Large-volume injection combined with gas chromatography/isotope ratio mass spectrometry for the analysis of polycyclic aromatic hydrocarbons.

RATIONALE: Compound-specific stable isotope analyses of carbon require relatively large amounts of sample for reliable analyses. Commonly applied injections of 1 µL may thus be insufficient for samples with low concentrations of pollutants (e.g. air particulate matter) or when the amount of a sample is limited. METHODS: A Large-Volume Injection (LVI) method for carbon stable isotope ratio analysis of Polycyclic Aromatic Hydrocarbons (PAHs) was optimized in this study. Gas chromatography/combustion/isotope ratio mass spectrometry (GCCIRMS) and ion trap mass spectrometry (ITMS) were used for the determination of stable carbon isotope ratios and quantification of compounds, respectively. RESULTS: The optimized method resulted in very good reproducibility, even for the most volatile PAH, naphthalene, when a small amount of higher boiling co-solvent was used. No significant fractionation of isotope ratios could be seen and the recoveries of analytes were similar to or better than that of a splitless cold injection. CONCLUSIONS: Injection of 100 µL, instead of the commonly used 1 µL, increases the detection limit for PAHs significantly and/or simplifies the sample preparation step. Using our optimized method, stable carbon isotope ratios can be reliably measured in samples with concentrations of PAHs down to 0.05-0.1 ng µL(-1).

Analysis of polycyclic aromatic hydrocarbons extracted from air particulate matter using a temperature programmable injector coupled to GC-C-IRMS.

Compound specific isotopic analysis (CSIA) can provide information about the origin of analysed compounds - in this case, polycyclic aromatic hydrocarbons (PAHs). In the study, PAHs were extracted from three dust samples: winter and summer filter dust and tunnel dust. The measurement was performed using the method validated in our laboratory using pure, solid compounds and EPA 610 reference assortment. CSIA required an appropriate clean-up method to avoid an unresolved complex in the gas chromatographic analysis usually found in the chromatography of PAHs. Extensive sample clean-up for this particular matrix was found to be necessary to obtain good gas chromatography-combustion-isotope ratio mass spectrometry analysis results. The sample purification method included two steps in which the sample is cleaned up and the aliphatic and aromatic hydrocarbons are separated. The concentration of PAHs in the measured samples was low; so a large volume injection technique (100 microl) was applied. The delta(VPDB)(13)C was measured with a final uncertainty smaller than 1 per thousand. Comparison of the delta(VPDB)(13)C signatures of PAHs extracted from different dust samples was feasible with this method and, doing so, significant differences were observed.

Use of a temperature-programmable injector coupled to gas chromatography-combustion-isotope ratio mass spectrometry for compound-specific carbon isotopic analysis of polycyclic aromatic hydrocarbons.

Compound-specific isotopic analysis (CSIA) can provide information about the origin of analysed compounds; for instance, polycyclic aromatic hydrocarbons (PAHs) in aerosols. This could be a valuable tool in source apportionment of particulate matter (PM) air pollution. Because gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) analysis requires an amount of at least 10 ng of an individual PAH, a high concentration of PAHs in the injected extract is needed. When the concentration is low a large volume injector creates the possibility of introducing a satisfactory amount of individual PAHs. In this study a temperature-programmable injector was coupled to GC-C-IRMS and injection parameters (solvent level, transfer column flow, transfers time) were optimised using six solid aromatic compounds (anthracene, fluoranthene, pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene) dissolved in n-pentane and EPA 610 reference mixture. CSIA results for solid PAHs were compared with results obtained for the single components analysed by elemental analysis-isotope ratio mass spectrometry. The injection method was validated for two sample injection volumes, 50 and 100 microL. This method was also compared with commonly used splitless injection. To be included in the study, measurements had to have an uncertainty lower than 0.5 per thousand for delta(VPDB)13C and a minimum peak height of 200 mV. The lower concentration limits at which these criteria were fulfilled for PAHs were 30 mg/L for 1 microL in splitless injection and 0.3 and 0.2 mg/L for 50 and 100 microL, respectively, in large volume injection.

