Rapid, Sensitive and Highly Selective (15)N Analysis of (15)N Enriched Nitrite in Water Samples and Soil Extracts by Nitric Oxide Production and CF-QMS Measurement

Abstract Nitrite is a very important intermediate in many microbiological N transformations in soils and water. The stable isotope (15)N is often used to investigate these processes. The determination of (15)N in low concentrations of nitrite in the presence of large concentrations of nitrate is very difficult. Methods used so far for the isotope analysis of nitrite are unsatisfactory, because the nitrite must be calculated as the difference between nitrate plus nitrite and nitrate alone. More useful are mehods by which the nitrite is selectively converted into a chemical form that is suitable for (15)N analysis and that is free from interference from other N species, particularly nitrate. Using this principle in the present study we developed a method where the nitrite is reduced to nitric oxide by iodide in acid medium. This reaction is fast and quantitative, and the (15)N abundance of NO can be precisely measured by continuous flow mass spectrometry. This method is used for samples from tracer experiments with artificially enriched nitrogen 15. Therefore, the use of simple quadrupole mass spectrometers directly linked to the reaction unit is possible with sufficient precision (Reaction-Continuous Flow Quadrupole Mass Spektrometry-RCFQMS). Using the technique developed sample volumes up to 10ml containing at least 1.0 μg nitrite-N (0, 1 μg/ml) with a (15)N abundance of ? 0.42 at.% gave a precision of RSD ? ± 3%.     

A New Approach to Determining the Content and 15N Abundance of Total Dissolved Nitrogen in Aqueous Samples: TOC Analyser-QMS Coupling

Abstract

The standard method for determining the ¹⁵N abundance of total dissolved nitrogen (TDN) in aqueous samples (e.g., soil leachate, sewage, urine) is currently Kjeldahl digestion followed by steam distillation or diffusion to isolate the ammonium, and then ¹⁵N measurement using IRMS. However, this technique is both time-consuming and laborious. One way of overcoming these disadvantages could be to couple a TOC analyser to determine the TDN with a sufficient quadrupole MS to determine the ¹⁵N abundance. The highTOC analyser (Elementar Analysensysteme Hanau, Germany), which catalytically oxidises the sample's total nitrogen with a high, constant yield to nitrogen monoxide (NO), appeared particularly suitable. The quadrupole-MS ESD 100 (InProcess Instruments Bremen, Germany) proved to be a suitable mass spectrometer for the ¹⁵N determination of NO. This combination of instruments was found to provide a workable method in numerous measurements of standard and actual samples. The detection limit concerning the N amount required per analysis is 2 μg, corresponding to an N concentration of 0.7mg/l in a maximum sample volume of 3ml. Depending on the N concentration, ¹⁵N abundances starting from 0.5 at.% can be measured with the required precision of better than 3% (simple standard deviation). For example, measuring the abundance of 0.5 at.% requires about 50 μg N, whereas for 1 at.% or more only about 5 μg N is needed per analysis.     

The Fate of [(15)N]Ammonium and [(15)N]Nitrate in the Soil of a 140-Year-Old Spruce Stand (Picea Abies) in the Fichtelgebirge (NE-Bavaria)

Abstract A (15)N tracer-experiment was carried out in a 140-year-old spruce stand (Picea abies (L.) Karst.) in the Fichtelgebirge (NE-Bavaria, Germany). Highly enriched (98 at%) [(15)N]ammonium and [(15)N]nitrate were applied as tracers by simulation of a deposition of 41.3 mol N ha(-1) with 11 water m(-2). To examine seasonal variations of uptake by spruce and understorey vegetation, different plots were labelled in spring, summer and autumn 1994. One aim of the present study was to perfect a method of preparation of soil extracts for isotope ratio mass spectrometry (IRMS) measurements. Ammonium and nitrate from soil extracts were prepared for IRMS measurements by steam distillation and subsequent freeze drying. Additionally, tracer distribution and transformations in the soil nitrogen pools were examined. Ammonium, nitrate and total nitrogen were examined in the organic layer and the upper 10 cm of the mineral soil during 3 months after the first tracer application in spring 1994. In July 1994, three months after tracer application, 40% of the [(15)N]ammonium label and 29% of the [(15)N]nitrate label, respectively, were recovered in the total N pool of the investigated soil horizons. In the organic layer the L/Of horizon retained most of the recovered tracers. Nitrification, immobilisation and mineralisation occurred even under the conditions of high soil acidity at the study site.     

