Isotope-Ratio-Monitoring Liquid Chromatography Mass Spectrometry (IRM-LCMS): First Results from a Moving Wire Interface System

Abstract

A Liquid Chromatography-Combustion (LC-C) Interface, based on a moving wire technique, has been built and tested. The LC effluent is deposited onto a transport wire, which carries the sample through solvent evaporation and combustion ovens. CO₂ from the combustion step is analysed in an isotope ratio mass spectrometer. Performance of the interface was tested by loop injections of sucrose and glucose into a liquid flow of methanol/water (80/20). Accuracy and precision of δ¹³C_PDB < 1‰ was achieved for sample concentrations > 500 ng/ul (5μl loop), sufficient for studies at natural isotope ratios. In case of ¹³C tracer applications the detection limit was determined to be about 20 pg carbon tracer (on wire).     

Breath water-based doubly labelled water method for the noninvasive determination of CO2 production and energy expenditure in mice

ABSTRACT

We explored a novel doubly labelled water (DLW) method based on breath water (BW-DLW) in mice to determine whole body CO₂ production and energy expenditure noninvasively. The BW-DLW method was compared to the DLW based on blood plasma. Mice (n?=?11, 43.5?±?4.6?g body mass (BM)) were administered orally a single bolus of doubly labelled water (1.2?g H₂¹⁸O kg BM^?1 and 0.4?g ²H₂O kg BM^?1, 99 atom% (AP) ¹⁸O or ²H). To sample breath water, the mice were placed into a respiration vessel. The exhaled water vapour was condensed in a cold-trap. The isotope enrichments of breath water were compared with plasma samples. The ²H/¹H and ¹⁸O/¹⁶O isotope ratios were measured by means of isotope ratio mass spectrometry. The CO₂ production (RCO₂) was calculated from the ²H and ¹⁸O enrichments in breath water and plasma over 5 days. The isotope enrichments of breath water vs. plasma were correlated (R²?=?0.89 for ²H and 0.95 for ¹⁸O) linearly. The RCO₂ determined based on breath water and plasma was not different (113.2?±?12.7 vs. 111.4?±?11.0?mmol?d^–1), respectively. In conclusion, the novel BW-DLW method is appropriate to obtain reliable estimates of RCO₂ avoiding blood sampling.     

Changes in the magnetic structure of (La0.25Pr0.75)0.7Ca0.3MnO3 upon the isotopic substitution of 18O for 16O

A giant isotope effect, wherein the low-temperature metallic state is replaced by an insulator state, was recently observed when ¹⁸O was substituted for ¹⁶O in (La_0.25Pr_0.75)_0.7Ca_0.3MnO₃ [N. A. Babushkina, L. M. Belova, O. Yu. Gorbenko et al., Nature (London) 391, 159 (1998)]. In the present work, the temperature evolution of the magnetic structure of two samples of this compound is studied by neutron diffraction. One sample contained a natural mixture of oxygen isotopes, 99.7% ¹⁶O, while the other was enriched to 75% with the isotope ¹⁸O. It is established that the samples are crystallographically identical at room temperature. As the temperature decreases, the sample with ¹⁶O undergoes successive antiferromagnetic (T _AFM=5150 K) and ferromagnetic (T _FM=110 K) transitions, which lead to the establishment of a noncollinear ferromagnetic structure, while a purely antiferromagnetic (T _AFM=150 K) order arises in the sample with ¹⁸O. The temperature dependences of the intensities of the diffraction peaks associated with the charge ordering differ substantially in samples with ¹⁶O and ¹⁸O, and they correlate with the behavior of their electric resistance and magnetic structure. Pis’ma Zh. éksp. Teor. Fiz. 69, No. 1, 46–51 (10 January 1999)     

Canopy Gradients in δ18O of Organic Matter as Ecophysiological Tool

Abstract

We determined vertical oxygen isotope gradients of leaf organic matter for a grassland in Switzerland and a mountain beech forest (Fagus sylvatica) in Northern Italy. A distinctly positive ¹⁸O/¹⁶O gradient with height above ground was found for the grassland (7.9‰m^?1, p < 0.001), whereas the gradient was negative for the forest (–0.077‰m^?1, p < 0.001). The results are consistent with microclimatic measurements, although large isotope variations between the species have to be taken into account for the grassland. A conceptual scheme is shown which relates the isotope enrichment to the canopy density, considering the effects of transpiration and canopy structure. We conclude that the analysis of the within canopy variation in δ¹⁸O of organic matter can be used to provide long-term estimates of leaf water isotope composition, thus improving existing isotope methods to determine the gas-exchange between vegetation and atmosphere.     

