Measurement of Nitrogen and Nitrogen Isotopic Ratios Using Reduction Pyrolysis Coupled with Mass Spectrometry

Abstract

An analytical system for measuring total nitrogen and its isotopic abundance in a variety of environmental samples has been developed. A reductive pyrolysis system and a directional focusing 6-inch gas mass spectrometer were combined into the analytical system. In the reductive part of the system, nitrogen species are converted to ammonia with an atmosphere of hydrogen in the presence of a heated nickel catalyst. Five percent of the gas stream is split away for measuring total nitrogen by a conductivity detector. The ammonia is removed from the gas stream employing a cold finger reaction vessel.

The hydrogen-free ammonia is decomposed thermally to nitrogen and hydrogen at 1000°C, employing a hot rhenium filament. The N₂ produced from the decomposition is used for measuring the abundance of masses 28 and 29 by mass spectrometry. From this ratio, the ¹⁵N atom fraction is calculated.

Standard samples of N₂, ammonia, orchard leaves and urea have been successfully analyzed to determine isotopic compositions. Samples containing as little as 20 μg of total nitrogen can be analyzed by this system. By the addition of multi-reaction vessels, three samples may be completed per hour.     

The determination of the nitrogen content and nitrogen-15 abundance by means of the nitrogen gas generated from inorganic and organic materials

A method is described for the determination of the total nitrogen content, after conversion to nitrogen gas, of various types of samples before the mass-spectrometric analysis of the ¹⁵N abundance. The sample is burned with a mixture of copper oxide, copper and lime in a closed tube under vacuum (modified Dumas method). The gas is released into an evacuated chamber of known volume and the nitrogen pressure is used to determine the nitrogen content of the sample. The gas is then admitted into a mass spectrometer. In order to obtain complete combustion of the material and to generate pure nitrogen gas, various combustion temperatures, combustion times and oxidizing agents were compared.     

Sensitive determinations of stable nitrogen isotopic composition of organic nitrogen through chemical conversion into N2O

We present a method for high-sensitivity nitrogen isotopic analysis of particulate organic nitrogen (PON) in seawater and freshwater, for the purpose of determining the aquatic nitrogen fixation rate through the 15N2 tracer technique for samples that contain a low abundance of organisms. The method is composed of the traditional oxidation/reduction methods, such as the oxidation of PON to nitrate (NO3*) using persulfate, the reduction of NO3* to nitrite (NO2*) using spongy cadmium, and further reduction of NO2* to nitrous oxide (N2O) using sodium azide. Then, N2O is purged from the water and trapped cryogenically with subsequent release into a gas chromatography column to analyze the stable nitrogen isotopic composition using continuous-flow isotope ratio mass spectrometry (CF-IRMS) by simultaneously monitoring the NO+ ion currents at masses 30, 31, and 32. The nitrogen isotopic fractionation was consistent within each batch of analysis. The standard deviation of sample measurements was less than 0.3 per thousand for samples containing PON of more than 50 nmolN, and 0.5 per thousand for those of more than 20 nmolN, by subtracting the contribution of blank nitrogen, 8 +/- 2 nmol at final N2O. By using this method, we can determine delta15N for lower quantities of PON better than by other methods, so we can reduce the quantities of water samples needed for incubation to determine the nitrogen fixation rate. In addition, we can expand the method to determine the nitrogen isotopic composition of organic nitrogen in general, such as that of total dissolved nitrogen (TDN; sum of NO3*, NO2*, ammonium, and DON), by applying the method to filtrates.     

Determination of the natural abundance δ15N of taurine by gas chromatography-isotope ratio measurement mass spectrometry

The measurement of the nitrogen isotope ratio of taurine (2-aminoethanesulphonic acid) in biological samples has a large number of potential applications. Taurine is a small water-soluble molecule which is notoriously difficult to analyze due to its polarity and functionality. A method is described which allows the determination of the natural abundance δ(15)N values of taurine and structural analogues, such as 3-amino-1-propanesulphonic acid (APSA), by isotope ratio mass spectrometry interfaced to gas chromatography (GC-irm-MS). The one-step protocol exploits the simultaneous derivatization of both functionalities of these aminosulphonic acids by reaction with triethylorthoacetate (TEOA). Conditions have been established which ensure quantitative reaction thus avoiding any nitrogen isotope fractionation during derivatization and workup. The differences in the δ(15)N values of derivatized and non-derivatized taurine and APSA all fall within the working range of 0.4‰ (-0.02 to 0.39‰). When applied to four sources of taurine with various δ(15)N values, the method achieved excellent reproducibility and accuracy. The optimized method enables the determination of the natural abundance δ(15)N values of taurine over the concentration range 1.5-7.84 μmol.mL(-1) in samples of biological origin.     

