Unveiling Lipidomics Quantitative Analysis: What, Why, and How

Lipids are indispensable biomolecules in living organisms, extensively involved in critical biological processes such as energy storage, cell signaling, and membrane structure formation. It’s widely reported that imbalances in lipid metabolism are closely associated with various diseases, including obesity, hypertension, diabetes, Alzheimer's disease, cardiovascular disease, Parkinson's disease, and cancer. However, the complexity and diversity of lipids make their study challenging. Lipidomics, a branch of systems biology, has emerged to analyze lipids within organisms comprehensively. This article details lipidomics from classification, its research significance, and the best practices for targeted lipidomics analysis based on the LC-MS/MS platform.

Figure 1. Abnormal lipid metabolism and associated diseases ^[1]

What is Lipidomics

Lipidomics is the systematic study of the types, functions, and dynamic changes of lipids within organisms, aiming to reveal their roles in physiological and pathological processes. Combining various analytical techniques, lipidomics is widely applied in exploring disease mechanisms, discovering biomarkers, and clinical diagnosis and treatment. Its research focuses include:

1. The types and quantities of lipids.

2. Lipid metabolic pathways and their regulatory mechanisms.

3. The role of lipids in cell signaling and disease.

4. Lipid bioanalysis and statistics.

Biological systems contain hundreds of thousands of different lipids. Natural lipids are typically composed of hydrophobic fatty acyl chains and polar head groups combined through different backbone structures (e.g., glycerol, sphingoid bases). Their structural diversity arises from variations in head group type, fatty acyl chain length, degree of unsaturation, double bond position, cis/trans geometric isomerism, acyl chain branching functional groups, and the type of linkage (ester, ether, vinyl ether) to the head group. Based on these chemical structure differences, the International Lipid Classification and Nomenclature Committee (ILCNC) proposed the "Comprehensive Classification System for Lipids" in 2005, categorizing lipids into 8 major classes: fatty acids, glycerolipids, sphingolipids, glycerophospholipids, prenols, sterols, saccharolipids, and polyketides.

Figure 2. The 8 major lipid categories and their representative structures ^[2]

These 8 categories constitute a vast lipid family. To address the field's need for standardized, systematic data resources, the National Institutes of Health (NIH) created the LIPID MAPS Structure Database (LMSD), which contains over 40,000 unique lipid structures across the eight lipid types. The specific data curated is shown in the table below.

Table 1. Lipid types and quantities in the LIPID MAPS structure database

Why Lipidomics Matters

As a crucial branch of metabolomics, lipidomics focuses on systematically deciphering the diversity, dynamic changes, and biological functions of lipid molecules in organisms. Its research significance is primarily reflected in the following dimensions:

• Unraveling the Complexity of Lipid Metabolism: The vast variety and diverse functions of lipid molecules necessitate comprehensive analysis by lipidomics to reveal their metabolic pathways and regulatory mechanisms, providing a scientific basis for understanding lipid metabolic complexity.

• Discovering Disease Biomarkers: Abnormal lipid metabolism is closely linked to various diseases. Lipidomics analyzes lipid changes in disease samples to discover potential lipid biomarkers, offering new avenues for early disease diagnosis.

• Deciphering the Role of Lipids in Cell Signaling: Lipid molecules play key roles in cell signaling. By studying the dynamic changes of lipid molecules, lipidomics reveals their regulatory mechanisms in cell growth, differentiation, and apoptosis.

• Advancing Precision Medicine and Personalized Therapy: Lipidomics analysis can identify individual differences in lipid metabolism, providing a scientific basis for precision medicine and personalized treatment.

• Promoting Bioenergy and Biomaterial Development: Lipidomics research provides theoretical support for developing bioenergy and biomaterials.

The complexity of lipid molecules—in terms of chemical structure (e.g., chain length, unsaturation, modifying groups) and dynamic range (concentrations spanning multiple orders of magnitude)—poses significant technical challenges to the sensitivity, resolution, and throughput of analytical methods. To address these challenges, modern lipidomics, centered around mass spectrometry (MS) and liquid chromatography (LC), has developed targeted lipidomics and untargeted lipidomics approaches. These approaches are used to identify and quantify lipids in cells, tissues, or organisms, thereby creating comprehensive lipid molecular profiles, investigating lipid biological functions, identifying disease-associated lipid metabolic changes, and developing therapeutic strategies targeting lipid metabolic pathways. The characteristics of the two lipidomics approaches are shown in Table 2.

