Identification and quantification of polyfunctionalized hopanoids by high temperature gas chromatography–mass spectrometry

Abstract

Hopanoids are triterpenoids produced mainly by bacteria, are ubiquitous in the environment, and have many important applications as biological markers. A wide variety of related hopanoid structures exists, many of which are polyfunctionalized. These modifications render the hopanoids too involatile for conventional gas chromatography (GC) separation, so require either laborious oxidative cleavage of the functional groups or specialized high temperature (HT) columns. Here we describe the systematic evaluation and optimization of a HT-GC method for the analysis of polyfunctionalized hopanoids and their methylated homologs. Total lipid extracts are derivatized with acetic anhydride and no further treatment or workup is required. We show that acid or base hydrolysis to remove di-and triacylglycerides leads to degradation of several BHP structures. DB-XLB type columns can elute hopanoids up to bacteriohopane-tetrol at 350 °C, with baseline separation of all 2-methyl/desmethyl homologs. DB-5HT type columns can additionally elute bacteriohopaneaminotriol and bacteriohopaneaminotetrol, but do not fully separate 2-methyl/ desmethyl homologs. The method gave 2-to 7-fold higher recovery of hopanoids than oxidative cleavage and can provide accurate quantification of all analytes including 2-methyl hopanoids. By comparing data from mass spectra with those from a flame ionization detector, we show that the mass spectromet (MS) response factors for different hopanoids using either total ion counts or m/z 191 vary substantially. Similarly, 2-methyl ratios estimated from selected-ion data are lower than those from FID by 10-30% for most hopanoids, but higher by ca. 10% for bacteriohopanetetrol. Mass spectra for a broad suite of hopanoids, including 2-methyl homologs, from Rhodopseudomonas palustris are presented, together with the tentative assignment of several new hopanoid degradation products.

Investigating molecular changes in organic matter composition in two Holocene lake‐sediment records from central Sweden using pyrolysis‐GC/MS

“…However, within this Py‐compound class, the sterol forms are a useful proxy for OM degradation status because of the high susceptibility to degradation [ Mackenzie et al , ]. Hopanoids are known to derive from the cell membranes of prokaryotes, especially bacteria [ Meredith et al , ; Sessions et al , ], but have not been determined directly with Py‐GC/MS and are rarely reported [ Buurman et al , ]. Similarly, the tocopherols are abundant in most photosynthetic organisms, because of their fundamental role in the photosynthetic process [ Maeda and DellaPenna , ].…”

A distinct pathway for tetrahymanol synthesis in bacteria

“…C-30 hopanoids, methylsterols, and tetrahymanol were analyzed via hightemperature gas chromatography-mass spectrometry (GC-MS) (52). Tetrahymanol was quantified by comparison with a cholestanol standard added to samples before derivatization and was normalized to the largest hopene peak detected in the sample.…”

“…The GC was coupled to a 5977A Series MSD with the source at 230°C and operated at 70 eV in EI mode scanning from 50 to 850 Da in 0.5 s. Aminohopanoids were detected via liquid chromatography-mass spectrometry at the Vincent Coates Foundation Mass Spectrometry Laboratory, Stanford University (mass-spec.stanford.edu). All lipids were identified based on their retention time and comparison with previously published spectra (33,52,53).…”

Diverse capacity for 2-methylhopanoid production correlates with a specific ecological niche

“…These samples appear in Figure 2 with either an identifier, if hopanoids were analyzed, or blank, if hopanoids were not analyzed. We also attempted to quantify hopanoids, specifically unextended hopanoids that cannot be identified by liquid chromatography-mass spectrometry, using standard GC-MS techniques (Sessions et al, 2013), but we were unable to unambiguously detect hopanoids due to a high background. Samples Figure S2).…”

“…were extracted as previously reported (Sessions et al, 2013). Methylated and non-methylated bacteriohopanepolyols were identified by liquid chromatographymass spectrometry as previously described (Welander et al, 2012).…”