Lipids are a class of organic molecules found in nature that are hydrophobic or amphiphilic, insoluble in water but soluble in nonpolar solvents. They are present in most biological systems and serve as the structural material for cell membranes and the secondary source of energy. Lipids also participate in many important cellular functions. Many major human diseases are related to disorders in lipid metabolism, such as diabetes, obesity, cancer, Alzheimer's disease, and some infectious diseases.
As an important branch of metabolomics, lipidomics focuses on studying all lipid molecules in organisms and inferring changes in other biomolecules interacting with lipids, thereby revealing the important role of lipids in various life activities. Lipidomics is an overall study of lipid compounds related to these diseases, aiming to identify biomarkers that indicate these diseases.
The lipidomics workflow (Züllig et al.,2020).
Lipids exist in cells, organelles, and extracellular body fluids such as plasma, bile, milk, intestinal fluid, and urine. To study lipids in a specific location, the tissue or cells need to be isolated first. Since lipids are insoluble in water, organic solvents are usually used for extraction. The traditional extraction agent is a mixture of chloroform, methanol, and water. The required samples are extracted in this mixture to extract all lipids. Excess water is added to the extraction solution to separate it into two phases, with methanol and water on top and chloroform below. Lipids remain in the chloroform phase, and after evaporation and concentration, the desired lipids are obtained. This lipid extraction method can extract total lipids from tissue samples, reducing lipid loss, being easy to operate, and achieving good extraction results.
For the detection of only specific lipids in total lipids, solid-phase extraction (SPE) is a preferable method. It uses solid adsorbents to adsorb target compounds from liquid samples, separating them from the sample matrix and interfering substances. Then, the target compounds are eluted with elution solution or desorbed by heating, achieving the purpose of separation and enrichment of target compounds. Of course, due to the development of sample preparation techniques in chemical analysis, there are many other available sample preparation methods.
Solvent Extraction in Lipidomics
Liquid-liquid extraction is the most common method used in lipidomics research. This method involves the use of two immiscible organic solvents, with the most commonly used being chloroform, methanol, and water. Its aim is to achieve maximum extraction efficiency for key lipid classes, ranging from phospholipids and glycolipids to fatty acids, triglycerides (TAGs), and diacylglycerols (DAGs). Initially, the Folch lipid extraction method (chloroform/methanol/water at 8:4:3 v/v/v) was employed, followed by the Bligh and Dyer lipid extraction method (chloroform/methanol/water at 1:2:0.8 v/v/v).
Folch Lipid Extraction Method
(Folch et al., J Biol Chem 1957, 226: 497)
The sample tissue is homogenized in a 2:1 chloroform/methanol mixture, with the final solvent volume being 20 times that of the tissue (1g of sample in 20mL of solvent). The homogeneous mixture is then shaken on a rotary shaker at room temperature for 15-20 minutes. The homogenate is filtered through folded filter paper in a funnel or centrifuged to recover the liquid phase.
The liquid phase is washed with 0.2 volumes of water (4mL of water for 20mL of liquid phase), preferably with a 0.9% NaCl solution. After vortexing for a few seconds, the mixture is centrifuged at low speed (2000 rpm), the upper liquid phase is discarded using a pipette, and the interface is washed twice with 1:1 methanol/water if necessary (for removing labeled molecules), without mixing the entire preparation.
After centrifugal separation, the upper liquid phase is removed by pipetting, leaving behind the chloroform containing lipids, which are evaporated under vacuum in a rotary evaporator or blown with nitrogen to a volume of 2-3 mL.
Bligh and Dyer Lipid Extraction Method
(Can J Biochem Physiol 37: 911-917)
a. Add 3.75 mL of 1:2 (v/v) chloroform
per 1 mL of sample and vortex well. If GC analysis is to be performed, the solvent should contain an internal standard (e.g., 0.5 μg of cholesterol).
b. Add 1.5 mL of chloroform and vortex well.
c. Finally, add 1.25 mL of distilled water and vortex well.
d. Centrifuge at room temperature at 1000 rpm for 5 minutes to obtain a two-phase separation (aqueous phase on top, organic phase at the bottom) of the liquid.
e. Recover the organic phase: Using a Pasteur pipette, carefully draw off 90% of the lower organic phase solution by gently applying positive pressure through the upper aqueous phase, ensuring that the tip of the pipette reaches the bottom of the centrifuge tube without allowing the upper aqueous phase to enter the pipette.
Solid Phase Extraction (SPE)
SPE stands as a highly mature sample preparation technique, utilizing columns packed with a stationary phase and various mobile phases to selectively retain specific molecules that interact with the stationary phase. Typically, SPE is employed in conjunction with other methods like SOSE and LLE, serving as an additional purification step or enriching specific target lipid classes from biological fluids or solid samples. There is a wide array of extraction columns available on the market. SPE columns for lipid extraction include normal phase silica gel columns, reverse phase columns (C8 and C18), and ion exchange columns (amine columns). Silica gel and amine columns are often used for separating neutral and polar lipids by altering elution solvents to achieve separation. C8 and C18 columns, on the other hand, are used for separating phospholipids (PC), cerebrosides, glycolipids, and fatty acids from aqueous samples.
