer Cachexia.

Skeletal muscle wasting and weight loss are characteristic features of cancer cachexia and contribute to impaired function, increased morbidity, and poor tolerance of chemotherapy. This study used a novel technique to measure habitual myofibrillar protein synthesis in patients with cancer compared with healthy controls. An oral heavy water (87.5 g deuterium oxide) tracer was administered as a single dose. Serum samples were taken over the subsequent week followed by a quadriceps muscle biopsy. Deuterium enrichment was measured in body water, serum alanine, and alanine in the myofibrillar component of muscle using gas chromatography-pyrolysis-isotope ratio mass spectrometry and the protein synthesis rate calculated from the rate of tracer incorporation. Net change in muscle mass over the preceding 3 months was calculated from serial CT scans and allowed estimation of protein breakdown. Seven healthy volunteers, 6 weight-stable, and 7 weight-losing (≥5% weight loss) patients undergoing surgery for upper gastrointestinal cancer were recruited. Serial CT scans were available in 10 patients, who lost skeletal muscle mass preoperatively at a rate of 5.6%/100 days. Myofibrillar protein fractional synthetic rate was 0.058%, 0.061%, and 0.073%/hour in controls, weight-stable, and weight-losing patients, respectively. Weight-losing patients had higher synthetic rates than controls (P = 0.03). Contrary to previous studies, there was no evidence of suppression of myofibrillar protein synthesis in patients with cancer cachexia. Our finding implies a small increase in muscle breakdown may account for muscle wasting.

A novel methodological approach for δ18O analysis of sugars using gas chromatography-pyrolysis-isotope ratio mass spectrometry

Although the instrumental coupling of gas chromatography-pyrolysis-isotope ratio mass spectrometry (GC-Py-IRMS) for compound-specific δ18O analysis has been commercially available for more than a decade, this method has been hardly applied so far. Here we present the first GC-Py-IRMS δ18O results for trimethylsilyl-derivatives of plant sap-relevant sugars and a polyalcohol (glucose, fructose, sucrose, raffinose and pinitol). Particularly, we focus on sucrose, which is assimilated in leaves and which is the most important transport sugar in plants and hence of utmost relevance in plant physiology and paleoclimate studies. Replication measurements of sucrose standards and concentration series indicate that the GC-Py-IRMS δ18O measurements are not stable over time and that they are amount (area) dependent. We, therefore, suggest running sample batch replication measurements in alternation with standard concentration series of reference material. This allows for carrying out (i) a drift correction, (ii) a calibration against reference material and (iii) an amount (area) correction. Tests with 18O-enriched water do not provide any evidence for oxygen isotope exchange reactions affecting sucrose and raffinose. We present the first application of GC-Py-IRMS δ18O analysis for sucrose from needle extract (soluble carbohydrate) samples. The obtained δ18Osucrose/ Vienna Standard Mean Ocean Water (VSMOW) values are more positive and vary in a wider range (32.1–40.1 ‰) than the δ18Obulk/ VSMOW values (24.6–27.2 ‰). Furthermore, they are shown to depend on the climate parameters maximum day temperature, relative air humidity and cloud cover. These findings suggest that δ18Osucrose of the investigated needles very sensitively reflects the climatically controlled evaporative 18O enrichment of leaf water and thus highlights the great potential of GC-Py-IRMS δ18Osucrose analysis for plant physiology and paleoclimate studies.

Oxygen isotope ratios (18O/16O) of hemicellulose-derived sugar biomarkers in plants, soils and sediments as paleoclimate proxy I: Insight from a climate chamber experiment

