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bioRxiv · 10.1101/2025.09.26.678775

A Universal Method for Quantifying Elemental Sulfur and Selenium in Biological Matrices

Eldakra, N. E.·York, E. J.·Switzer, C. H.
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Abstract

Elemental sulfur occupies a central position in biological sulfur chemistry as a thermodynamically stable endpoint of reactive sulfur species (RSS) interconversion, yet its role in biology has remained poorly defined due to the lack of robust analytical methods for its direct measurement. Here, we describe a selective, accessible chemical workflow for the detection and quantification of zero-valent elemental sulfur (S0) and elemental selenium (Se0) in complex biological systems, enabling systematic interrogation of these species as biological redox metabolites. Elemental sulfur is isolated from biological samples as the cyclo-octasulfur (S8) allotrope via phase-selective organic solvent extraction and structurally confirmed by Raman spectroscopy. Extracted S8 is subsequently derivatized with triphenylphosphine (TPP) to form triphenylphosphine sulfide (TPP=S), which is quantified using complementary 31P-nuclear magnetic resonance (NMR) spectroscopy and GC-MS, providing sensitivity across nanomolar to millimolar concentration ranges. The method exhibits strong chemical selectivity against competing sulfur species and is compatible with a wide range of biological matrices, including conditioned media, cultured cells, tissues, and solid biological materials. Extension of the workflow to selenium chemistry enables selective detection and quantification of elemental selenium (Se0) following chemical or cellular reduction of selenite. Collectively, these results establish a practical and transferable analytical platform that renders zero-valent chalcogen species experimentally accessible in biological contexts. By enabling direct measurement of elemental sulfur across diverse biological systems, this method reveals S0 as a previously inaccessible, redox-active sulfur metabolite and provides a foundation for defining its formation, distribution, and function in cellular and tissue redox biology. Highlights{circ} S0 and Se0 are predicted redox endpoints, yet selective detection is limited. {circ}A broadly accessible chemical method is introduced to quantify S0 and Se0. {circ}Quantification spans nM-mM ranges in diverse matrices, e.g., cells and media. {circ}Reveals S0 as a redox-active metabolite present in multiple biological matrices.

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Eldakra, N. E., York, E. J., Switzer, C. H.. 2025-09-28. A Universal Method for Quantifying Elemental Sulfur and Selenium in Biological Matrices. https://doi.org/10.1101/2025.09.26.678775

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