Desorption of oxygen and nitrogen from iron surfaces Application of X-ray photoelectron spectroscopy to surface analysis

The patterns of desorption of oxygen and nitrogen from metal surfaces were examined and applied in the determination of gaseous elements in metal samples. The abundance of gaseous elements on metal surfaces is controlled by thermal desorption and diffusion to or from the surface. A heat treatment suitable for the separation of adsorbed gases from those in the bulk sample is discussed. Preheating of the metal samples at ca. 300 degrees in a vacuum reduces the residual adsorbates to a minimum and retards the diffusion of the gaseous elements from the bulk to the surface.     

Differentiation of the geographical origin of durum wheat semolina samples on the basis of isotopic composition

The identification of the geographical origin of foods is attracting great interest from consumers and producers since it may be used as a criterion for guaranteeing quality and authenticity. Stable isotope techniques can provide useful information on the origin of food products. The natural abundance isotopic ratios of carbon, oxygen and nitrogen were determined and a comparison made between the isotopic content of samples originating from Italy and those of samples originating from other countries (Canada, Turkey, Australia). A correlation was found between the isotopic composition and geographical origin of the samples. The relationship between isotopic content and the latitude has been confirmed.     

Solid-state nitrogen-15 NMR and quantum chemical study of N,N-dimethylaniline derivatives

In this study the components of the nitrogen chemical shift (CS) tensor are examined for a series of para substituted N,N-dimethylaniline derivatives. This is done through measurement of the 15N NMR spectra of powder samples and through quantum chemical calculations on the isolated molecules. Experiments and calculations show that the isotropic CS, delta(iso), decreases with increasing electron donating ability of the para substituent, in agreement with previous solution studies. More importantly, this study shows that this decrease in the isotropic (solution) CS is due to decreasing values of the CS tensor component delta(11) and component delta(33). The component delta(22) is essentially invariant to the electron donating/withdrawing ability of the para substituent. Through Ramsey's theory of nuclear magnetic shielding, it can be seen that the variation in delta(11) and delta(33), and hence delta(iso), is due to changes in the n-pi* and the sigma-pi* energy gaps in N,N-dimethylaniline. This, in turn, is a result of the change in the energy of the pi* molecular orbital with change in the pi-electron donating ability of the para substituent. The effects of nitrogen inversion on the components of the nitrogen CS tensor components are also discussed. This study also shows the feasibility of performing 15N cross-polarization experiments on nonspinning powder samples at natural isotopic abundance.     

External-source contamination during extraction—distillation in isotope-ratio analysis of soil inorganic nitrogen

The sources of contamination introduced during the extraction, distillation and drying phases of isotope-ratio analysis of soil inorganic nitrogen (ammonium and nitrite + nitrate) were identified, and the individual amounts of contaminants were quantified by isotope dilution. The procedure involves addition of internal standard solutions of ¹⁵N-labelled ammonium and nitrite to reagent blanks which are carried through each stage of the analysis at the same time as the test samples. Potassium chloride extractants, filter-papers, distillation reagents and atmospheric ammonia all contributed to dilution of the sample ¹⁵N. Some materials tested contained sufficient contaminants to cause large errors in the determination of sample ¹⁵N abundance. Both the amount and isotopic composition of contaminants can be determined by the isotope-dilution procedure, which permits the measured sample ¹⁵N abundance to be corrected for contamination.     

New method for prefractionation of plasma for proteomic analysis

The depth of proteome analysis is severely limited in complex samples with a wide dynamic range of protein abundance such as plasma. Removal of high‐abundance proteins should reveal the signal of lower abundance plasma proteins. However, smaller proteins may be part of larger protein complexes and hence the removal of proteins involved in complexes with high‐abundance proteins such as albumin may inhibit the search for disease biomarkers. Prefractionation of a sample divides it into fractions of reduced complexity, allowing improved detection of lower abundance proteins. Using a prefractionation device, which employs Gradiflow? technology, we were able to separate small volume plasma samples into multiple fractions based on the molecular weight and/or charge. The resulting samples of reduced complexity were directly compatible with 2‐DE. The use of this prefractionation machine may therefore be useful in the hunt for disease biomarkers.     

ZoomQuant: An application for the quantitation of stable isotope labeled peptides

The main goal of comparative proteomics is the quantitation of the differences in abundance of many proteins between two different biological samples in a single experiment. By differentially labeling the peptides from the two samples and combining them in a single analysis, relative ratios of protein abundance can be accurately determined. Protease catalyzed (18)O exchange is a simple method to differentially label peptides, but the lack of robust software tools to analyze the data from mass spectra of (18)O labeled peptides generated by common ion trap mass spectrometers has been a limitation. ZoomQuant is a stand-alone computational tool that analyzes the mass spectra of (18)O labeled peptides from ion trap instruments and determines relative abundance ratios between two samples. Starting with a filtered list of candidate peptides that have been successfully identified by Sequest, ZoomQuant analyzes the isotopic forms of the peptides using high-resolution zoom scan spectrum data. The theoretical isotope distribution is determined from the peptide sequence and is used to deconvolute the peak areas associated with the unlabeled, partially labeled, and fully labeled species. The ratio between the labeled and unlabeled peptides is then calculated using several different methods. ZoomQuant's graphical user interface allows the user to view and adjust the parameters for peak calling and quantitation and select which peptides should contribute to the overall abundance ratio calculation. Finally, ZoomQuant generates a summary report of the relative abundance of the peptides identified in the two samples.     