The oxygen isotopic composition of cellulose is a valuable proxy in paleoclimate research. However, its application to sedimentary archives is challenging due to extraction and purification of cellulose. Here we present compound-specific δ18O results of hemicellulose-derived sugar biomarkers determined using gas chromatography–pyrolysis–isotope ratio mass spectrometry, which is a method that overcomes the above-mentioned analytical challenges. The biomarkers were extracted from stem material of different plants (Eucalyptus globulus, Vicia faba and Brassica oleracea) grown in climate chamber experiments under different climatic conditions.The δ18O values of arabinose and xylose range from 31.4‰ to 45.9‰ and from 28.7‰ to 40.8‰, respectively, and correlate highly significantly with each other (R=0.91, p<0.001). Furthermore, δ18Ohemicellulose (mean of arabinose and xylose) correlate highly significantly with δ18Oleaf water (R=0.66, p<0.001) and significantly with modeled δ18Ocellulose (R=0.42, p<0.038), as well as with relative air humidity (R=−0.79, p<0.001) and temperature (R=−0.66, p<0.001). These findings confirm that the hemicellulose-derived sugar biomarkers, like cellulose, reflect the oxygen isotopic composition of plant source water altered by climatically controlled evapotranspirative 18O enrichment of leaf water. While relative air humidity controls most rigorously the evapotranspirative 18O enrichment, the direct temperature effect is less important. However, temperature can indirectly exert influence via plant physiological reactions, namely by influencing the transpiration rate which affects δ18Oleaf water due to the Péclet effect. In a companion paper (Tuthorn et al., this issue) we demonstrate the applicability of the hemicellulose-derived sugar biomarker δ18O method to soils and provide evidence from a climate transect study confirming that relative air humidity exerts the dominant control on evapotranspirative 18O enrichment of leaf water.Finally, we present a conceptual model for the interpretation of δ18Ohemicellulose records and propose that a combined δ18Ohemicellulose and δ2Hn-alkane biomarker approach is promising for disentangling δ18Oprecipitation variability from evapotranspirative 18O enrichment variability in future paleoclimate studies.

Lignin methoxyl hydrogen isotope ratios in a coastal ecosystem

Stable hydrogen isotope ratios of plant lignin methoxyl groups have recently been shown to record the hydrogen isotopic composition of meteoric water. Here we extend this technique towards tracing water source variations across a saltwater to freshwater gradient in a coastal, subtropical forest ecosystem. We measure the hydrogen isotopic composition of xylem water (δDxw) and methoxyl hydrogen (δDmethoxyl) to calculate fractionations for coastal mangrove, buttonwood and hammock tree species in Sugarloaf Key, as well as buttonwoods from Miami, both in Florida, USA. Prior studies of the isotopic composition of cellulose and plant leaf waxes in coastal ecosystems have yielded only a weak correlation to source waters, attributed to leaf water effects. Here we find δDmethoxyl values range from −230‰ to −130‰, across a 40‰ range in δDxw with a regression equation of δDmethoxyl ‰=1.8*δDxw−178‰ (R2=0.48, p<0.0001, n=74). This is comparable within error to the earlier published relationship for terrestrial trees which was defined across a much larger 125‰ isotopic range in precipitation. Analytical precision for measurements of δD values of pure CH3I by gas chromatography–pyrolysis–isotope ratio mass spectrometry (GC–P–IRMS) is σ=6‰ (n=31), which is considerably better than for CH3I liberated through cleavage with HI from lignin with σ=18‰ (n=26). Our results establish that δDmethoxyl can record water sources and salinity incursion in coastal ecosystems, where variations sufficiently exceed method uncertainties (i.e., applications with δD excursions >50‰). For the first time, we also report yields of propyl iodide, which may indicate lignin synthesis of propoxyl groups under salt-stress.

Analytical challenges in the quantitative determination of 2H/1H ratios of methyl iodide

A published method to determine the hydrogen isotope ((2)H/(1)H) ratios of methoxyl groups, cleaved from lignin by hydroiodic acid (HI), calls for the analysis of methyl iodide (CH(3)I). However, analysis of halogenated compounds by gas chromatography/pyrolysis/isotope ratio mass spectrometry (GC/P/IRMS) yields incomplete conversion into H(2) and unwanted HX products post-pyrolysis. We tested analytical capabilities when measuring a pure CH(3) I standard using a GC Isolink pyrolysis furnace at 1420 °C and a DELTA V(Plus) ™ isotope ratio mass spectrometer, both by Thermo Scientific. We added and tested the efficacy of either a secondary reduction furnace containing heated metals or a cold trap to eliminate unwanted HI prior to IRMS analysis for (2)H/(1) H determinations. While 94% of the hydrogen in CH(3) I is converted into H(2) in GC/P/IRMS, 6% yields HI. The use of either a secondary reduction furnace or cold trap can eliminate HI. However, secondary reduction is untenable given changes in reduction/transmission efficiency and (2)H/(1) H drift. A cold trap provides a pragmatic solution: the δ(2)H values of -97 ‰ were measured from injections of 23-57 µg of CH(3)I to a precision of 4.5 ‰ (n = 26), within the error of the offline measurements. We have refined an earlier method to measure the δ(2)H values of CH(3)I by GC/P/IRMS by adding a cold trap to remove HI. This approach should facilitate future experimentation with the measurement of CH(3)I liberated from natural samples for potentially diverse applications including forensic sourcing of wood and tree-wood-based reconstructions of source water.