Headspace solid phase microextraction--GC/C-IRMS for delta13CVPDB measurements of mono-aromatic hydrocarbons using EA-IRMS calibration.

This work aims at comparing the delta(13)C(VPDB) of mono-aromatic hydrocarbons benzene, toluene, ethylbenzene and xylene isomers (BTEX) measured by elemental analyser (EA)-isotope ratio mass spectrometer (IRMS) with the delta(13)C(VPDB) measured on the same compounds by headspace solid phase microextraction - GC/C-IRMS (hSPME - GC/C-IRMS) with the final goal of using these compounds as internal standards on the latter system. The EA-IRMS measurements were done using calcium and lithium carbonate isotopic reference materials: NBS19 and L-SVEC for establishing the delta(13)C(VPDB) scale. The EA-IRMS measurements with helium dilution of a set of five reference materials (USGS40, USGS41, IAEA-CH-6, IAEA-CH-3 and IAEA-601) show systematic bias of 1 per thousand relative to their assigned values. This bias due to the dilution mechanism in the used ConfloII interface device could not be avoided. As the selected hydrocarbons: BTEX could not be analysed by EA-IRMS without helium dilution, their delta(13)C(VPDB) must be corrected from this observed bias using an external calibration. The CO(2) gas calibrated using EA-IRMS without helium dilution, was used as an in-house reference for the delta(13)C(VPDB) measurements of the BTEX by the hSPME - GC/C-IRMS system. The comparison made between the delta(13)C(VPDB) measured on the same BTEX compounds by EA-IRMS (with external calibration) and by hSPME - GC/C-IRMS techniques showed good agreement.

Methods to reduce interference effects in thermal conversion elemental analyzer/continuous flow isotope ratio mass spectrometry delta18O measurements of nitrogen-containing compounds.

On-line determination of the oxygen isotopic composition (delta(18)O value) in organic and inorganic samples is commonly performed using a thermal conversion elemental analyzer (TC-EA) linked to a continuous flow isotope ratio mass spectrometry (IRMS) system. Accurate delta(18)O analysis of N-containing compounds (like nitrates) by TC-EA-IRMS may be complicated because of interference of the N(2) peak on the m/z 30 signal of the CO peak. In this study we evaluated the effectiveness of two methods to overcome this interference which do not require any hardware modifications of standard TC-EA-IRMS systems. These methods were (1) reducing the amount of N(2) introduced into the ion source through He dilution of the N(2) peak and (2) an improved background correction on the CO m/z 30 sample peak integration. Our results show that He dilution is as effective as diverting the N(2) peak in order to eliminate this interference. We conclude that the He-dilution technique is a viable method for the delta(18)O analysis of nitrates and other N-containing samples (which are not routinely measured using He dilution) using TC-EA-IRMS, since it can easily be programmed in the standard software of IRMS systems. With the He-dilution technique delta(18)O values of the nitrate isotope standards USGS34, IAEA-N3 and USGS35 were measured using the shortest possible traceability chain to the VSMOW-SLAP scale, and the results were -28.1 +/- 0.1 per thousand, +25.5 +/- 0.1 per thousand and +57.5 +/- 0.2 per thousand, respectively. An improved background correction was also an effective method, but required manual correction of the raw data.

Assessment of age-related glucose oxidation rates of broiler chickens by using stable isotopes.

