A guide to precise measurements of isotope abundance by ESI-Orbitrap MS

Stable isotopes of carbon, hydrogen, nitrogen, oxygen and sulfur are widespread in nature. Nevertheless, their relative abundance is not the same everywhere. This is due to kinetic isotope effects in enzymes and other physical principles such as equilibrium thermodynamics. Variations in isotope ratios offer unique insights into environmental pollution, trophic relationships in ecology, metabolic disorders and Earth history including climate history. Although classical isotope ratio mass spectrometry (IRMS) techniques still struggle to access intramolecular information like site-specific isotope abundance, electrospray ionization&ndash;Orbitrap mass spectrometry can be used to achieve precise and accurate intramolecular quantification of isotopically substituted molecules (&lsquo;isotopocules&rsquo;). This protocol describes two procedures. In the first one, we provide a step-by-step beginner&rsquo;s guide for performing multi-elemental, intramolecular and site-specific stable isotope analysis in unlabeled polar solutes by direct infusion. Using a widely available calibration solution, isotopocules of trifluoroacetic acid and immonium ions from the model peptide MRFA are quantified. In the second approach, nitrate is used as a simple model for a flow injection routine that enables access to a diverse range of naturally occurring isotopic signatures in inorganic oxyanions. Each procedure takes 2&ndash;3 h to complete and requires expertise only in general mass spectrometry. The workflows use optimized Orbitrap IRMS data-extraction and -processing software and are transferable to various analytes amenable to soft ionization, including metabolites, peptides, drugs and environmental pollutants. Optimized mass spectrometry systems will enable intramolecular isotope research in many areas of biology. &nbsp;</p

Measurement of pulmonary surfactant disaturated-phosphatidylcholine synthesis in human infants using deuterium incorporation from body water

The aim of the study was to determine surfactant palmitate disaturated-phosphatidylcholine (DSPC-PA) synthesis in vivo in humans by the incorporation of deuterium from total body water into DSPC-PA under steady state condition. We studied three newborns and one infant (body weight (BW) 4.6 \ub1 2.9 kg, gestational age 37.5 \ub1 2 weeks, age 9 \ub1 9 days) and four preterm newborns (BW 1.3 \ub1 0.6 kg, gestational age 30.3 \ub1 2.5 weeks, postnatal age 8.8 \ub1 9.2 h). All infants were mechanically ventilated during the study and the four preterm infants received exogenous surfactant at the start of the study. We administered 0.44 g 2H 2O/kg BW as a bolus intravenously, followed by 0.0125 g 2H2O/kg BW every 6 h to maintain deuterium enrichment at plateau over 2 days. Urine samples and tracheal aspirates (TA) were obtained prior to dosing and every 6 h thereafter. Isotopic enrichment curves of DSPC-PA from sequential TA and urine deuterium enrichments were analyzed by Gas Chromatography-Isotope Ratio-Mass Spectrometry (GC-IRMS) and normalized for Vienna Standard Mean Ocean Water. Enrichment data were used to measure DSPC-PA fractional synthesis rate (FSR) from the linear portion of the DSPC-PA enrichment rise over time, relative to plateau enrichment of urine deuterium. Secretion time (ST) was defined as the time lag between the start of the study and the appearance of DSPC-PA deuterium enrichment in TA. Data were given as mean \ub1 SD. All study infants reached deuterium-steady state in urine. DSPC-PA FSR was 6.5 \ub1 2.8%/day (range 2.6-10.2). FSR for infants who did not receive exogenous surfactant was 5.7 \ub1 3.5%/day (range 2.6-9.9%/day) and 7.3 \ub1 2.1%/day (range 5.1-10.2%/day) in the preterms, whereas DSPC-PA ST was 10 \ub1 10 h and 31 \ub1 10 h respectively. Surfactant DSPC-PA synthesis can be measured in humans by the incorporation of deuterium from body water. This study is a simpler and less invasive method compared to previously published methods on surfactant kinetics by means of stable isotope