Headspace analyses of 2H labeling of acetone: Enabling studies of fatty acid oxidation in vivo

We demonstrate that one can measure low levels of 2H labeling (e.g., <0.025% excess 2H) by exchanging hydrogen (deuterium) in water with acetone and subjecting samples to gas chromatography–pyrolysis–isotope ratio mass spectrometry. This analytical method circumvents the need to use typical off-line reduction methods that convert water to hydrogen gas prior to isotope ratio mass spectrometry or the need to purchase extra peripheral devices that would permit the direct analysis of water labeling. This method enables routine measurements of fatty acid oxidation in rodents; that is, one administers a 2H-labeled fatty acid(s) and then quantifies the production of 2H-labeled water.

Isolation of malignant B cells from patients with chronic lymphocytic leukemia (CLL) for analysis of cell proliferation: Validation of a simplified method suitable for multi-center clinical studies

Background Heavy water ( 2H 2O) labelling of DNA enables the measurement of low-level cell proliferation in vivo, using gas chromatography/pyrolysis isotope ratio mass spectrometry (GC/P/IRMS), but the methodology has been too complex for widespread use. Here, we report a simplified method for measuring proliferation of malignant B cells in patients with chronic lymphocytic leukemia (CLL). Design and methods Patients were labelled with 2H 2O for 6 weeks; blood samples were obtained at 0, 3, and 6 weeks during 2H 2O labelling and 9, 12, and 16 weeks thereafter. Bone marrow was sampled at week 6. Phlebotomy was performed at multiple, non-research clinical sites. CLL cells were isolated in a central laboratory, using a novel RosetteSep™-based method; DNA labelling was analyzed by GC/P/IRMS. Results In 26 of 29 patients, CLL cell isolation resulted in ≥95% purity for malignant CD5+ B cells; in one patient, malignant cells expressed marginal levels of CD5, and in two others, further sorting of CD5hi malignant cells was required. Cell yields correlated with white blood cell counts and exceeded GC/P/IRMS requirements (≈10 7 cells) >98% of the time; high-quality DNA labelling data were obtained. RosetteSep isolation achieved adequate CLL cell purity from bone marrow in only 64% of samples, but greatly reduced subsequent sort time for impure samples. Conclusion This method enables clinical studies of CLL cell proliferation outside of research settings, using a shorter 2H 2O intake protocol, a minimal sampling protocol, and centralised sample processing. The CLL cell isolation protocol may also prove useful in other applications. (clinicaltrials.gov identifier: NCT00481858).

Compound‐specific δ18O analyses of neutral sugars in soils using gas chromatography–pyrolysis–isotope ratio mass spectrometry: problems, possible solutions and a first application

Although gas chromatography-pyrolysis-isotope ratio mass spectrometry (GC-Py-IRMS) has allowed us to make online compound-specific delta18O measurements for about the last ten years, this technique has hardly been applied. We tested different pyrolysis reactor designs using standards (vanillin, ethylvanillin, a fatty acid methyl ester and alkanes) in order to optimize the GC-Py-IRMS delta18O measurements. The method was then applied to methylboronic acid (MBA) sugar derivatives (pentoses, 6-deoxyhexoses and hexoses). Plant- and microbial-derived monosaccharides were extracted hydrolytically from litter and topsoils before derivatization. The measured delta18O values of samples and co-analyzed reference material were first drift-corrected by use of regularly discharged pulses of CO reference gas. Secondly, they were corrected for the amount dependence of the delta18O values. Thirdly, the delta18O values were calibrated using the reference material (principle of 'Identical Treatment'), and, finally, a correction was applied by taking the hydrolytically introduced and water-exchangeable oxygen atoms into account. Our results suggest that the delta18O values of plant-derived monosaccharides in litter reflect the climatic conditions of the last year, whereas delta18O values of the respective topsoils reflect the averaged climate signal of the last decades or even centuries. This demonstrates the high potential of the method for palaeoclimate reconstructions.

