While both branched-chain fatty acids (BCFAs) and straight-chain fatty acids (SCFAs) were analyzed, the profiling of BCFAs was considered especially important for future metabolomics and lipidomics studies.
A study from the University of Tübingen in Germany evaluated the performance of seven commercial ultrahigh-pressure liquid chromatography (UHPLC) columns prior to untargeted analysis of isomer-selective branched chain fatty acids (BCFAs) using UHPLC coupled to electrospray ionization (ESI) mass spectrometry with a quadrupole time-of-flight instrument (QTOF-MS) (1). QTOF-MS determination was further aided by sequential window acquisition of all theoretical mass spectra (SWATH, or SWATH-MS).
saturated and unsaturated fatty acids, triglyceride, triacylglycerol, triacylglyceride lipid, glycerol and 3 fatty acids, body fat in humans, structure formula, ball-and-stick model, 3d render | Image Credit: © MohammedElAmine - stock.adobe.com
The seven UHPLC columns were as follows: six Chiralpak polysaccharide columns (IA-U, IB-U, IC-U, ID-U, IG-U, and IH-U) and one Acquity charged surface hybrid (CSH) C18 column. The systematic retention and isomer selectivity study performed on these columns showed that the IG-U was the most effective in selectivity of short- and medium-chain BCFA isomers, while the C18 was preferred for long-chain isomers and was used for the liquid chromatography (LC) analysis of BCFAs with differing branching types and positions (1). Altogether, the method was used for the profiling of BCFAs in Staphylococcus aureus lipid extracts as well as pooled human platelets and plasma.
This study of fatty acids, appearing in the Journal of Chromatography A, explained the differences between straight chain fatty acids (SCFAs) and BCFAs by detailing BCFAs as some of the most abundant constituents of microbial lipids, used in bacteria to increase lipid fluidity and lower phase transition temperature (1). BCFAs have been found in human adipose and fetus biofilm tissue, and biofluids such as milk and serum, according to the study (1). Though fatty acids have traditionally been quantified by gas chromatography (GC) coupled to mass spectrometry (GC–MS) or flame ionization detection (GC-FID), their determination in lipidomics studies has more often been made by liquid chromatography (LC)-based methods which the researchers said often do not distinguish between SCFAs and BCFAs.
In the experiment, equivalent chain length numbers were measured for both the IG-U and C18 columns, and those calculations compared favorably to the derived fatty acid methyl esters (FAMEs) traditionally quantified by GC analysis. But regarding the preference for the C18 column over the other six that were tested, the researchers said silica-based polysaccharide chiral stationary phases (CSPs) showed potential in “uncommon applications,” namely lipidomic isomer separations as sought here, and had also previously demonstrated enantioselectivity for BCFAs (1). The C18 column itself had been used for fatty acid-focused lipid profiling, and to specifically detect triacylglycerol (TG) isomers in BCFAs.
For the bacterial samples studied, the researchers found that their optimized UHPLC–ESI-QTOF-MS method had a “strong” potential to detect different BCFA isomers, singling out anteiso and iso-BCFAs among the multiple branching positions that were tested (1). The utility of the mammalian platelet and plasma pool samples was to provide an evaluation of assay specificity in the presence of unsaturated fatty acids, and the isomer-selective LC methods showed an improvement over GC in preparative applications. The study concluded that, especially in conjunction with ESI-MS, the attention paid to the chromatographic approach here could be applied more broadly in subdisiciplines of both lipidomics and metabolomics.
(1) Fu, X.; Hafza, N.; Götz, F.; Lämmerhofer, M. Profiling of branched chain and straight chain saturated fatty acids by ultra-high performance liquid chromatography–mass spectrometry. J. Chromatogr. A 2023, 1703, 464111. DOI: 10.1016/j.chroma.2023.464111
Carol Robinson Awarded 2024 Lifetime Achievement European Inventor Award
July 24th 2024Carol Robinson of the University of Oxford has received the European Inventor Award 2024 for Lifetime Achievement from the European Patent Office for her work bringing mass spectrometry to structural biology.
Microseparations and Analysis Covered at HPLC 2024
July 23rd 2024At HPLC 2024 in Denver, Colorado, various sessions will be led by experienced scientists, covering different aspects of chromatography techniques and maximizing their effectiveness. Here is some information about tomorrow’s sessions.
Automated LC Method Development: An HTC-18 Interview with Bob Pirok
July 23rd 2024At HTC-18 in Leuven, Executive Editor of LCGC International, Alasdair Matheson, spoke to this year’s winner of the 2024 HTC-18 Innovation Award, Bob Pirok from the University of Amsterdam, about his innovative research on automated LC method development.