by Dieter Juchelka, Senior Product Specialist, Thermo Fisher Scientific and Nils Kuhlbusch, R&D Scientist, Thermo Fisher Scientific
Isotope ratio analysis delivers unique insights into the natural processes all around us. The highly valuable approach allows actions that take place on the atomic level to be related to effects on much larger scales. Many fields currently benefit from isotope ratio analysis, including biochemistry, paleoclimate reconstruction and food authenticity, as the method determines subtle isotopic variations in a sample. These variations enable detailed information on past and present events to be ascertained. Key to unlocking this information, however, is measuring the isotope ratios with high precision.
Isotope ratio mass spectrometry (IRMS) using electron impact ionization and magnetic sector instruments is the current gold standard for isotope ratio analysis. Fundamentally, it determines the isotope ratios after the analyte has been converted into a low molecular weight gas (e.g., CO2, SO2, or N2) by combustion, pyrolysis, microbial fermentation or chemical treatment. All the isotope information is obtained in very few signals, giving a high precision analysis. But crucially, intramolecular isotopic information is lost, leaving important compositional questions unanswered.
New isotope ratio methodology can overcome this limitation and answer challenging questions in isotope ratio analysis. Here, soft electrospray ionization (ESI) ionizes polar compounds in liquid samples, which are then transferred into a Fourier transform mass spectrometer (FT-MS, i.e. Orbitrap) as intact molecular ions to acquire high resolution, accurate mass spectra. This enables the determination of isotope ratios from the analysis of intact isotopologues, conserving the intramolecular isotope information. So, how does ESI-FT-MS work, and what insights can the technique reveal?
Expanding Isotope Ratio Analysis with FT-MS
FT-MS for isotope ratio determination leverages its capabilities to resolve the peaks of a compound’s different isotopologues — molecules that differ only in their isotopic composition. Polar compounds dissolved in liquid solutions can be directly introduced into the system, requiring no preparatory chemical derivatization or conversion.
After being transferred into the mass spectrometer by the electrospray ionization source, the intact molecular ions are subsequently filtered by quadrupole technology to remove matrix interferences. This way, only a selected package of ions — including the compounds of interest — is processed by the mass analyzer, resulting in a mass spectrum such as that displayed in Figure 1 for nitrate. The peaks of the different isotopologue ions can be used to determine multiple isotope ratios directly from one mass spectrum.
Figure 1: Schematic overview of isotope ratio analysis workflow utilizing ESI-FT-MS technology.
The presented workflow for the analysis of polar analytes in liquid samples using ESI-FT-MS technology incorporates dual inlet methodology to determine highly accurate isotope ratios of unknown samples relative to a reference. The workflow also features a new data evaluation framework that enables conversion of isotopologue intensities to isotope ratios and the corresponding δ values, which are commonly used in the isotope field.
The Benefits of IRMS with ESI-FT-MS
A plethora of applications are available for IRMS with ESI-FT-MS: the technology enables the simultaneous acquisition of all major and some minor isotopologues (enabling abundance determination of 13C, 15N, 18O, 17O, 2H, 34S, 33S, 36S, and more). As FT-MS optionally achieves controlled fragmentation, position-specific isotope ratio data can be acquired for organic compounds. In addition, the abundances of isotopologues with multiple rare isotope substitutions, or so-called “clumped isotopes,” can be measured.
As well as directly measuring isotope ratios, ESI-FT-MS technology enables:
- Reliable data reporting: It facilitates the reporting of accurate results relative to international standards.
- Increased productivity: It requires less sample preparation and lower sample amounts then conventional methodology.
- Flexibility: IRMS with ESI-FT-MS technology can extract accurate isotopic information from singly- and multi-substituted isotopologues simultaneously.
The power of IRMS with ESI-FT-MS technology has been illustrated in a range of applications. Here we discuss how the technique can be used for oxyanion analysis, and to obtain the δ2H of biomolecules.
Investigating the Isotopic Composition of Intact Oxyanions
Oxyanions are vital constituents that play the role of both products and reactants in ecologically significant metabolic reactions in the ocean and other aquatic systems. Insights gained from the isotopic composition of oxyanions are highly useful in studying the biogeochemical cycles of sulfur and nitrogen. Sulfate (SO42-), for example, is abundant in the ocean and provides detailed information about microbial sulfate reduction in anoxic seabeds. Nitrate (NO3-), another important oxyanion, is the predominant form of bioavailable nitrogen in the ocean. Isotope ratio analysis of this constituent provides a way to study vital processes such as N2-fixation, assimilation, and denitrification.
