The accumulation of dissolved organic matter (DOM) in the ocean has been associated with linear terpenoid structures, and carotenoid degradation products, which are considered potential precursors (Arakawa, et al., 2017; Semitsoglou-Tsiapou, et al., 2022). Steroidal compounds, particularly those resembling Carboxylic-rich Alicyclic Material (CRAM), are significant in marine biogeochemistry due to their role in DOM formation (Hertkorn et al., 2006; Liu et al., 2023). Phytosterols, such as fucosterol, β-sitosterol, campesterol, cholesterol, stigmasterol, ergosterol, and brassicasterol, are prevalent in brown macroalgae and marine environments (Hannan, et al., 2020). This study explores the photochemical degradation of stigmasterol (C₂₉H₅₀O) as a model for the formation of CRAM-like structures in marine DOM.
We conducted in vitro photooxidation of stigmasterol under UVA irradiation (365 nm, 5 μmole/m²/s) for three days and characterized the photodegraded products using Negative Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (ESI-FT-ICR MS). The goal was to identify water-soluble compounds with structural features similar to those observed in marine DOM samples isolated by the PPL resin-based solid-phase extraction (SPE) method.
Stigmasterol was dissolved in n-hexane and subjected to UVA irradiation. The photodegraded products were extracted with ultrapure water and purified using a Bond Elut-PPL resin cartridge. A dark control was included for comparative analysis. We identified thirty-one new CHO-class ions [C_8-29H_7-45O_2-7, m/z 179-505; DBE = 2–8, average DBE 5.65; O/C ratio 0.15–0.57; H/C ratio 0.82–1.79]. Notably, eight molecular formulas met the criteria for CRAM structures, including C₉H₇O₄, C₁₁H₁₁O₄, C₁₃H₁₃O₄, C₁₃H₁₃O₅, C₁₂H₁₅O₆, C₁₅H₁₉O₅, C₁₄H₁₉O₆, and C₁₇H₂₃O₅. Intensity variations were observed among these ions, with significant intensities noted for C₂₉H₄₃O₆ (DBE 8), C₂₈H₄₃O₆ (DBE 7), C₂₈H₄₃O₇ (DBE 7), C₂₈H₄₁O₇ (DBE 8), C₁₇H₂₅O₅ (DBE 5), and C₁₇H₂₃O₅ (DBE 6).
Comparative analysis with PPL-DOM from Coastal Seawater (CSW) and Deep-Sea Water (DSW) at 2000 m depth in the Mariana Trench revealed the presence of eighteen and seven molecules, respectively, with six and three molecular formulas aligning with CRAM criteria. These findings suggest that photochemical oxidation of stigmasterol leads to carboxylation reactions, contributing to the pool of recalcitrant marine DOM. The FT-ICR MS data provide molecular evidence supporting the role of photodegraded sterols in the formation of CRAM-like structures in marine environments.
 

Dissolved organic matter,CRAM,phytosterol,photochemical oxidation,precursor,recalcitrant organic matter,FT-ICR MS