Forensic Studies by EA-IRMS

Abstract The authenticity of natural and synthetic matter can be checked by measurement of the isotope ratios of C, N and S. Controlled substances like drugs of abuse (cocaine, heroin) and explosives (TNT) or, simply, traces of paint can hold information in their isotope pattern. Total combustion of samples in an elemental analyzer followed by on-line determination of the isotopes of the combustion products (CO(2), N(2), SO(2)) in an isotope ratio mass spectrometer (EA-IRMS) provides high sample throughout with a minimum of sample preparation.     

Can an IR Diode Laser Spectrometer be Used for the Determination of Natural Isotope Ratios in Biological Samples?

Abstract

An IR diode laser spectrometer can detect the enrichment of stable isotopes (¹³C [1] and ¹⁵N[2]) in tracer studies. However our system cannot detect differences of the natural abundances of these isotopes. This problem is not a principal limitation of the IR diode laser spectroscopy, but is inherent to our standard system. A new isotope ratio IR diode laser spectrometer has an accuracy high enough for most biological applications. Some advantages like flexibility in measuring different isotopes, insensitivity against other gases in the sample and price are correlated to this system.     

Possibility of mechanically stimulated transmutation of carbon nuclei in ultradisperse deuterium-containing media

The variation of the concentration of ¹⁴C in graphite subjected to vibratory milling in the presence of heavy (or light) water is investigated using methods of radiocarbon β-spectral analysis. It is discovered that the C+D₂O system exhibits a statistically significant increase in the β counting rate in comparison to the C+H₂O system milled under similar conditions. A quantitative model of the transmutation of the ¹³C isotope into the ¹⁴C isotope involving an interaction of multiphonon excitations with deuterons in the graphite crystal lattice in the presence of background thermal neutrons is proposed. Zh. Tekh. Fiz. 67, 100–107 (June 1997)     

The Measurement of Muscle Protein Synthesis in Broilers with a Flooding Dose Technique: Use of 15N-Labelled Phenylalanine,GC-MS and GC-C-IRMS

Abstract

An experiment was carried out to measure fractional muscle protein synthesis rates (k _s ) in broilers with injection of a flooding dose of phenylalanine (1 ml/100 g body weight of 150 mM phenylalanine; 38 atom percent excess (APE) [¹⁵N]phenylalanine). K _s was calculated from the [¹⁵N] enrichment in phenylalanine of tissue-free and protein-bound phenylalanine using both gas chromatography mass spectrometry (GC-MS) and gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) for measurements after a 10 min isotope incorporation period.

The tertiary-butyldimethylsilyl (t-BDMS) derivatives of phenylalanine were used for gas chromatographic separation in both systems. GC-MS and GC-C-IRMS were calibrated for a range of 7 to 37 [¹⁵N]APE and 0 to 0.62 [¹⁵N]APE, respectively, and for sample sizes of 0.45 to 4.5 nmol phenylalanine and 7 to 40 nmol phenylalanine, respectively. Reproducibility of standards as a measure of precision varied from 0.06 to 0.29 [¹⁵N]APE and from 0.0004 to 0.0018 [¹⁵N]APE in GC-MS and GC-C-IRMS, respectively.

K _s was measured in the m. pectoralis major of broilers fed rye based diets (56%) which were provided either unsupplemented (-) or supplemented (+) with an enzyme preparation containing xylanase. K _s in breast muscles was significantly increased from 21.8%/d to 23.9%/d due to enzyme supplementation.

It can be concluded from the study that the measurement of protein synthesis in broilers with the flooding dose technique can be carried out by using [¹⁵N]phenylalanine, GC-MS and GC-C-IRMS.     

Pressure dependence of the lattice constant of diamond: Isotopic effects

The pressure dependence of lattice constants of ¹³C-enriched diamond and diamond of natural isotopic composition have been studied by using x-ray synchrotron radiation and the imaging plate technique. The compression curves reveal features which indicate an inversion of the isotope effect at high pressure. Pis’ma Zh. éksp. Teor. Fiz. 63, No. 2, 73–77 (25 January 1996) Published in English in the original Russian journal. Edited by Steve Torstveit.     

Use of the optogalvanic effect (OGE) for isotope ratio spectrometry of 13CO2 and 14CO2

The use of isotopic carbon dioxide lasers for determination of carbon (and oxygen) isotope ratios was first demonstrated in 1994. Since then a commercial device called LARA^?, has been manufactured and used for Helicobacter pylori breath tests using ¹³C-labelled urea. The major advantages of the optogalvanic effect compared with other infrared absorption isotope ratio measurement techniques are its lack of optical background and its high sensitivity resulting from a signal gain proportional to laser power. Continuous normalisation using two cells, a standard and sample, lead to high accuracy as well as precision. Recent advances in continuous flow measurement of ¹³C/¹²C ratios of CO₂ in air and extensions of the technique to ¹⁴C, which can be analysed as a stable isotope, are described.     