A rapid and precise method for determination of D/H ratios of plant methoxyl groups

Stable hydrogen isotope ratio measurements of specific plant components are increasingly used in numerous fields of research, including sample origin verification and climate research. A recently suggested method with considerable potential in this context is the D/H isotope ratio (delta(2)H value) analysis of plant matter methoxyl groups. The method entails ether or ester cleavage with hydriodic acid (HI) to form the gaseous compound methyl iodide (CH(3)I) and measurement of the delta(2)H value of this gas. Here we describe a method for the rapid and precise delta(2)H analysis of plant matter methoxyl groups using gas chromatography/pyrolysis/isotope ratio mass spectrometry (GC/P/IRMS). The conditions for sample preparation were investigated for isotope discrimination effects, the GC conditions were optimized, the reproducibility of the measurement of standards was studied, and the precision of the method was defined. The reproducibility of delta(2)H values determined for a CH(3)I standard on 20 consecutive measurements was found to be 2 per thousand. The method was also tested on four methoxyl-rich plant components: vanillin, lignin, wood and pectin. The analytical precision obtained, when expressed as the average standard deviation for these compounds, was better than 1.6 per thousand. The described method is rapid, allowing preparation and analysis of a sample within 1 h, and produces accurate and reproducible isotopic measurements.

Measurement of very low rates of cell proliferation by heavy water labeling of DNA and gas chromatography/pyrolysis/isotope ratio–mass spectrometric analysis

DNA replication during S-phase represents a biochemical metric of cell division. We present here a protocol for measuring very low rates of cell proliferation, on the basis of the incorporation of deuterium ((2)H) from heavy water ((2)H(2)O) into the deoxyribose moiety of purine deoxyribonucleotides in DNA of dividing cells, by use of gas chromatography/pyrolysis/isotope ratio-mass spectrometry (GC/P/IRMS). Very low levels of label incorporation (>or=0.002% atom percent excess (2)H) can be quantified by GC/P/IRMS. This protocol thereby permits shorter periods or lower amounts of (2)H(2)O administration than would be required using standard GC/MS techniques for measuring cell proliferation kinetics (see accompanying protocol in this issue). A disadvantage of this approach compared to GC/MS is the requirement for larger numbers of cells (> approximately 10(7)). This protocol enables definitive in vivo studies of the fraction or absolute number of newly divided cells and their subsequent survival kinetics in animals and humans, even when turnover rates are very low. Indolent hematologic malignancies, such as chronic lymphocytic leukemia, and other relatively quiescent cells represent promising areas of application.

Deuterium/hydrogen ratio analysis of thymol, carvacrol, γ-terpinene and p-cymene in thyme, savory and oregano essential oils by gas chromatography–pyrolysis–isotope ratio mass spectrometry

Isotope ratio mass spectrometry online coupled with capillary gas chromatography (GC–Py–IRMS) on column INNOWAX is used in the origin specific analysis and the authenticity control of the phenolic essential oils (EOs). Isotopic data δ 2H V-SMOW of thymol and carvacrol in natural essential oils were evidently more depleted than synthetic products (from −49 to 7‰ for thymol and −61‰ for carvacrol). δ 2H V-SMOW values of p-cymene, γ-terpinene and thymol in authentic thyme oils ( Thymus vulgaris L. and Thymus zygis L.) were found from −300 to −270‰, from −285 to −248‰ and from −259 to −234‰, respectively. δ 2H V-SMOW values of carvacrol and p-cymene in authentic oregano oils ( Origanum heracleoticum L., Coridothymus capitatus L. and Origanum compactum L.) varied from −223 to −193‰ and from −284 to −259‰, respectively. For authentic Satureja montana subsp. montana essential oils, the mean δ 2H V-SMOW value for aromatic compounds were found to be the following: γ-terpinene −273‰ (SD = 4.6‰) and p-cymene −283‰ (SD = 3.0‰), thymol −245‰ (SD = 1.8‰) and carvacrol −226‰ (SD = 1.7‰). In addition, p-cymene was previously found as a precursor of the biosynthesis of thymol and carvacrol in thyme oil, thus, we considered p-cymene as an endogenous reference compound (ERC) for D/H ratio analysis. The isotopic fractionation factors α thymol/ p-cymene = 1.05 and α carvacrol/ p-cymene = 1.08 were obtained and also used to control the authenticity of the phenolic EOs.