Different SPE columns are employed for different lipids. For instance, in the study of phospholipids implicated in atherosclerosis by Stübiger, a C18 purification column is utilized to extract and enrich oxidized phospholipids (OxPLs) from plasma lipids. The procedure involves pouring the lipid extraction solution into micro-preparative high-efficiency solid-phase extraction (mHP-SPE) C18 spin-columns (PepClean, Pierce). These columns are pre-washed with 500 mL of MeOH: 0.2% formic acid (70:30 w/w), followed by elution with 700 mL of MeOH: 0.2% formic acid (82:18 w/w) once, then with 800 mL of MeOH: 0.2% formic acid (92:2 w/w) once more. Finally, the columns are regenerated with 500 mL of 2-propanol to thoroughly remove lipids (i.e., neutral lipids) from the columns. The purity of the purified OxPLs is checked by thin-layer chromatography, and the obtained oxidized lipids are analyzed using LC-ESI-MS/MS.
In another study on skin ceramide lipidomics by Ruben t'Kindt, an amine-bonded silica gel column is used for purifying the lipid extraction solution. The method involves washing a 100 mg, 3.0 mL amine-bonded silica gel column with 2 mL of hexane. The dried lipids are dissolved in 300 μL of hexane: isopropanol (11:1 v/v), followed by elution of ceramides with 2 mL of hexane/methanol/chloroform (80/10/10 v/v). After drying with nitrogen gas, the ceramides are dissolved in 300 μL of isopropanol/chloroform (50/50 v/v) for HPLC/MS analysis.
Solid Phase Microextraction (SPME)
SPME, pioneered by the Pawliszyn research group in 1991 and commercialized in 1993, has become a widely adopted sample preparation technique. It combines extraction, concentration, desorption, and injection into a single process. SPME, rooted in solid phase extraction (SPE), retains SPE's benefits while eliminating the need for column packing materials and organic solvents for desorption. SPME employs a stationary phase (polymeric coating or adsorbent) coated onto a quartz fiber, serving as the absorption medium. It extracts and concentrates target analytes and directly thermally desorbs them in the gas chromatography injection port (or flushes them into a liquid chromatography column with the HPLC mobile phase, or even directly for mass spectrometry analysis). This technique is suitable for handling and analyzing volatile and semi-volatile organic compounds. SPME offers eight major advantages: simplicity of operation, versatility, low-cost equipment, rapid extraction, solvent-free, online and in vivo sampling capability, automation, and direct desorption in analytical systems. SPME can be used for detecting pollutants in the environment, such as pesticide residues, phenols, polychlorinated biphenyls, polycyclic aromatic hydrocarbons, fatty acids, amines, aldehydes, benzene derivatives, nonionic surfactants, organic metal compounds, inorganic metal ions, etc. It is also applicable in similar fields such as food, pharmaceuticals, clinical analysis, forensic analysis, etc. Naturally, SPME is also used in lipidomics research.
The charm of SPME lies in its ability to extract analytes from live samples, making it suitable for metabolomics and lipidomics research.
Supercritical Fluid Extraction (SFE)
Supercritical fluids possess unique physicochemical properties, with viscosities ranging from 1% to 10% of regular fluids, diffusion coefficients approximately 10 to 100 times that of ordinary liquids, and densities 100 to 1,000 times greater than atmospheric gases. As a result, supercritical fluids combine the large solubility capacity of liquids with the ease of diffusion and movement characteristic of gases, resulting in significantly higher mass transfer rates compared to liquid-phase processes. Therefore, they are favored for their extraction efficiency and environmental friendliness.
The most commonly used supercritical fluid is supercritical carbon dioxide (SF-CO2), characterized by its low critical pressure and temperature of only 7.4 MPa and 32°C, respectively. SF-CO2 is non-toxic and easily removed from samples, with a polarity similar to that of pentane, making it suitable for extracting hydrophobic compounds such as lipid compounds. To extract polar compounds, modifiers such as methanol are added to carbon dioxide. While lipid extraction from plant materials has been predominant in the past, recent advancements have extended the application to animal tissues. For instance, researchers utilized SFE devices to extract fatty acids from ostrich fat. The extraction setup included stainless steel extraction vessels, separators, an injection pump, and a condenser. Pressure was regulated using a pressure regulator, temperature controlled with an adjustable water bath, and carbon dioxide flow rate adjusted by pump frequency. Liquid carbon dioxide was pumped into the extraction vessel, reaching supercritical conditions. Adjusting pressure and temperature in the separators allowed the extracted components to be released. In one experiment, ostrich fat tissue was extracted with carbon dioxide for 5 hours, at pressures ranging from 15 to 30 MPa, temperatures from 40 to 50°C, and carbon dioxide flow rates from 15 to 35 L/h, to evaluate the extraction effectiveness.