Limitations in detection of 15N incorporation by mass spectrometry in protein-based stable isotope probing (protein-SIP)

The method of protein-based stable isotope probing (protein-SIP) has previously been shown to allow the modeling of carbon fluxes in microbial communities, thus tackling one of the key questions in microbial ecology. The method allows the analysis of stable isotope distribution in peptides, revealing metabolic activities of the species present in an ecosystem. Besides carbon, an application of protein-SIP with nitrogen is of interest for resolving the nitrogen fluxes in microbial communities. Thus, the sensitivity and reliability of a protein-SIP approach employing ¹⁵N was analyzed. For this, cultivations of Pseudomonas fluorescens ATCC 17483 with different ratios of ¹⁴N/¹⁵N were performed, from 10 % down to 0.1 % ¹⁵N. After incubation leading to complete labeling of biomass, proteins were extracted and separated by one-dimensional gel electrophoresis, followed by tryptic digest and UPLC Orbitrap MS/MS analysis. ¹⁵N relative isotope abundance (RIA) was calculated based on isotopic patterns from identified peptides in mass spectra. Proteomics data have been deposited to ProteomeXchange with identifier PXD000127. The distribution of ¹⁵N RIA values among peptides was analyzed in samples with different ¹⁵N amount, and potential causes for variations within individual samples of either technical or biological origin were investigated. Using a number of 50 peptides, significant differences (p?≤?0.05) in ¹⁵N incorporation were found between samples of different ¹⁵N RIA down to 0.1 %. The study demonstrates that protein-SIP using ¹⁵N is sufficiently sensitive for quantitative investigation of microbial activity in nitrogen cycling processes.     

高效液相色谱-四极杆/静电场轨道阱高分辨质谱对水产品中未知污染物的非定向快速筛查与测定

建立了高效液相色谱-四极杆/静电场轨道阱高分辨质谱对水产品中污染物的非定向快速筛查与测定的方法。筛查时样品用乙腈提取、氮气浓缩吹干、甲醇-水溶液定容，采用全扫描数据依赖二级扫描模式进样分析。利用Trace Finder软件对水产样品中未知污染物的精确质量数、同位素丰度比、二级碎片离子进行数据库检索匹配。定量时样品采用优化的QuEChERS方法净化，对筛查过程确认的三环唑、咖啡因和乙氧基喹啉3种污染物进行目标离子二级扫描模式定量分析。鱼和虾中3种化合物在5~1000 μg/L范围内线性关系良好，相关系数均大于0.99；方法检出限（LOD）为1 μg/kg，定量限（LOQ）为5 μg/kg，平均回收率为70.5~90.9%，相对标准偏差为5.4%~12.8%。筛查方法具有快速、准确、高通量等优点，结合定量方法能够用于实际水产品中未知污染物的筛查与测定。     

The protein profile of Theobroma cacao L. seeds as obtained by matrix-assisted laser desorption/ionization mass spectrometry

The water-soluble protein profile of the seeds of green, red, and yellow Theobroma cacao L. fruits has been determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS). The seeds were powdered under liquid nitrogen and defatted. The residues were dialyzed and lyophilized. The obtained samples were suspended in the matrix solution of sinapinic acid. The obtained MALDI mass spectra showed the presence of a wide number of proteins with molecular weight ranging from 8000 to 13,000 Da and a cluster of peaks centered at 21,000 Da that were attributed to albumin. The abundance of this peak was found to depend on the different portion of the seed (husk, apical and cortical parts); however, the MALDI mass spectra obtained from the different varieties of cocoa were practically superimposable. Changes in the protein profiles were also observed after the cocoa seeds were treated by fermentation and roasting, which are processes usually employed for the commercial production of cocoa.     

快速微波法制备掺氮石墨烯用于碱性氧还原电催化剂

采用微波法在氨气气氛下快速加热石墨烯（G）制备了含氮量在4.05 wt%-5.47 wt%的掺氮石墨烯（NG）. 将上述的掺氮石墨烯用作碱性电解质条件下的氧还原电催化剂,起始还原电势为0.17 V（vs SHE）,接近商用碳载铂催化剂的0.21 V（vs SHE）. 采用透射电子显微镜、拉曼光谱和X射线光电子能谱研究了掺氮石墨烯的形貌、结构和掺杂氮原子的键合方式. 结果发现,掺氮石墨烯的氧还原起始电位随着石墨氮原子含量的提高而上升,说明石墨类型的氮含量是影响其氧还原催化活性的关键因素. 实验结果表明,微波法快速制备的掺氮石墨烯在碱性条件下表现出较高的氧还原催化活性,具有作为碱性燃料电池阴极催化剂的潜力.     

