Methodological Development and Theoretical Framework for the Isotopic Analysis of Mass Spectral Fragments of Phytol...

Recent advances in Fourier Transform Orbitrap mass spectrometry have enabled precise position-specific isotope analysis (PSIA) of organic molecules at natural abundance by resolving isotopologues in molecular fragments. Here, we present a novel Orbitrap-based method to resolve intramolecular ¹³C isotopic patterns in a long-chain, apolar lipid at natural abundance. The method uses atmospheric pressure chemical ionization (APCI) with higher-energy collisional dissociation (HCD) for fragmentation, achieving analytical measurement precision of ∼3‰ across 26 fragment ions, measured simultaneously, using <1.2 μg of analyte within a 45 min analysis. We demonstrate the method on phytol, a ubiquitous long-chain, branched isoprenoid alcohol, mostly derived from the side chain of the chlorophyll molecule, and which is the biosynthetic precursor of phytane, a fossil lipid ubiquitously found in sediments. Our results reveal systematic differences in δ¹³C values of fragment ions indicative of intramolecular ¹³C heterogeneity within phytol from different biosynthetic origins. This approach provides a robust isotopic fingerprinting tool for tracing the metabolic and environmental history of phytol and other isoprenoid lipids.