Table 2. Targeted lipidomics vs. untargeted lipidomics

Targeted lipidomics, by precisely quantifying specific lipid molecules, provides a bridge connecting basic research and clinical applications. Its research and clinical applications are mainly reflected in the following aspects:

Figure 3. Targeted lipidomics bridges basic research and clinical applications

How to Conduct Quantitative Analysis for Targeted Lipidomics

Given the multi-dimensional research value of targeted lipidomics, achieving its scientific goals heavily relies on precise and efficient lipid analysis platforms. The combination of mass spectrometry and chromatography significantly enhances the ability for lipid identification, quantification, and functional analysis. The entire workflow includes sample collection, lipid extraction, mass spectrometry-based lipidomics analysis, data processing, and mass spectrometry-based lipidomics applications.

Figure 4. Workflow for mass spectrometry-based lipidomics analysis ^[3]

Targeted lipidomics is an analytical method focusing on specific lipid molecules or metabolic pathways, utilizing LC-MS/MS technology for high-sensitivity, high-specificity quantitative analysis of specific lipids. However, targeted lipid analysis faces multiple methodological challenges:

• Complexity of Lipid Chemical Structures: The presence of isomers and homologs in biological systems creates complexity in structural interpretation and separation.

• Interference from Endogenous Matrix Effects: Complex matrices in biological samples (e.g., plasma, tissue) can suppress or enhance target signals, affecting quantitative accuracy.

• Differences in Ionization Efficiency: Different lipids have vastly different ionization efficiencies and ionization responses (e.g., glycerophospholipids respond well in positive ion mode, while acidic phospholipids require negative ion mode).

• Limitations in Quantitative Dynamic Range: Insufficient dynamic range when high-abundance and low-abundance lipid signals coexist.

• Lipid Stability Issues: The abundant unsaturated bonds in lipids are prone to oxidation and hydrolysis by endogenous enzymes.

The core of targeted lipidomics lies in the precise analysis of key lipid molecules related to disease, metabolic regulation, or functional studies. Representative molecules analyzed based on their classification are shown in Table 3:

Table 3. Common categories and representative molecules in targeted quantitative analysis

Due to the importance of targeted lipidomics research, quantitative analysis will propel lipid metabolism research into a new era of precision, dynamics, and systematization.

WuXi AppTec DMPK has established quantitative analysis methods covering the 8 major lipid categories, based on Ultra-Performance Liquid Chromatography tandem Mass Spectrometry (UPLC-MS/MS) systems, offering a full range of targeted lipidomics analysis services. The following sections will systematically elaborate on the targeted quantitative analysis of several common lipids, including fatty acids, glycerophospholipids, sterols, and their metabolites.

Fatty Acid Analysis in LC-MS/MS-Based Lipidomics Research

Fatty acids serve as key metabolic hubs in lipid metabolism, performing multiple biological functions in organisms: acting as high-density energy carriers participating in the tricarboxylic acid (TCA) cycle for energy supply, forming the structural basis of the cell membrane phospholipid bilayer, regulating inflammatory signaling pathways through arachidonic acid metabolic cascades, and functioning as nuclear receptor (e.g., PPARs) ligands to regulate the expression of lipid metabolism-related genes.

Figure 5. Interconversion relationships between fatty acids and several other lipids in vivo ^[4]

Epidemiological studies confirm significant pathophysiological associations between abnormal circulating fatty acid profiles and insulin resistance, type 2 diabetes (T2DM), non-alcoholic fatty liver disease (NAFLD), and atherosclerotic cardiovascular disease (ASCVD). Accurate quantification of in vivo fatty acid concentrations is particularly important for disease monitoring.

Fatty acid LC-MS/MS analysis faces the following challenges:

• Interference from Structural Diversity: Isomer groups formed by carbon chain length (C2-C30), number of double bonds (0-6), and positional isomers (ω-3/ω-6) lead to insufficient chromatographic resolution and decreased specificity in MS identification.

