Determination of stable carbon isotope ratios for molecules in natural organic matter using ESI FT-ICR MS

Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) enables nontargeted identification of molecular formulas in natural organic matter (NOM), yet molecular-level isotope analysis at natural abundance remains limited. Here, we present a comprehensive workflow for molecular formula–specific isotope analysis (MSIA) in NOM using FT-ICR MS, including calibration against universal reference materials (RMs). The central step of MSIA is an intensity-tuning strategy that, with sufficient spectral averaging, achieves sub–per mil (‰) precision and accuracy and enables robust isotopic comparison (Δδ 13 C) across samples. Between terrestrial and marine NOM, we observed Δδ 13 C values of 17.3, 8.8, and 10.1‰ for marine-enriched formulas C 19 H 22 O 10 , C 20 H 26 O 9 , and C 20 H 24 O 9 , respectively, whereas an invariant formula, C 18 H 22 O 6 , showed a nonsignificant difference (Δδ 13 C = 2.7‰, P = 0.296). Matrix effects for RMs spiked into NOM were within the method uncertainty, rendering existing RMs suitable for MSIA when matched in carbon number, peak intensity, and mass (<50 daltons). Under these conditions, molecular formula–level δ 13 C values were obtained, e.g., −46.8‰ for C 18 H 22 O 6 in terrestrial NOM.