During their relatively short commercial lifespan of six weeks, broiler chickens undergo very pronounced age- or body weight-related changes in metabolic rate and body composition. The present study was aimed to assess the age-related changes in glucose oxidation rate of broiler chickens by using 13C-labeled glucose. The methodology for this breath test needed to be established first. Broiler chickens aged from two to six weeks were placed in open-circuit respiration cells and received a single intubation of U-13C6-glucose, followed by breath sampling for 4 hours and mass spectrometric analysis of 13C: 12C ratio in the exhaled air. Simultaneously, CO2 concentration in the respiration cell air was continuously monitored in order to calculate the cumulative percentage dose recovery (CPDR). With respect to the methodology, an oral dose of 2 mg U-13C6-glucose per kg body weight while maintaining a CO2 in the concentration of 0.4 to 0.5% was considered to be optimal. The three-parameter Gompertz curve fitted the CPDR values very well. Pronounced age-related changes in exogenous glucose oxidation rates in rapidly growing meat-type chickens were assessed. Young broiler chickens spend only a relatively low percentage of ingested glucose for immediate oxidation. In contrast, broiler chickens approaching the age of maximal absolute growth rate oxidize a greater proportion of the recently ingested glucose relative to the non-oxidative disposal pathways. This shift in the exogenous partitioning is discussed in relation to age-dependent changes in glucose turnover, lipid oxidation and deposition and metabolic heat production.

Influence of SDZ RAD vs. MMF on gastric emptying in renal transplant recipients.

SDZ RAD and mycophenolate mofetil (MMF) are increasingly used in the prevention of renal allograft rejection. SDZ RAD, having a macrolide structure, and MMF, known with gastrointestinal side-effects, may have gastric motility modifying properties. Gastric emptying was examined 1 yr after renal transplantation in eight patients taking corticosteroids (CS), cyclosporin A (CsA) and SDZ RAD and six patients treated with CS, CsA and MMF. Comparing the two groups, no significant differences in gastric emptying of solids and liquids were noted. Compared with normal volunteers, solid gastric emptying was faster in the SDZ RAD group and similar in the MMF group. It is concluded that in stable renal transplant recipients treated with MMF, gastric emptying was normal. Because of the impact on drug absorption and gastrointestinal symptoms, further studies are indicated to corroborate the potential prokinetic properties of SDZ RAD.

An inter- and intra-laboratory comparison of breath ¹³CO2analysis.

BACKGROUND: ¹³C breath test analysis requires accurate ¹³CO2measurements. AIM: To perform a multicentre study to evaluate the repeatability and reproducibility of breath ¹³CO2analysis. METHODS: Two series of 25 paired randomly coded tubes (each consisting of 23 ¹³CO2-enriched breath samples and two samples of standard reference pure CO2with certified δ ¹³C(PDB)) were sent to participating centres for ¹³CO2measurement. Each series of tubes was analysed 10 days apart. The repeatability and reproducibility of ¹³C measurements was assessed by Mandel's k and h statistics. RESULTS: Twenty-two centres participated in the study: 18 showed good inter- and intra-laboratory variability, whilst four showed abnormally high inter- or intra-laboratory variability. Breath test results were also significantly affected by the accuracy of the ¹³C analytical procedures. CONCLUSIONS: A low accuracy of ¹³C measurements may significantly affect the results of breath tests, leading to inappropriate clinical decisions. Standardization of ¹³C analysis is required to guarantee optimal ¹³C measurements and accurate ¹³C breath test results.

Symptoms associated with impaired gastric emptying of solids and liquids in functional dyspepsia.

OBJECTIVE: The relationship between functional dyspepsia and delayed gastric emptying of solids or liquids is still unclear. The aim of the present study was to investigate in dyspeptic patients the prevalence of delayed gastric emptying for solids or for liquids and to investigate the relationship to the dyspepsia symptom pattern. METHODS: In 392 and 330 patients with functional dyspepsia, the solid and liquid gastric emptying, respectively, was measured using breath tests, and the severity of eight dyspeptic symptoms was scored. RESULTS: Gastric emptying of solids and liquids were delayed in 23% and 35% of the patients. Multivariate analysis showed that the presence of vomiting and postprandial fullness was associated with delayed solid emptying (OR 2.65, 95% CI = 1.62-4.35 and OR 3.08, 95% CI = 1.28-9.16, respectively). Postprandial fullness was also associated with the risk of delayed liquid emptying when symptom was present (OR 3.5, 95% CI = 1.57-8.68), relevant or severe (OR 2.504, 95% CI = 1.41-4.65), and severe (OR 2.214, 95% CI = 1.34-3.67). Severe early satiety was associated with the risk of delayed liquid emptying (OR 1.902, 95% CI = 1.90-3.30). CONCLUSIONS: A subset of dyspeptic patients has delayed gastric emptying of solids or of liquids. Delayed gastric emptying of solids was constantly associated with postprandial fullness and with vomiting. Delayed emptying for liquids was also associated with postprandial fullness and with severe early satiety.

