Purification of dinosterol for hydrogen isotopic analysis using high-performance liquid chromatography–mass spectrometry

A semi-preparative normal-phase high-performance liquid chromatography–mass spectrometry (HPLC–MS) method is presented for the purification of various alcohol fractions from total lipid extracts derived from sediments, for the purpose of hydrogen isotopic measurement by gas chromatography–isotope ratio mass spectrometry (GC–IRMS). 4-Methylsterols, including the dinoflagellate-specific marker dinosterol (4,23,24-trimethylcholestan-22-en-3β-ol), were successfully separated from notoriously co-eluting plant-derived pentacyclic triterpenoid alcohols and alkyl alcohols. We find that substantial hydrogen isotope fractionation occurs during chromatographic separation, demonstrating the importance of recovering the entire peak when subsequent hydrogen isotope analyses are to be performed. This is the first report of such hydrogen isotopic fractionation for a natural unlabelled compound.