• Wide Range of Physicochemical Properties: Short-chain fatty acids (SCFAs, C2-C5) have weak retention in reversed-phase chromatography due to high polarity, requiring optimization with hydrophilic interaction chromatography (HILIC); long-chain fatty acids (LCFAs) are prone to adsorption in tubing/columns due to strong hydrophobicity, requiring high-temperature column ovens and ammonium formate buffer systems to improve peak shape.

• Matrix Effects: Phospholipids in biological samples can inhibit ionization efficiency via steric hindrance, and high salt concentrations cause charge competition, necessitating an optimized solid-phase extraction (SPE) method during sample preparation and fine-tuning of ion source parameters.

• Dynamic Range Coverage Challenge: The concentration span from picomolar SCFAs to millimolar LCFAs requires techniques such as segment scanning or dynamic exclusion (DDA) to improve detection sensitivity.

• Limitations in Ionization Efficiency: Carboxyl groups easily form deprotonated [M-H]- ions in the ESI source; introducing derivatization reagents (e.g., AMP+ kit) can enhance ionization response.

Addressing the characteristics and analytical difficulties of fatty acids, WuXi AppTec DMPK has achieved comprehensive full-coverage analysis service for long- and short-chain fatty acids through chromatographic column selection, mobile phase, and gradient optimization; resolved carryover interference issues through needle wash solvent selection, additive types, and concentration interactions; and achieved chromatographic separation and accurate quantification in various animal matrices.

Figure 6. Chromatogram of two fatty acids separated in an animal matrix using a UPLC-MS/MS system

The challenges in fatty acid analysis stem from their structural diversity (chain length, double bonds), differences in physicochemical properties (polarity/hydrophobicity), and biological sample complexity. Solution strategies require combining chromatographic optimization (e.g., coupling HILIC/RPLC), derivatization/isotope labeling, high-resolution mass spectrometry, and software-assisted data processing to achieve accurate identification and quantification.

Glycerophospholipid Analysis in LC-MS/MS-Based Lipidomics Research

Glycerophospholipids are major components of cell membranes involved in signal transduction. Based on the nature of their polar head groups, the phospholipid family is further classified into phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA), cardiolipin (CL), etc. These compounds can be sequentially metabolized in vivo and play important roles in physiological processes, including participation in cell membrane formation, signaling, membrane protein regulation, organelle generation, and disease association. In LC-MS/MS analysis, based on compound properties, PA, PS, PI, etc., are typically analyzed in negative ion mode, while PC, PE, etc., are analyzed in positive ion mode.

Figure 7. Major phospholipid types in glycerophospholipids and their associated diseases ^[5]

As biomarkers, lysophospholipids play important roles in the glycerophospholipid metabolic pathway. For example, lysophosphatidylcholine (Lyso-PC) acts as an inflammatory mediator, regulating endothelial cell proliferation and apoptosis, thereby influencing the development of atherosclerosis. Lysophosphatidic acid (Lyso-PA) affects both cardiomyocyte apoptosis and fibroblast proliferation and is crucial in the occurrence and development of coronary heart disease. These two types of lysophospholipids are frequently detected as biomarkers in biological samples.

Lyso-PA contains both a hydrophilic phosphate group (polar) and a hydrophobic monoacyl chain (non-polar), leading to unstable retention behavior in reversed-phase chromatography, presenting analytical challenges such as peak tailing, significant carryover, and low signal in negative ion mode. Based on the physicochemical properties of lysophospholipid compounds and drug development needs, WuXi AppTec DMPK has optimized the analysis for Lyso-PA regarding its solubility, strong polarity, low abundance, and stability through the following methods:

• Select appropriate solvents to ensure compound dissolution and long-term storage stability.

• Chosen suitable precipitation agents and surrogate matrices, adding appropriate concentrations of stabilizers to ensure compound stability during analysis, and ensuring that surrogate matrix spike recovery meets requirements.

• Adjust mobile phase pH value and volatile salt concentration to achieve symmetric peak shape, low carryover, and stable retention time for lysophosphatidic acid compounds on the column.

• Adjust the mobile phase gradient to separate compound isomer interferences.