However, the most significant application of SFE is in extracting lipid molecules from dried blood plasma spots. Studies have shown that SFE exhibits better selectivity for phospholipids than liquid-liquid extraction methods, including phosphatidylcholine (PC), lysophosphatidylcholine (lysoPC), phosphatidylethanolamine (PE), and sphingomyelin (SM).
Microwave-Assisted Extraction (MAE)
Microwave-Assisted Extraction (MAE) is a method that utilizes microwave energy to enhance the efficiency of solvent extraction, accelerating the extraction process of target compounds from solid samples through microwave heating. MAE rapidly and efficiently generates heat in both the sample and the solvent under the influence of high-frequency microwave energy, inducing dipole rotation, ionic conduction, and high-frequency friction. This leads to the rapid generation of heat within a short period. The rotation of dipole molecules results in the breaking of weak hydrogen bonds and accelerated penetration of solvent molecules into the sample matrix, facilitating the dissolution of analytes and significantly reducing the extraction time.
Microwave heating exhibits selective characteristics, as it selectively heats materials with different dielectric properties. Materials with low dielectric constants and minimal dielectric loss are essentially "transparent" to microwaves. Compounds with higher polarity, both solutes and solvents, absorb more microwave energy, leading to faster heating and promoting extraction speed. Conversely, non-polar solvents that do not absorb microwaves experience minimal heating. Therefore, when selecting extraction solvents, it is essential to consider their polarity to achieve optimal results.
MAE also demonstrates a biological effect (non-thermal effect). Since most biological entities contain polar water molecules, the action of microwaves induces strong polar oscillations, resulting in relaxation of intermolecular hydrogen bonds, breakdown of cell membrane structures, and acceleration of solvent penetration into the matrix and dissolution of the target components. Therefore, when extracting analytes from biological matrices using MAE, extraction efficiency is improved.
Ultrasound-Assisted Extraction (UAE)
Ultrasound waves, with frequencies above 20 kHz, represent mechanical vibrations propagating through a medium. During propagation, ultrasound interacts with the medium, causing changes in the phase and amplitude of the ultrasound waves. High-power ultrasound can induce alterations in the state, composition, structure, and functionality of the medium.
Applications of ultrasound-assisted extraction (UAE) can be categorized into two types: high-frequency, low-energy ultrasound waves (typically below 1 W/cm2) with frequencies mostly in MHz, and low-frequency, high-energy ultrasound waves (usually 10–100 W/cm2) with frequencies in kHz.
UAE is a method known for its good repeatability and high extraction quality. Unlike microwave-assisted extraction (MAE), UAE does not raise the temperature of the extraction system, which is advantageous for extracting metabolites with poor thermal stability. UAE can also be combined with liquid-liquid extraction (LLE) to improve the efficiency of lipid extraction from biological samples. Besides extracting biological fluids, UAE-LLE is also utilized for extracting fatty acids from various types of samples.
Other Available Extraction Methods
In chemical analysis sample processing, there are two important sample preparation methods: Accelerated Solvent Extraction (ASE) and Matrix Solid Phase Dispersion (MSPD), which can be used for sample preparation in lipidomics research.
Accelerated Solvent Extraction (ASE) is an automated method of extraction using organic solvents under elevated temperature and pressure conditions. Compared to other liquid extraction methods, its prominent advantages include minimal organic solvent usage, speed, and high recovery rates. Researchers such as Spiric et al. utilized ASE to extract fatty acid profiles and cholesterol content from carp flesh and compared it with an improved Soxhlet extraction method, demonstrating the viability of ASE extraction (Anal Chim Acta, 2010, 672:66–71). Jansen et al. employed ASE to extract lipid biomarkers from soil, showing comparable extraction efficiency to other methods (Appl Geochem, 2006, 21:1006–1015). Balasubramanian et al. conducted lipid extraction from marine microalgae cells in seawater using ASE and other methods, indicating the suitability of ASE as an extraction technique (Chem Engineering J, 2013, 215–216:929–936).
Matrix Solid Phase Dispersion (MSPD) was first proposed in 1989 as a method for processing animal tissue samples. The sample is ground together with various polymer carrier solid phase extraction materials impregnated with substances like C18, obtaining a semi-dry mixture which is then used as packing material in a column. Different solutions are then used to elute the column, extracting various analytes. The method relies on the use of lipophilic materials (C18) to disrupt cell membranes and disperse tissues, with C18 acting as a dispersant. Organic phases bonded to the surface of silica gel solid phase extraction materials act similarly to adsorbents used in traditional methods. During the stirring process of the sample with solid materials, shear forces disperse the tissues. The bonded organic phase acts like a solvent or detergent, dissolving and dispersing sample components on the surface of the support material. This significantly increases the surface area of the extracted sample, with components distributing according to their polarity in the organic phase. Non-polar components disperse in non-polar organic phases, while small polar molecules bond with silanol groups on the silica gel, and large weakly polar molecules disperse on the surface of the multiphase material.
Reference
- Züllig, T., Trötzmüller, M., & Köfeler, H. C. (2020). Lipidomics from sample preparation to data analysis: a primer. Analytical and bioanalytical chemistry, 412(10), 2191-2209.
