Phosphorus Analyzed in Soil Using Two-Dimensional Ion Chromatography-Tandem Mass Spectrometry

Scientists from the University of Saskatchewan in Saskatoon, Canada used two-dimensional ion chromatography-tandem mass spectrometry (2D-IC-MS/MS) to analyze phosphorus compounds in various soil samples. Their findings were published in the Journal of Chromatography A (1).

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Phosphorus (P) is a vital biotic macronutrient in various biochemical reactions. While soil P compositions can vary by environment due to biological, chemical, and physical factors, at most 80% can be present as organic phosphorus (P_o). Organic P species are defined by the phosphate link to a carbon moiety and include species such as phosphate monoesters, phosphate diesters, and phosphonates. While not available directly for uptake, Po mobilization can support soil processes when soil labile P concentrations are low. However, the proportions and solubilities of P_o impact the amount utilized by organisms. Phospholipids and nucleic acids rapidly degrade in soils while inositol phosphates generally build up in soils and stay unavailable for long periods of time. Understanding the composition of soil P_o can help scientists understand the roles of individual species during environmental processes.

For this study, 2D-IC-MS/MS was used to estimate soil P_o concentrations. Techniques like ion chromatography (IC) are usually hyphenated with MS. Further, chromatographic separation minimizes MS’s inability to resolve isobars or structurally similar compounds that share exact precursor and product ions. Chromatographic separation coupled with MS detection for P compounds has been successfully applied in various matrices, including human cells and plants. Further, IC-MS has been used to successfully analyze sugar phosphates and in metabolic profiling. However, MS-based methodologies for measuring P compounds in soil samples are scarce due to large matrix complexities. Multidimensional separation is ideal for multi-component separation, with 2D chromatography effectively improving resolution and sensitivity.

In the first dimension, early eluting anions were diverted to waste while P compounds were preconcentrated in a trap column; this was followed by chromatographic separation and detection in the second dimension. A mass spectrometer was used for detection, with quantification being performed using the multiple reaction monitoring scan (MRM) method, an MS-based technique used for accurately measuring the relative or absolute concentrations of particular molecules, such as peptides and proteins, that are identified in proteomic profiling studies (2).

The linear range of the studied P compounds, mostly consisting of nucleotides, was 0.05–50 ng/mL. Most P compounds were detected and quantified in calcareous subsoil samples in the ranges of 1.00–51.78 ng/g and 0.7–12.5 ng/g for active and stalled soil, respectively. The developed method achieved chromatographic separation that enabled unambiguous identification of isobars, isomers, and isotopologues contributing to MS detection interferences. With these findings, P compound speciation has been deemed possible compared to applying liquid ³¹P nuclear magnetic resonance (NMR) spectroscopy that relies on P compound grouping. However, improvements must be made to the extraction method and post-clean-up procedures, due to the complexity of soil extract composition, extreme matrix effect, and potential loss of analytes during preconcentration. Applying a cartridge to clean fulvic acid, humic acid, and other interferents did not show any improvement in reducing the matrix effect. For future works, the extraction method will be revised, and other solid-phase extraction (SPE) clean-up strategies will be used in hopes of reducing the matrix effect.

References

(1) Gachumi, G.; Schryer, A.; Siciliano, S. D. TWO-DIMENSIONAL ION CHROMATOGRAPHY TANDEM-MASS SPECTROMETRIC (2D-IC-MS/MS) METHOD FOR THE ANALYSIS OF PHOSPHORUS COMPOUNDS IN SOIL. J. Chromatogr. A 2024, 465287. DOI: 10.1016/j.chroma.2024.465287

(2) Multiple Reaction Monitoring. Elsevier B.V. 2024. https://www.sciencedirect.com/topics/medicine-and-dentistry/multiple-reaction-monitoring (accessed 2024-8-27)