Uptake of [(15)N] Ammonium and [(15)N]Nitrate in a 140-Year-Old Spruce Stand (Picea abies) in the Fichtelgebirge (NE Bavaria)

Abstract In April 1994 a (15)N tracer pulse study was started in a 140-year-old spruce stand (Picea abies [L.] Karst.) located at the Fichtelgebirge (NE Bavaria). Highly enriched (98%) [(15)N]ammonium and [(15)N]nitrate were applied simulating wet deposition. For two growing seasons the pathways and dynamics of the tracer were followed in all compartments of spruce (needles and twigs of all age classes, stem wood and bark, roots) and understorey vegetation and in soils of the organic (L/Of and Oh) and mineral horizons (A(0-5) and A(5-10)). By variations of the application time on different plots within the growing season (spring, summer and autumn) a seasonal effect of labelling on uptake and distribution patterns was tested. First results of this tracer study indicate that young and old spruce stands do not differ basically in pattern of uptake and distribution of mineral nitrogen. There are indications that spruce uses preferentially ammonium versus nitrate and that the ratio of ammonium/nitrate which is being consumed depends on the ammonium/nitrate ratio in the soil solution. The uptake rates decrease within the growing season.     

The impact of ozone on the (15)n incorporation and nitrogen assimilation of wheat and maize

Abstract Young wheat (C3) and maize (C4) plants were exposed to near-ambient concentrations of ozone in open-top chambers in order to investigate the possible effects of ozone on nitrogen metabolism. Nitrogen was supplied to the plants by adding (15)N-labelled tracer substances via the soil substrate. Enzyme activities (NADH nitrate reductase, nitrite reductase, glutamine synthetase and NADH glutamate dehydrogenase) and the incorporation of (15)N were determined. The findings show that nitrogen metabolism was affected by O(3), however, there were distinct differences between the two species. In plants treated with O(3), NADH nitrate reductase activity in maize leaves was reduced, while NR activity in wheat leaves only slightly declined. Only minor changes were observed with respect to the activities of nitrite reductase, glutamine synthetase and NADH glutamate dehydrogenase. Feeding experiments using (15)NO(3) (-) showed that the incorporation of nitrate nitrogen in wheat plants exposed to ozone remains virtually unchanged, whereas in maize plants reduced incorporation rates were observed for nitrate nitrogen. The incorporation of ammonium nitrogen was distinctly increased in wheat and maize by the impact of ozone. When investigating pigment contents, reduced levels of chlorophyll a and b and carotenoids were observed, whereas the pigment content of wheat leaves remained unchanged. These results indicate that young maize plants are more susceptible than wheat plants to short-term ozone exposure.     

The Fate of [15N]Ammonium and [15N]Nitrate in the Soil of a 140-Year-Old Spruce Stand (Picea Abies) in the Fichtelgebirge (NE-Bavaria)

Abstract

A ¹⁵N tracer-experiment was carried out in a 140-year-old spruce stand (Picea abies (L.) Karst.) in the Fichtelgebirge (NE-Bavaria, Germany). Highly enriched (98 at%) [¹⁵N]ammonium and [¹⁵N]nitrate were applied as tracers by simulation of a deposition of 41.3 mol N ha^?1 with 11 water m^?2. To examine seasonal variations of uptake by spruce and understorey vegetation, different plots were labelled in spring, summer and autumn 1994.

One aim of the present study was to perfect a method of preparation of soil extracts for isotope ratio mass spectrometry (IRMS) measurements. Ammonium and nitrate from soil extracts were prepared for IRMS measurements by steam distillation and subsequent freeze drying. Additionally, tracer distribution and transformations in the soil nitrogen pools were examined. Ammonium, nitrate and total nitrogen were examined in the organic layer and the upper 10 cm of the mineral soil during 3 months after the first tracer application in spring 1994.

