BACKGROUND Traditional investigation of bacteriohopanepolyols (BHPs) has relied on derivatisation by acetylation prior to gas chromatography/mass spectrometry (GC/MS) or liquid chromatography/MS (LC/MS) analysis. Here, modern chromatographic techniques (ultrahigh-performance liquid chromatography (UPLC)) and new column chemistries were tested to develop a method for BHP analysis without the need for derivatisation. METHODS Bacterial culture and sedimentary lipid extracts were analysed using a Waters Acquity Xevo TQ-S triple quadrupole mass spectrometer in positive ion atmospheric pressure chemical ionisation (APCI) mode. Waters BEH C18 and ACE Excel C18 were the central columns evaluated using a binary solvent gradient with 0.1% formic acid in the polar solvent phase in order to optimise performance and selectivity. RESULTS Non-amine BHPs and adenosylhopane showed similar performance on each C18 column; however, BHP-containing terminal amines were only identified eluting from the ultra-inert ACE Excel C18 column. APCI-MS/MS product ion scans revealed significant differences in fragmentation pathways from previous methods for acetylated compounds. The product ions used for targeted multiple reaction monitoring (MRM) are summarised. CONCLUSIONS UPLC/MS/MS analysis using an ACE Excel C18 column produced superior separation for amine-containing BHPs and reduced run times from 60 to 9 min compared with previous methods. Unexpected variations in fragmentation pathways between structural subgroups must be taken into account when optimising MRM transitions for future quantitative studies. Copyright © 2016 John Wiley & Sons, Ltd.