The Hydrogen Kinetic Isotope Effects of the Reactions of n-Alkanes with Chlorine Atoms in the Gas Phase

The stable-hydrogen kinetic isotope effects (KIEs) for a series of n-alkanes in reaction with chlorine atoms in the gas phase were studied in a 25-L PTFE reaction chamber at 298 K. The time dependence of both the stable hydrogen isotope ratios and the concentrations was determined using a gas chromatography pyrolysis isotope ratio mass spectrometry (GC-P-IRMS) system. The following KIE values, in per mil (‰), were obtained: 39.6 ± 2.7 (n-butane), 28.2 ± 0.9 (n-pentane), 24.6 ± 1.0 (n-hexane), 24.0 ± 1.2 (n-heptane), 17.9 ± 3.3 (n-octane), 15.1 ± 0.7 (n-nonane), and 14.9 ± 1.8 (n-decane). The errors given are the ±1σ standard errors. These measured values were used to derive structure–reactivity relationship (SRRs), which allow for the calculation of the KIEs for the reaction of n-alkanes with Cl atoms. The results of the calculations agree with the measurements within few per mil or better. The site specific stable hydrogen isotope fractionation effects for methyl groups are approximately a factor of 3 larger than those for methylene group, a finding which is qualitatively similar to site-specific stable hydrogen isotope effects reported in literature for reactions of alkanes with the OH radical. Because n-alkanes with close to natural isotope ratios (i.e. neither artificially labeled, nor enriched or depleted) were used, the KIE data are directly applicable to atmospheric studies. Based on these KIE values, the impact of Cl-atom reactions of the stable hydrogen isotope ratio on alkanes are estimated for different levels of Cl-atom concentrations. On average in the troposphere, the impact of Cl-atom reactions of the stable hydrogen isotope ratio of n-alkanes will be small. However, in regions of the troposphere with high concentrations of Cl atoms, such as the tropospheric ozone depletion episodes during polar sunrise, the impact of Cl-atom reactions is substantial.

Online determination of 2 H/ 1 H and 13 C/ 12 C isotope ratios of cinnamaldehyde from different sources using gas chromatography isotope ratio mass spectrometry

The combination of gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) and gas chromatography-pyrolysis-isotope ratio mass spectrometry (GC-P-IRMS) is applied to the authenticity assessment of cinnamaldehyde from various sources. For that reason, cinnamon oils were self-prepared by steam distillation from three different varieties of cinnamon bark on the market, C. ceylanicum (ceylon), C. cassia (cassia) and C. burmanii (cassia vera). Furthermore, the so-called wood cinnamon was investigated, which is produced from the outer bark of older branches of cinnamon of minor quality. Self-prepared oils were analysed from commercial cinnamon powder. In addition several commercial samples of cinnamon oil and cinnamaldehyde, some of them declared to be natural, were investigated. δ 2ΗV-SMOW and δ 13CV-PDB values of cinnamaldehyde were determined and characteristic authenticity ranges were deduced, allowing the differentiation between synthetic and natural samples. By correlation of both the δ 2ΗV-SMOW and δ 13CV-PDB values, characteristic authenticity ranges were defined for ceylon, cassia and wood cinnamon. The δ 2ΗV-SMOW and δ 13CV-PDB values of cassia vera samples are in the range of cassia. By comparing the δ 2ΗV-SMOW values of different self-prepared samples (ground bark, distillate) of cinnamon determined by TC/EA-IRMS with the corresponding GC-IRMS values, online GC-IRMS methods are proved to be essential in the authentication of complex natural products.

Measurements by gas chromatography/pyrolysis/mass spectrometry: fundamental conditions in (2)H/(1)H isotope ratio analysis.

Gas chromatography/pyrolysis/isotope ratio mass spectrometry (GC/P/IRMS) is a relatively new method for on-line determination of (2)H/(1)H isotope ratios. The influence of different parameters on the (2)H/(1)H isotope ratios obtained in GC/P/IRMS has been thoroughly studied using several flavor compounds, such as 5-nonanone, linalool, menthol, linalyl acetate and 4-decanolide. The requirement of "conditioning" the pyrolysis reactor to obtain reliable delta(2)H(V-SMOW) values is discussed. Furthermore, the influence of the carrier gas flow of the gas chromatograph on the completeness of pyrolysis and subsequently on the delta(2)H(V-SMOW) values is investigated in detail. The linear range of the compounds investigated is determined. The results show that calibrating the GC/P/IRMS system with secondary standard substances is absolutely necessary in order to obtain reliable delta(2)H(V-SMOW) values. In view of interlaboratory comparability, validation procedures are recommended.