Mass spectrometric method for the absolute calibration of the intramolecular nitrogen isotope distribution in nitrous oxide

A mass spectrometric method to determine the absolute intramolecular (position-dependent) nitrogen isotope ratios of nitrous oxide (N2O) has been developed. It is based on the addition of different amounts of doubly labeled 15N2O to an N2O sample of the isotope ratio mass spectrometer reference gas, and subsequent measurement of the relative ion current ratios of species with mass 30, 31, 44, 45, and 46. All relevant quantities are measured by isotope ratio mass spectrometers, which means that the machines inherent high precision of the order of 10–5 can be fully exploited. External determination of dilution factors with generally lower precision is avoided. The method itself can be implemented within a day, but a calibration of the oxygen and average nitrogen isotope ratios relative to a primary isotopic reference material of known absolute isotopic composition has to be performed separately. The underlying theoretical framework is explored in depth. The effect of interferences due to 14N15N16O and 15N14N16O in the 15N2O sample and due to 15N 2 + formation are fully accounted for in the calculation of the final position-dependent nitrogen isotope ratios. Considering all known statistical uncertainties of measured quantities and absolute isotope ratios of primary isotopic reference materials, we achieve an overall uncertainty of 0.9 (1). Using tropospheric N2O as common reference point for intercomparison purposes, we find a substantially higher relative enrichment of 15N at the central nitrogen atom over 15N at the terminal nitrogen atom than measured previously for tropospheric N2O based on a chemical conversion method: 46.3±1.4 as opposed to 18.7±2.2. However, our method depends critically on the absolute isotope ratios of the primary isotopic reference materials air–N2 and VSMOW. If they are systematically wrong, our estimates will also necessarily be incorrect

Discovering Nature’s Fingerprints: Isotope Ratio Analysis on Bioanalytical Mass Spectrometers

For a generation or more, the mass spectrometry that developed at the frontier of molecular biology was worlds apart from isotope ratio mass spectrometry, a label-free approach done on optimized gas-source magnetic sector instruments. Recent studies show that electrospray-ionization Orbitraps and other mass spectrometers widely used in the life sciences can be fine-tuned for high-precision isotope ratio analysis. Since isotope patterns form everywhere in nature based on well-understood principles, intramolecular isotope measurements allow unique insights into a fascinating range of research topics. This Perspective introduces a wider readership to current topics in stable isotope research with the aim of discussing how soft-ionization mass spectrometry coupled with ultrahigh mass resolution can enable long-envisioned progress. We highlight novel prospects of observing isotopes in intact polar compounds and speculate on future directions of this adventure into the overlapping realms of biology, chemistry, and geology

Purification and Gas Chromatography–Combustion–Isotope Ratio Mass Spectrometry of Aroma Compounds from Green Tea Products and Comparison to Bulk Analysis

A method for carbon isotope ratio (δ¹³C) analysis was developed for compound-specific isotope analysis of tea volatiles, and the values were compared with the δ¹³C value from bulk isotope analyses. The δ¹³C value of 2-phenylethanol liberated via enzymatic hydrolysis of the 2-phenylethyl β-primeveroside standard was examined first. Isotope fractionations for 2-phenylethyl β-primeveroside from preparative high-performance liquid chromatography (HPLC) were also analyzed. The enzymatic treatment and the preparative HPLC process did not cause carbon isotope fractionations, substantiating the strategies available for δ¹³C analysis of volatile compounds. On the basis of the gas chromatography–combustion–isotope ratio mass spectrometry data from 2-phenylethanol, it was possible to derive the conditions for enzyme treatment and preparative HPLC of the glycoconjugates of 2-phenylethanol, (<i>Z</i>)-3-hexenol, and benzyl alcohol isolated from green tea leaves. Larger variations in δ¹³C were found for individual volatile compounds compared with bulk analytical data from the leaves, indicating the potential to utilize this strategy in assigning the geographical origin of green tea