In July 1994, three months after tracer application, 40% of the [¹⁵N]ammonium label and 29% of the [¹⁵N]nitrate label, respectively, were recovered in the total N pool of the investigated soil horizons. In the organic layer the L/Of horizon retained most of the recovered tracers. Nitrification, immobilisation and mineralisation occurred even under the conditions of high soil acidity at the study site.     

Measuring nitrification in sediments--comparison of two techniques and three 15NO(-)3 measurement methods

Nitrification is a crucial process in sediment nitrogen cycling. We compared two (15)N tracer-based nitrification measurement techniques (isotope pairing technique (IPT) combined with (15)N nitrate pool dilution and (15)N ammonium oxidation) and three different (15)N analyses from bottom water nitrate (ammonia diffusion, denitrifier and SPINMAS) in a sediment mesocosm. The (15)N nitrate pool dilution technique combined with IPT can be used to quantify the in situ nitrification, but the minimum detection limit for the total nitrification is higher than that in the (15)N ammonium oxidation technique. The (15)N ammonium oxidation technique, however, is not applicable for sediments that have high ammonium content. If nitrate concentration and the amount of (15)N label in the sample are low, the (15)N nitrate analysis should be done with the denitrifier method. In higher (15)N concentrations, the less sensitive SPINMAS method can also be applied. The ammonia diffusion method is not suitable for bottom water (15)N nitrate analyses.     

A new approach to determining the content and 15N abundance of total dissolved nitrogen in aqueous samples: TOC analyser-QMS coupling

The standard method for determining the 15N abundance of total dissolved nitrogen (TDN) in aqueous samples (e.g., soil leachate, sewage, urine) is currently Kjeldahl digestion followed by steam distillation or diffusion to isolate the ammonium, and then 15N measurement using IRMS. However, this technique is both time-consuming and laborious. One way of overcoming these disadvantages could be to couple a TOC analyser to determine the TDN with a sufficient quadrupole MS to determine the 15N abundance. The high TOC analyser (Elementar Analysensysteme Hanau, Germany), which catalytically oxidises the sample's total nitrogen with a high, constant yield to nitrogen monoxide (NO), appeared particularly suitable. The quadrupole-MS ESD 100 (InProcess Instruments Bremen, Germany) proved to be a suitable mass spectrometer for the 15N determination of NO. This combination of instruments was found to provide a workable method in numerous measurements of standard and actual samples. The detection limit concerning the N amount required per analysis is 2 microg, corresponding to an N concentration of 0.7 mg/l in a maximum sample volume of 3ml. Depending on the N concentration, 15N abundances starting from 0.5 at.% can be measured with the required precision of better than 3% (simple standard deviation). For example, measuring the abundance of 0.5 at.% requires about 50 microg N, whereas for 1 at.% or more only about 5 microg N is needed per analysis.     

Isolation of NO(3)(-)-N as 1-phenylazo-2-naphthol (Sudan-1) for measurement of delta(15)N

The conversion of nitrate (NO(3)(-)) to 1-phenylazo-2-naphthol (Sudan-1) has been examined as a method for natural abundance measurement of delta(15)N of NO(3)(-). The reaction results in dilution of NO(3)(-)-N with only one reagent-derived N and the product is readily concentrated from dilute samples by reverse phase chromatography. There is systematic isotopic fractionation during the reaction, but this can be allowed for by analysing known NO(3)(-) standards along with each sample set. Sudan-1 prepared from surface water samples containing approximately 50 &mgr;g NO(3)(-)-N can be analysed by automated continuous flow isotope ratio mass spectrometry with a precision of 0.2 per thousand (one standard deviation) and the accuracy is not affected by interference from other nitrogenous species in the sample or reagents. Copyright 1999 John Wiley & Sons, Ltd.     