Fiber laser intracavity absorption spectroscopy for in situ multicomponent gas analysis in the atmosphere and combustion environments

Intracavity absorption spectroscopy with a broadband Er³⁺-doped fiber laser is applied for the measurements of several molecular species revealing quantitative information about the gas concentration, temperature and chemical reactions in flames. The spectral range of measurements extends from 6200 cm⁻¹ to 6550 cm⁻¹ with the proper choice of the fiber length and by moving an intracavity lens. With a pulsed laser applied in this experiment, the sensitivity to absorption corresponds to an effective absorption path length of 3 km assuming the cavity is completely filled with the sample. For a cw laser, the effective absorption path length is estimated to be 50 km. Absorption spectra of various molecules such as CO₂, CO, H₂O, H₂S, C₂H₂ and OH were recorded separately in the cell and/or in low-pressure methane and propane flames. The presented measurements demonstrate simultaneous in situ detection of three molecular products of chemical reactions at different flame locations. Variation of the relative strengths of OH absorption lines with the temperature enables the estimation of the local flame temperature. The sensitivity of this laser does not depend on the broadband cavity losses and it can be used for in situ measurements of absorption spectra in hostile environments such as contaminated samples, flames or combustion engines. The presented technique can be applied for various diagnostic purposes, such as in environmental, combustion and plasma research, in medicine and in the determination of stable isotope ratios.     

Stable isotope determination of ester and ether methyl moieties in plant methoxyl groups

Plant methoxyl groups of lignin and pectin have both distinct stable hydrogen isotope (δ²H) and carbon isotope (δ¹³C) values that can be used for studying environmental processes and for investigating the origin and authenticity of biomaterials. Up to now, the reported methods have been applied only to determine isotope values of the bulk plant methoxyl pool. In this work, we have applied several methods to distinguish between stable isotope ratios of methoxyl groups of pectin and the bulk plant methoxyl pool. Our results demonstrate that by applying alkaline hydrolysis to specifically cleave off the ester methyl moiety (pectin-like), we can distinguish δ²H and δ¹³C values of the pectin methoxyl pool from the bulk methoxyl pool. No measureable isotope discrimination was observed either during sample preparation or during analytical measurement. Furthermore, using this method, no major isotope difference in either the hydrogen or carbon isotope signature of the methoxyl groups of plant pectin and bulk matter from plant species such as leaves from trees, apples, carrots and potatoes was noted. We show the methanol released during alkaline hydrolysis of plant material and subsequently treated with hydriodic acid to be an excellent procedure to measure specifically and precisely the δ¹³C and δ²H isotope values of plant pectin-like methoxyl groups. This method is particularly advantageous when plant matter with a low methoxyl content has to be analysed.     

Microcombustion of ng Amounts of Carbon in Non-Volatile Materials for isotope Ratio Evaluation

Abstract

A novel microcombustion technique for carbon isotopic analysis of nanogram amounts of carbon in non-volatile materials based on isotope ratio monitoring (irm) mass spectrometry is described. Liquid or solid samples placed in a quartz sleeve are combusted at 1000°C in a continuous stream of helium and oxygen. CO₂ removed from the carrier gas stream by cryogenic trapping is transferred onto a GC column. Following GC separation, the CO₂ is transferred via an open split to the ion source of a gas isotope ratio mass spectrometer. Reproducibility for samples >25 nmol carbon is <1‰. Problems associated with blanks from various sources and with reproducible deposition of small sample amounts led to variable accuracy, which was dependent on the compound class being analysed. Minimum sample size was in the range from 5 to 10 nmol carbon. Measurements of dissolved organic carbon (DOC) of groundwater from Germany yielded consistent values of δ¹³C = -28.8‰.     