A cost-effective detection of low-abundance mutation with DNA three-way junction structure and lambda exonuclease

We presented a low-abundance mutation detection method with lambda exonuclease and DNA threeway junction structure.The assistant strand in the DNA three-way junction structure could regulate the reaction system from the kinetics and thermodyna mics aspects.The optimization of the assista nt strand helps to improve the selectivity of the mutant-type DNA to the wild-type DNA about 35 times.Moreover,the cost of the optimization process could be saved by about 90%.The method was applied to the detection of a human ovarian cancer-related gene mutation BRCA1(rs1799949,c.2082 CT).The limit of detection to the mutation abundance in the DNA three-way junction structure system(0.2%) was one order lower compared with that in the double-stranded DNA structure system(2%).The mutation abundance in different standard samples was quantitively measured,and the results were consistent with the initial abundance in the standard samples.     

钇的预注入对Ti-6Al-4V合金氮等离子体浸没离子注入行为的影响

为了提高氮等离子体浸没离子注入Ｔｉ－６Ａｌ－４Ｖ合金时氮原子的峰值深度,利用钇来增加氮在基体中的注入深度和扩散深度,先用２０ｋＶ的电压进行钇等离子体浸没离子注入处理,然后用３０ｋＶ的电压进行氮等离子体浸没离子注入处理。俄歇能谱测试表明氮原子的峰值深度有明显提高,注钇时间为３０ｍｉｎ的试件,氮原子的峰值深度从５０ｎｍ上升到１００ｎｍ。摩擦性能式表明处理后试件的抗磨损能力大大提高。     

Instrumental neutron activation analysis of some rock samples from Akwana and Arufu areas,Middle Benue Trough,Nigeria

Twenty nine (29) major, minor and trace elements including rare earth elements (REEs) were determined by INAA in rock samples from Akwana and Arufu areas, Middle Benue Trough, Nigeria. The rock units are located within the lead-fluorite and baryte mineralisation there. The concentrations of the major elements in the tertiary basalt (TB) and dolerite are similar while the elemental abundance in the granite gnesses is completely different. The elemental concentrations in the rock samples do not show any known relationship with fluorite abundance. The concentrations of some elements in both tertiary basalt and dolerite show them to be derived from alkali basaltic liquid. The chondritic normalised REE distribution patterns for the TB are similar to those of BCR-1 but the chondritic relative REE abundance is more enriched in TB than in the BCR-1. The chondritic normalised REE distribution patterns for the granite gneisses are also compared to those of the standard granite samples.     

Mesoionic trifluoroacetylated 5-imino oxazolines: a re-examination of the structure using 13C NMR spectroscopy and cycloaddition reactions

The position of the trifluoroacetyl group in mesoionic 2,3-diaryl-5-imino oxazolines has been disputed. The natural abundance ¹³C NMR spectra and the 1,3-dipolar cycloaddition product of the mesoionic oxazoline with the dimethylacetylenedicarboxylate are compatible with a structure in which the trifluoroacetyl group is bonded to the exocyclic 5-imino nitrogen.     

Mass spectra of some azoles

The principal electron-impact fragmentation patterns of 3,4,5-triphenyl-1,2,4-triazole, 2,5-diphenyl-1,3,4-thiadiazole, their perfluorinated analogues and 2,5-di(pentafluorophenyl)-1,3,4-oxadiazole have been established from metastable ion evidence and precise mass measurements. Although ions produced by expulsion of nitrogen from the molecular-ions of these compounds are of low abundance, the simultaneous expulsion of nitrogen and a C₇X₅ radical (X = H or F) gives rise to abundant ions.     

Sodium-lead alloy in the decomposition of organic samples for the detection of heteroelements

The decomposition of organic samples using open-tube fusion with sodium-lead alloy was demonstrated to be safe, complete, and controlled, in most cases. The detection of heteroelements in the fusion mixture of common organic samples was successful with samples of 10 to 20 mg. Detection of chlorine and nitrogen by classical methods in “difficult” compounds was secured, and the detection of fluorine in the fusion mixture of fluorocompounds extended the use of the alloy into this area. The use of the pyrazolone color reaction in the detection of nitrogen allowed the detection of more minute amounts of nitrogen than was previously detected using Prussian blue formation. The detection limits for heteroelements in organic samples were in the low microgram region. The application of sodium-lead alloy in quantitative work was briefly studied and will be considered in greater detail in the future.