Analysis of the coexisting pathways for NO and N2O formation in Chernozem using the 15N-tracer SimKIM-Advanced model

The nitrogen (N) cycle consists of a variety of microbial processes. These processes often occur simultaneously in soils, but respond differently to local environmental conditions due to process-specific biochemical restrictions (e.g. oxygen levels). Hence, soil nitrogen cycling (e.g. soil N gas production through nitrification and denitrification) is individually affected through these processes, resulting in the complex and highly dynamic behaviour of total soil N turnover. The development and application of methods that facilitate the quantification of individual contributions of coexisting processes is a fundamental prerequisite for (i) understanding the dynamics of soil N turnover and (ii) implementing these processes in ecosystem models. To explain the unexpected results of the triplet tracer experiment (TTE) of Russow et al. (Role of nitrite and nitric oxide in the processes of nitrification and denitrification in soil: results from ¹⁵N tracer experiments. Soil Biol Biochem. 2009;41:785–795) the existing SimKIM model was extended to the SimKIM-Advanced model through the addition of three separate nitrite subpools associated with ammonia oxidation, oxidation of organic nitrogen (N_org), and denitrification, respectively. For the TTE, individual treatments with ¹⁵N ammonium, ¹⁵N nitrate, and ¹⁵N nitrite were conducted under oxic, hypoxic, and anoxic conditions, respectively, to clarify the role of nitric oxide as a denitrification intermediate during N₂O formation. Using a split nitrite pool, this analysis model explains the observed differences in the ¹⁵N enrichments in nitric oxide (NO) and nitrous oxide (N₂O) which occurred in dependence on different oxygen concentrations. The change from oxic over hypoxic to anoxic conditions only marginally increased the NO and N₂O release rates (1.3-fold). The analysis using the model revealed that, under oxic and hypoxic conditions, N_org-based N₂O production was the dominant pathway, contributing to 90 and 50 % of the total soil N₂O release. Under anoxic conditions, denitrification was the dominant process for soil N₂O release. The relative contribution of N_org to the total soil NO release was small. Ammonia oxidation served as the major pathway of soil NO release under oxic and hypoxic conditions, while denitrification was dominant under anoxic conditions. The model parameters for soil with moderate soil organic matter (SOM) content were not scalable to an additional data set for soil with higher SOM content, indicating a strong influence of SOM content on microbial N turnover. Thus, parameter estimation had to be re-calculated for these conditions, highlighting the necessity of individual soil-dependent parameter estimations.     

15N-Isotopenmessung mit dem Emissionsspektrometer NOI-6PC

Die emissionsspektroskopische ¹⁵N-Analyse ist eine leistungsfähige Methode für ¹⁵N-Tracerexperimente in Landwirtschaft, Biologie und Medizin. Vorteile des NOI-6 ¹⁵N-Analysatorsystems sind die geringe Probemenge (10 μg), die kurze MeBzeit und der hohe Bedienungskomfort. In Koppulung mit einer Verbrennung nach Dumas ist die gleichzeitige Bestimmung der Gesamt-Stickstoffkonzentration und des prozentualen ¹⁵N-Anteiles möglich.

The emission spectroscopic ¹⁵N analysis is a powerful tool for ¹⁵N tracer experiments in agriculture, biology, and medicine. Advantages of the NOI-6 ¹⁵N analyzer systems are the low sample amount, the short measuring time, and the high handling comfort.

In the future, methods for the simultaneous determination of total nitrogen and of nitrogen-IS will get increasing importance.     

Automated and rapid online determination of 15N abundance and concentration of ammonium, nitrite, or nitrate in aqueous samples by the SPINMAS technique

On the basis of the principle of reaction continuous-flow quadrupole mass spectrometry, an automated sample preparation unit for inorganic nitrogen (SPIN) species was developed and coupled to a quadrupole Mass Spectrometer (MAS). The SPINMAS technique was designed for an automated, sensitive, and rapid determination of 15N abundance and concentration of a wide variety of N-species involved in nitrogen cycling (e.g. NH4+, NO3-, NH2OH etc.). In this paper, the SPINMAS technique is evaluated with regard to the determination of 15N abundance and concentration of the most fundamental inorganic nitrogen compounds in ecosystems such as NH4+, NO2-, and NO3-. The presented paper describes the newly developed system in detail and demonstrates the general applicability of the system. For a precise determination of 15N abundance and concentration, a minimum total N-amount of 10 microg NH4+ - N, 0.03 microg NO2- - N, or 0.3 microg NO3- - N has to be supplied. Currently, the SPINMAS technique represents the most rapid and only fully automated all-round method for a simultaneous determination of 15N abundance and total N-amount of NH4+, NO2-, or NO3- in aqueous samples.     