Precise δ13C-determination in the Range of Natural Abundance on Amino Acids from Protein Hydrolysates by Gas Chromatography - Isotope Ratio Mass Spectrometry

Abstract

Routine on-line ¹³C-analysis by coupled gas chromatography - combustion – isotope ratio - mass spectrometry of individual amino acids out of mixtures in the natural abundance range demands their strictly standardized derivatization. For the N-acetyl-propylesters of amino acids from protein hydrolysates and blood serum a mean precision of 0.5% ° (1 SD) for derivatization, separation and measurement could be attained. From δ¹³C-values of derivatives δ¹³C-values of amino acids could be calculated by a carbon balance equation, implying an isotope effect on the C-1 of acetate of about 1.04 for nine amino acids. The global δ¹³C-value of casein calculated from the individual amino acids differed by less than 1.5%° from the directly determined value. The δ¹³C-values of amino acids from plant protein hydrolysates analyzed by the on-line method were in agreement with results obtained by classical procedures. The method permits the δ-value determination of up to nine amino acids from 200 μg of a mixture in less than one hour. By this means the detection of ¹³C-labeled amino acids in nmole amounts diluted by the 5000-fold amount of carrier becomes possible.     

15N/14N Analysis of Amino Acids with GC-C-IRMS - Methodical Investigations and Ecotoxicological Applications

Abstract

In order to perform the ¹⁵N/¹⁴N analysis of amino acids using a gas chromatograph and isotope ratio mass spectrometer linked up via a combustion interface (GC-C-IRMS), the amino acids must be derivatized. Tert-butyldimethylsilylation is examined using various techniques (direct conversion to nitrogen gas, ConFlo-IRMS [1], GC-C-IRMS [2]) and subsequently applied to isotopic characterization of amino acids from wheat protein hydrolyzate obtained from plants exposed to ozone. The method provides a reliable tool for studying ecotoxicological effects on plants at a molecular level in addition to the investigation of the natural variations of different N fractions.     

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.     

Isotope effect for transport and magnetic properties of La 0.35 Pr 0.35 Ca 0.3 MnO 3 thin films

The effect of ¹⁶ O → ¹⁸ O isotope substitution on electrical resistivity, magnetoresistance, and ac magnetic susceptibility was studied for La_0.35Pr_0.35Ca_0.3MnO₃ epitaxial thin films deposited onto LaAlO₃ and SrTiO₃ substrates. For the films on LaAlO₃, the isotope substitution resulted in the reversible transition from a metal-like to insulating state. The applied magnetic field ( H ≥ 2 T) transformed the sample with ¹⁸O back to the metallic state. The films on SrTiO₃ remained metallic at low temperatures for both ¹⁶O and ¹⁸O, but the shift of the resistivity peak corresponding to onset of metallic state exceeded 63 K after ¹⁶ O → ¹⁸ O substitution. The temperature dependence of both resistivity and magnetic susceptibility was characterized by hysteresis, especially pronounced in the case of the films on LaAlO₃. Such a behavior gives certain indications of the phase separation characteristic of interplay between ferromagnetism and charge ordering. Received 11 February 2000 and Received in final form 13 September 2000     

Accuracy and precision of a laser-spectroscopy approach to the analysis of δ2H and δ18O in human urine

The isotope ratio analysis of body water often involves large sample numbers and lengthy sample processing. Here we demonstrate the ability of isotope ratio infrared spectroscopy (IRIS) to rapidly and accurately analyse the isotope ratios of water in urine. We analysed water extracted from human urine using traditional isotope ratio mass spectrometry (IRMS) and compared those values with IRIS-analysed extracted water and un-extracted urine. Regression analyses for δ²H and δ¹⁸O values between (1) extracted water analysed via IRMS and IRIS and (2) urine and extracted water analysed via IRIS were significant (R ²=0.99). These results indicate that cryogenic distillation of urine was not required for an accurate estimate of the isotopic composition of urine when using IRIS.     

Sauna,sweat and science II – do we sweat what we drink?

ABSTRACT

Inspired by a previous ‘Sauna, sweat and science’ study [Zech et al. Isot Environ Health Stud. 2015;51(3):439–447] and out of curiosity and enthusiasm for stable isotope and sauna research we aimed at answering the question ‘do we sweat (isotopically) what we drink’? We, therefore, pulse-labelled five test persons in a sauna experiment with beverages that were ²H-enriched at about +25,600?‰. Sweat samples were collected during six sauna rounds and the hydrogen isotope composition δ²H_sweat was determined using an isotope ratio mass spectrometer. Before pulse labelling, δ²H_sweat – reflecting by approximation body water – ranged from –32 to –22?‰. This is ～35?‰ enriched compared to usual mid-European drinking water and can be explained with hydrogen-bearing food as well as with the respiratory loss of ²H-depleted vapour. The absence of a clearly detectable ²H pulse in sweat after pulse labelling and δ²H_sweat results of ≤+250?‰ due to a fast ²H equilibration with body water are moreover a clearly negative answer to our research question also in a short-term consideration. Given that the recovery of the tracer based on an isotope mass balance calculation is clearly below 100?%, we finally answer the question ‘where did the rest of the tracer go?’