Isotope ratio analysis of acetate using Orbitrap Exploris Isotope Solutions

Significance of the Topic

Acetate is a central intermediate in the breakdown of organic matter in environmental settings and plays a critical role in global biogeochemical carbon cycling. Its rapid microbial turnover and low ambient concentration make it challenging to trace using conventional methods. Stable carbon and hydrogen isotopic fingerprints of acetate provide insights into its sources, transformation pathways, and the mechanisms of organic matter remineralization, especially under anoxic conditions.

Objectives and Study Overview

This study evaluates the performance of the Orbitrap Exploris Isotope Solutions platform for high-precision carbon and hydrogen isotope ratio measurements of acetate in complex environmental matrices. Key aims include demonstrating molecular-average and site-specific isotope analysis at nanomole sensitivity, assessing isotope ratio linearity over a range of concentrations, and validating an isolation workflow to remove isobaric interferences.

Methodology and Instrumentation

The workflow combines chromatographic purification of organic acids with electrospray ionization (ESI) Orbitrap mass spectrometry and advanced data processing. Sample introduction was achieved by:

• Dual Syringe Inlet system with diverter valve
• Automated in-flow injection via Vanquish Neo UHPLC

Purification of acetate involved:

• Precipitation of chloride and sulfate as AgCl and BaSO4
• Ion-exchange chromatography with pre-concentrator trap
• Neutralization of collected fractions and solvent exchange to methanol

Orbitrap Exploris 240 MS settings included negative-mode ESI, 60,000 resolution, 5 µL/min flow rate, and precise tuning of sweep gas, spray voltage, and AGC targets. Data were acquired with Xcalibur software and processed with IsoX, followed by R-based statistical evaluation.

Main Results and Discussion

High resolution accurate mass spectra resolved intact acetate isotopocules, enabling direct 13C/12C and 2H/1H ratio calculations. Isotope ratio measurements remained linear and accurate across 12.5–200 µM, with carbon ratios stable above a selected ion current (SIC) threshold of 1e8. Coeluting organic acids (propionate, butyrate) induced up to threefold ion suppression but did not bias isotope ratios within analytical precision. Mass-resolved carbonate contaminants within the 57–62 m/z window caused space-charge effects that suppressed 13C signals at high abundance; mitigation via reduced sweep gas, lower spray voltage, or AGC tuning restored accuracy.

Benefits and Practical Applications

The described strategy allows reliable isotope ratio analysis of acetate in saline brines, marine porewaters, and other challenging matrices at nanomolar levels. It supports studies of microbial metabolic pathways, carbon turnover in sediments, and greenhouse gas precursor fluxes. The platform also offers potential for site-specific hydrogen isotope mapping of methyl groups.

Future Trends and Opportunities

Advances may include integration of automated sample preparation, expansion to additional low-molecular-weight organic acids, and coupling with spatially resolved sampling technologies. Enhanced software algorithms for real-time interference correction and streamlined workflows will further improve throughput and accessibility for environmental monitoring and biogeochemical research.

Conclusion

This work demonstrates that Orbitrap Exploris Isotope Solutions combined with tailored chromatographic purification provides precise and accurate carbon and hydrogen isotope ratio measurements of acetate in complex environmental samples. The optimized protocols overcome ion suppression and contamination challenges, enabling routine nanomole-level isotope analysis for biogeochemical investigations.

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