15N, 13C-On-line Measurements with an Elemental Analyser (Carlo Erba,NA 1500), a Modified Trapping Box and a Gas Isotope Mass Spectrometer (Finnigan,MAT 251)

Abstract

An on-line method for the determination of ¹⁵N and ¹³C with a gas isotope mass spectrometer (Finnigan, MAT 251) was developed to improve the sensitivity and to reduce measurements time and the cost of the sample analysis. For this purpose an elemental analyser (Carlo Erba, NA 1500) was coupled to the mass spectrometer using parts of the capillary system of a trapping box (Finnigan, type CN). For the determination of samples with natural concentrations of ¹⁵N and ¹³C the uncertainty of the delta value is less than 0.2 δ‰. The detection limit is in the order of 10 μg (total N or total C) and 7 samples can be analysed per hour.     

J-multiplied HSQC (MJ-HSQC): a new method for measuring 3J(HNHalpha) couplings in 15N-labeled proteins

A new method for the measurement of homonuclear 3J(HNHalpha) coupling constants in 15N-labeled small proteins is described. The method is based on a modified sensitivity enhanced HSQC experiment, where the 3J(HNHalpha) couplings are multiplied in the f1-dimension. The J-multiplication of homonuclear 3J(HNHalpha) couplings is based on simultaneous incrementation of 15N chemical shift and homonuclear coupling evolution periods. The time increment for the homonuclear coupling evolution period is chosen to be a suitable multiple (2N x t1) of the corresponding increment for 15N-shift evolution. This results in the splitting of the HSQC correlation in the f1-dimension by 2N x 3J(HNHalpha). Because the pulse sequence has good sensitivity and water suppression properties, it is particularly useful for natural abundance samples.     

15N Analysis of nitric oxide and nitrous oxide by cryotrap enrichment using a gas chromatograph quadrupole mass spectrometer and its application to (15)N-tracer investigations of NO/N(2)O formation in soil

Nitric oxide (NO) and nitrous oxide (N(2)O) are two important trace gases in the atmosphere. Determining the concentration and (15)N abundance of NO and N(2)O in air is difficult owing to their very low concentration in the atmosphere (NO < 1 ppb(v); N(2)O approximately 0.32 ppm(v)). Although (15)N analysis of N(2)O in ambient concentrations can be carried out using a gas chromatograph quadrupole mass spectrometer system (GC-QMS) and a dosage of 2 mL of air by means of a sample loop, this system is not sensitive enough to measure the ambient concentration of NO and its (15)N abundance. Therefore the concentration of NO must be enriched by cryotrapping (cooling with liquid nitrogen). The (15)N analytical method developed enables the sensitive and sufficiently precise measurement of (15)N-enriched NO in air. Furthermore, the analytical equipment developed greatly improves existing (15)N(2)O analysis using the GC-QMS technique. An application of the (15)N analysis method will be shown for an investigation on the NO and N(2)O formation in black earth soil after (15)NH(4)(+), (15)NO(3)(-) and (15)NO(2)(-) labelling. Copyright 1999 John Wiley & Sons, Ltd.     

The Impact of Ozone on the 15N Incorporation and Nitrogen Assimilation of Wheat and Maize

Abstract

Young wheat (C3) and maize (C4) plants were exposed to near-ambient concentrations of ozone in open-top chambers in order to investigate the possible effects of ozone on nitrogen metabolism. Nitrogen was supplied to the plants by adding ¹⁵N-labelled tracer substances via the soil substrate. Enzyme activities (NADH nitrate reductase, nitrite reductase, glutamine synthetase and NADH glutamate dehydrogenase) and the incorporation of ¹⁵N were determined.

The findings show that nitrogen metabolism was affected by O₃, however, there were distinct differences between the two species. In plants treated with O₃, NADH nitrate reductase activity in maize leaves was reduced, while NR activity in wheat leaves only slightly declined. Only minor changes were observed with respect to the activities of nitrite reductase, glutamine synthetase and NADH glutamate dehydrogenase.

