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Recent Advances in Methods for Analyzing Lipids and Fatty Acids

Published: March 3, 2023


Lipids represent a large class of molecules that play vital roles in biological processes. Owing to the huge number and variety of lipid species among diverse biological systems, the fast and accurate quantification of lipids remains a difficult task, especially when the target molecules are low-abundance lipid species that are complex and rare. Recent advances in lipid extraction, identification, and quantification have greatly promoted the understanding of the functions of distinct lipid species. This methods collection aims to cover recent advances in the methods developed for the analysis of lipids originating from different resources, including advances in sample preparation, separation, identification, and quantification.

The aim of the method described by Parrish and Wells is to determine the total lipid content and distinguish the lipid species in marine samples1. The lipids and biological specimens in seawater are first collected by filtration and are then recovered by liquid-liquid extraction in non-polar organic solvents, which are composed of chloroform or mixtures of chloroform and methanol. The latter mixture is used when solids are present. After FAME derivatization with H2SO4 in MeOH, the lipids are measured by rod thin-layer chromatography and detected with flame ionization. The advantage of this method is that particulate lipid-containing fractions of marine samples can be isolated in the separation step using filters at a defined cutoff. In addition, liquid-liquid extraction facilitates the separation of the lipids from the other components in the aqueous matrix. The hydrophobic nature of lipids contributes to the isolation of lipids from marine samples and their enrichment. The protocol may have applications in ecological and biogeochemical studies aiming to evaluate ecosystem health, such as studies measuring the degree of impact of anthropogeny on an ecosystem.

Gangliosides are sialic acid-bearing glycosphingolipids, and they are extremely abundant in the brain. Gangliosides are physiological modifiers and therapeutic targets across cancer, metabolism, and brain function. In this context, Porter et al. describe bench-level methods that can be used for the isolation, purification, and semiquantitative or qualitative analysis of gangliosides2. These techniques, which include thin-layer chromatography analysis, allow the maximization of ganglioside yields at large and small scales while streamlining the purification, and they also ensure optimal recovery and accurate analyses. The method described here is useful for researchers who are interested in ganglioside isolation, purification, and analysis in biomedical discovery and therapeutic development.

The method developed by Angelini et al. is used to generate cardiolipin fingerprinting of leukocytes by MALDI-TOF mass spectrometry3. The method allows the measurement of the ratio between monolysocardiolipin and cardiolipin for the rapid diagnosis of Barth syndrome. MALDI-TOF mass spectrometry is used for obtaining the lipid profiles of small amounts of various biological samples to identify lipid biomarkers or pathological alterations. The main advantage of this technique is the detection of diagnostic lipid species by a single round of mass spectrometry immediately after the isolation of leukocytes from 1 mL of blood. The high efficiency and the eye sensitivity and specificity of this assay make it a suitable diagnostic test to be incorporated into the routine work of clinical laboratories for the screening of Barth syndrome.

Lipid-based excipients (LBEs) have certain advantages, such as low toxicity, good biocompatibility, and natural-based processing, and the application of LBEs contributes to the sustainable development of pharmaceutical manufacturing. Consequently, understanding solid-state alterations in LBEs and their effect on the performance of LBE pharmaceutical products is a key factor in manufacturing robust lipid-based dosage forms. The protocol described by Salar-Behzadi et al. shows that X-ray diffraction and differential scanning calorimetry can be referred to as gold standards in the investigation of solid-state LBEs4. When LBEs are used as a coating material or as an encapsulation matrix, differential scanning calorimetry (DSC) allows for the screening of the thermal behavior of lipids and the assessment of the miscibility of the potentially active pharmaceutical ingredients (API).

Although protocols for lipid analysis are well-documented, in order to be applied to different scientific domains, they often require modifications according to the research conditions and sample types. In aquatic sciences, determining the lipid species and content of aquatic organisms is a thriving field of study5. A Chromarod Iatroscan TLC-FID system can generate rapid synoptic lipid class data from small samples. Therefore, it can be used to screen marine samples before conducting detailed chromatographic studies. When combined with qualitative microscopic methods, this system can be a very useful tool to investigate micro-level crystallization.

To date, the characterization of isomeric lipids is still challenging due to the presence of species that have sophisticated structures, such as double bond positions, different orientations, and multiple acyl chain connectivity with distinct functional group stereochemistry6. In the future, combined approaches and/or advanced analytical techniques will be needed for effective lipid characterization and, more importantly, to better understand the novel lipid species that are continually being discovered7. Further analytical tools should be developed to match the selected sample preparation process. In order to design highly effective lipid-based pharmaceuticals, it is necessary to understand the structure–function–processability relationships of lipids. This understanding will, in turn, lead to the establishment of novel lipid function roles and the development of more efficient disease diagnostics and drugs. Furthermore, high-throughput methods for the fast screening of the cells producing lipids or fatty acids are highly desirable. In this context, this methods collection has demonstrated that optimized analytical techniques can characterize diverse lipid species with high accuracy and specificity, and that the optimization and standardization of current methods could promote the understanding of the biological mechanisms of lipids and their usage.


The authors have nothing to disclose.


The authors have no acknowledgments.


  1. Parrish, C. C., Wells, J. S. Determination of total lipid and lipid classes in marine samples. Journal of Visualized Experiments. (178), e62315 (2021).
  2. Porter, M. J., Zhang, G. L., Schnaar, R. L. Ganglioside extraction, purification and profiling. Journal of Visualized Experiments. (169), e62385 (2021).
  3. Angelini, R., Russo, S., Corcelli, A., Lobasso, S. Fingerprinting cardiolipin in leukocytes by mass spectrometry for a rapid diagnosis of Barth syndrome. Journal of Visualized Experiments. (181), e63552 (2022).
  4. Salar-Behzadi, S., Corzo, C., Laggner, P. A package of established analytical tools to investigate the solid-state alteration of lipid-based excipients. Journal of Visualized Experiments. (186), e63993 (2022).
  5. Couturier, L. I. E., et al. State of art and best practices for fatty acid analysis in aquatic sciences. ICES Journal of Marine Science. 77 (7-8), 2375-2395 (2020).
  6. Zheng, X., Smith, R. D., Baker, E. S. Recent advances in lipid separations and structural elucidation using mass spectrometry combined with ion mobility spectrometry, ion-molecule reactions and fragmentation approaches. Current Opinion in Chemical Biology. 42, 111-118 (2018).
  7. Merrill, A. H., Dennis, E. A., McDonald, J. G., Fahy, E. Lipidomics technologies at the end of the first decade and the beginning of the next. Advances in Nutrition. 4 (5), 565-567 (2013).

Cite this Article

Zhang, L., Guo, Z. Recent Advances in Methods for Analyzing Lipids and Fatty Acids. J. Vis. Exp. (193), e64976, (2023).More

Zhang, L., Guo, Z. Recent Advances in Methods for Analyzing Lipids and Fatty Acids. J. Vis. Exp. (193), e64976, (2023).

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