A recent study highlighted the strength of isotope ratio analysis by FT-MS technology and electrospray ionization through simultaneous measurement of multiple singly- and multiply substituted isotopic forms of diverse oxyanions. Here, the team investigated the isotopic composition of sulfate, nitrate, and phosphate using Orbitrap technology. As the measurement is direct, there is no need to convert the oxyanions into room temperature gases by means such as the combustion of sulfate into SO2 or the conversion of nitrate into N2 or N2O, saving time and effort.
The study showed that the FT-based isotope ratio MS approach was precise and accurate for the isotope ratio analysis of oxyanions. Specifically, the method demonstrated analytical precision near shot-noise limits, enabling the detection of isotope ratio variations at the sub-permil level. The approach was highly accurate as well: although there are currently no IUPAC-certified sulfate standards, the experiment setup comparing the classical IRMS approach and FT-based isotope ratio MS demonstrated that δ34S can be determined with an accuracy of 1−2% relative to an in-house standard. The study also measured δ15N of a potassium nitrate reference material mixture, giving a linear response (R2 > 0.99).
Figure 2: Mass spectrum of sulfate (analyzed as HSO4-) acquired by ESI-FT-MS technology to determine isotope ratios for multiple major and minor isotopologues.
Further experiments even showed the method’s suitability for rare singly- and doubly-substituted isotopologues, including the clumped isotopologues such as a sulfate molecule bearing 34S and 18O substitution (34S18O, Figure 2).
Based on all these findings, we can clearly see that FT-MS technology with isotope ratio methodology offers the high levels of accuracy and precision needed for environmental applications. This means that deeper, more comprehensive studies using previously inaccessible isotopologues are now possible.
Better Understanding of Biomolecular Cycles
Hydrogen, the simplest of elements, can hide a wealth of details in its isotope biogeochemistry. Uncovering this treasure trove of knowledge grants access to deeper understanding across many disciplines, including biogeochemical cycles, paleoclimate studies, and ecology.
Despite its existence in practically every biomolecule, hydrogen isotope analysis is challenging. Currently, targets are limited to molecules that are abundant and analytically tractable, such as lipids and hydrocarbons. Additionally, while hydrogen is commonplace, deuterium is much less so. In fact, it constitutes less than 0.015% of hydrogen atoms at natural abundance. Any isotope ratio analytical methods for environmental applications, therefore, must be highly sensitive to count enough 2H-isotopologues to achieve low uncertainty on isotope ratios, while minimizing the experimental duration.
A new study developed a method for the simultaneous measurement of δ13C and δ2H of analytes in solution. In this work, acetate was used as the target analyte for two reasons: first, acetate plays a significant role in biogeochemical cycles, making it highly relevant, and second, it acts as both a substrate and product of metabolic processes, showing the method’s versatility.
Here, we can see just how powerful the new isotope ratio methodology is for biomolecule analysis. Most crucially, natural abundance δ2H and δ13C values can be simultaneously quantified by the method, saving significant amounts of analytical time. The analysis itself has a run time of 15 minutes and only requires 15 nmol of acetate. The method is highly precise here, too. Precisions better than 0.5 and 3% were obtained for δ13C and δ2H, respectively, demonstrating the suitability of the technique for environmental applications. The acquisition error (AE) also remained consistently proportional to the shot noise limit.
The insights gained through this study are invaluable to ocean sediment studies. Most notably, the study identified a large biological signal between fermenting and autotrophic acetogenic bacteria, — the two main sources of acetate in anaerobic environments. But the approach is applicable beyond ocean studies, too: acetate production and consumption also play a role in terrestrial wetlands.
ESI-FT-MS: Highly Productive, Sensitive, and Accurate Analysis of Isotope Ratios
Isotope ratio data is the key to understanding the natural processes around us, and analysts don’t need to endure missing intricate details in their results any longer. IRMS with ESI-FT-MS technology is a productive solution that delivers accurate and precise isotope ratio information from intact samples such as oxyanions and biomolecules. Whether looking to uncover valuable answers in biogeochemical cycles or complex metabolic pathways, ESI-FT-MS with isotope ratio methodology can take IRMS analysis to new levels of insight.