Feeding experiments using ¹⁵NO₃ ^? showed that the incorporation of nitrate nitrogen in wheat plants exposed to ozone remains virtually unchanged, whereas in maize plants reduced incorporation rates were observed for nitrate nitrogen. The incorporation of ammonium nitrogen was distinctly increased in wheat and maize by the impact of ozone.

When investigating pigment contents, reduced levels of chlorophyll a and b and carotenoids were observed, whereas the pigment content of wheat leaves remained unchanged. These results indicate that young maize plants are more susceptible than wheat plants to short-term ozone exposure.     

Zum Stand der Stoffwechselforschung mit 15N- und 13C-markierten Tracersubstanzen in der Kinderheilkunde (Protein Turnover Research Using 15N and 13C Labelled Substrates in Pediatrics)

Abstract

Measurements in protein turnover and in metabolism of amino acids and their degradation products by means of stable isotope labelled substrates have been increasingly applied in clinical research over the last years. In spite of numerous studies dealing with this topic, quite a few important insufficiently clarified methodical aspects remain. This refers, for instance, to the choice of suitable tracer substances, the difficulties in the determination of the excretion plateau and the validation of the oxidation rates as measured with individual-labelled amino acids with regard to the whole body protein synthesis. Such problems may become of decisive importance in special subjects, such as preterm infants and critically-ill patients.

Investigations into these issues conducted by our group have revealed that the protein turnover in the very small preterm infant is by no means as intensive as previously claimed. The utilisation of urea nitrogen for the whole body protein synthesis of the infant may assume substantial proportions under the conditions of marginal protein intake and of catchup-growth. Studies conducted by means of ¹⁵N-labelled bifidobacteria have pointed at the intensive substrate exchange existing between microflora and host.

Pediatric research has to be non-invasive. Consequently, methods based on arterio-venous differences in tracer concentrations and on muscle biopsies do not have very high priority in pediatric research. A search for references published in the last five years has shown, that ¹⁵N-glycine is still the most frequently used tracer substance. There is a tendency towards a further increase of cell culture experiments run with stable isotope labelled amino acids.

Clinical research groups increasingly turn their attention to stable isotopes and mass spectrometry. This impressively demonstrates the continuing importance of tracerkinetic methods in all branches of medicine.     

ConFlo III - an interface for high precision delta(13)C and delta(15)N analysis with an extended dynamic range

A newly developed interface coupling a CHN combustion device (elemental analyser 'EA') to an isotope ratio mass spectrometer is described and evaluated. The purpose of the device is to extend the dynamic range of delta(13)C and delta(15)N analysis from less than 2 orders of magnitude to more than 3 orders of magnitude. Carbon isotope ratio measurements of atropine as a model compound have been performed analysing between 1 μg to 5 mg C with acceptable to excellent precision (0.6 to 0.06 per thousand, delta-notation). The correction due to the blank signal is critical for sample amounts smaller than 4 μg C. The maximum sample weight is determined by the combustion capacity of the EA. Larger sample amounts are measured using dilution of a small part of the EA effluent with helium. The dilution mechanism works virtually free of isotope fractionation. Copyright 1999 John Wiley & Sons, Ltd.     

Spectrophotometry Determination of Nitrate Nitrogen and Nitrite Nitrogen in Sewage Sludges

Extraction followed by spectrophotometry was employed to determine nitrate and nitrite in sewage sludge. Recovery of added nitrate and nitrite was complete. The procedure was suitable for the determination of nitrate nitrogen and nitrite nitrogen in this matrix.     

Simultaneous Determination of Plasma Enrichments of 1-13C- and 15N-Labelled Phenylalanine and Tyrosine

Abstract

A methylchloroformate derivative was used for the simultaneous determination of plasma enrichments of 1-¹³C-phenylalanine, 1-¹³C-tyrosine, ¹⁵N-phenylalanine and ¹⁵N-tyrosine by gas chromatography/mass spectrometry. All four tracer enrichments could be measured in a single GC run. A specific ion fragment was obtained for each tracer. This approach allowed an easy determination of the “tracer to tracee ratios”. Each ion fragment could be measured with an appropriate single-to-noise ratio and precision in samples obtained from 100 μl plasma. The derivatization consists of a fast one-step reaction. Therefore it is well suited for studies involving a large number of samples, such as non-steady state bolus studies.