Erosive enterocolitis in mycophenolate mofetil-treated renal-transplant recipients with persistent afebrile diarrhea.

BACKGROUND: Diarrhea is the most frequently reported adverse event in mycophenolate mofetil (MMF)-treated transplant patients. The aim of this study was to explore the gastrointestinal tract in MMF-treated renal transplant recipients with persistent afebrile diarrhea to characterize its nature and etiology. METHODS: Renal transplant recipients with persistent afebrile diarrhea (daily fecal output >200 g) were prospectively investigated for infections, morphologic, and functional (gastrointestinal motility and intestinal absorptive capacity) integrity of the gastrointestinal tract; 26 patients met the inclusion criteria. RESULTS: All but one patient had an erosive enterocolitis. Seventy percent of the patients had malabsorption of nutrients, contributing to the diarrhea. In +/-60%, an infectious origin was demonstrated and successfully treated with antimicrobial agents without changes in immunosuppressive regimen. In +/-40%, no infection occurred, but a Crohn's disease-like pattern of inflammation was noted. These patients also had a less pronounced bile-acid malabsorption but a significant faster colonic transit time, correlating with the trough level of mycophenolic acid (MPA). Cessation of MMF, however, was associated with allograft rejection in one third of these patients. CONCLUSIONS: Persistent afebrile diarrhea in renal transplant recipients is characterized by erosive enterocolitis, which is of infectious origin in +/-60%. In +/-40%, a Crohn's disease-like (entero-)colitis was present. Because reduction or cessation of MMF was the only effective therapy, MPA or one of its metabolites may be suggested as a possible cause. However, reduction or cessation of MMF was associated with an increased risk for rejection.

Stable isotopes and 13CO2 breath tests for investigating gastrointestinal functions.

In medical investigation there is a need for non-invasive methods. Moreover, patients ask for easy methods that are simple to perform and medical doctors demand reliable techniques. With the advent of stable isotopes a new area of tracer technology became available. In gastroenterology, 13CO2 breath tests are used which fulfill all the conditions needed in modern clinical research and investigation.

Anti-tumor necrosis factor treatment restores the gut barrier in Crohn's disease.

OBJECTIVES: A primary defect of the tight junctions and, hence, increased intestinal epithelial permeability has been proposed as a basic pathogenic event in Crohn's disease. Challenge of the mucosal immune system by the commensal gut flora would then result in chronic inflammation. Alternatively, increased permeability could be the result of inflammation. Our aim was to study intestinal permeability in refractory Crohn's disease before and after treatment with monoclonal chimeric antibodies directed against tumor necrosis factor (TNF) to investigate whether the abnormal permeability persists after control of inflammation. METHODS: Twenty-three patients with active Crohn's disease were evaluated before and 4 wk after a single infusion of 5 mg/kg infliximab. Intestinal permeability was studied by measurement of urinary excretion of 51Cr-EDTA after oral intake. RESULTS: The increased permeation of 51Cr-EDTA through the small intestine (1.63% interquartile range [IQR] 1.06-2.07) and the overall permeation (3.27% IQR 2.40-4.38) before therapy decreased significantly after infliximab infusion to values (1.04% IQR 0.74-1.54 and 2.42% IQR 2.03-2.80, respectively) in the range of those found in normal volunteers (1.12% IQR 0.85-1.58 and 2.28% IQR 1.88-2.86, respectively). CONCLUSION: Inhibiting the proinflammatory cytokine tumor necrosis factor dramatically reduces gut inflammation and largely restores the gut barrier in Crohn's disease. Our data confirm the central role of TNF in gut barrier modulation in inflammatory conditions in vivo.