• Optimize ion source parameters to improve compound response in the mass spectrometer.

Figure 8. LC-MS/MS analysis of multiple phosphatidic acids

By systematically optimizing the mobile phase composition, gradient, pH, and column type in LC, the analytical performance for phosphatidic acids can be significantly improved, specifically reflected in:

• Lower detection limit, achievable down to 1 ng/mL.

• Reduced column carryover, with overall carryover below 0.5%.

• Improved peak shape, with symmetric chromatographic peaks without tailing, capable of separating isomers caused by different double bond positions.

• Understanding the impact of carbon chain length and double bonds on compound retention time in chromatography aids in the qualitative analysis of unknown compounds.

Sterols and Metabolite Bile Acids Analysis in LC-MS/MS-Based Lipidomics Research

Sterols are a class of lipid molecules with a tetracyclic structure. In lipid metabolism, they not only serve as key components of cell membranes but also participate in signal transduction, hormone synthesis, and energy metabolism regulation, playing vital roles in maintaining life activities and physiological functions.

Sterols contain only one hydroxyl group (-OH) as a polar group, with the rest of the structure being a hydrophobic tetracyclic carbon skeleton and alkyl side chain. Their overall low polarity and lack of strong acidic or basic functional groups make it difficult to form stable charged ions via protonation or deprotonation, often resulting in low signals during LC-MS/MS analysis. Therefore, employing a derivatization strategy to chemically modify sterol compounds can effectively improve mass spectrometry detection performance.

Figure 9. Schematic diagram of sterol compound derivatization ^[6]

Under specific conditions, sterols undergo a derivatization reaction with reagents such as picolinic acid. Derivatization can significantly enhance the ionization efficiency of the target analytes in the electrospray ionization (ESI) source. Simultaneously, the increased molecular weight and structural rigidity of the derivative can reduce non-specific fragmentation during mass spectrometric cleavage, increasing the abundance of the precursor ion, thereby achieving a higher signal-to-noise ratio in Multiple Reaction Monitoring (MRM) mode. Furthermore, derivatization can optimize the chromatographic retention behavior of compounds by introducing hydrophilic or hydrophobic groups, further reducing matrix effect interference. This dual enhancement effect makes derivatization a key technique for improving the detection sensitivity, method reproducibility, and quantitative accuracy of sterol compounds, especially suitable for precise analysis in trace biological samples or complex matrices. Acylation reagents and their mechanisms of action are listed in Table 4.

Table 4. Sterol acylation reagents and their functions

Acylation of sterol compounds can enhance the overall polarity of the compound, reduce the retention and carryover of the derivative in chromatography, and improve ionization efficiency in mass spectrometry, simultaneously addressing the challenges of MS sensitivity and chromatographic separation. This provides a universal technical framework for high-precision analysis of sterol molecules in complex biological samples.

Figure 10. Signal enhancement of cholesterol after derivatization

Bile acids, as the end products of cholesterol catabolism in the liver, participate in lipid metabolism. Their physiological functions span the entire process of lipid "digestion-absorption-transport-excretion." These molecules exert important effects through a unique dual-functional mechanism: on one hand, as amphipathic molecules, they physically emulsify and promote the digestion and absorption of dietary lipids; on the other hand, they precisely regulate cholesterol synthesis, lipoprotein metabolism, and energy balance by activating signaling pathways involving nuclear receptors (FXR, LXR) and membrane receptors (TGR5). Their enterohepatic circulation system not only maintains cholesterol homeostasis but also builds a bridge for host-microbiota metabolic dialogue, as secondary bile acids generated through gut microbiota modification can reciprocally regulate host lipid metabolism.

From a clinical application perspective, disorders in bile acid metabolism are closely associated with metabolic syndromes such as obesity, NAFLD, and atherosclerosis, and have become key targets for the treatment of dyslipidemia.

Based on the chromatography-mass spectrometry platform, WuXi AppTec DMPK has developed detection methods with high sensitivity and excellent resolution, successfully overcoming the technical bottleneck of simultaneously detecting multiple biomarkers in complex biological matrices using traditional methods. This method employs simultaneous positive and negative ion acquisition mode and can complete the analysis of multiple bile acids in just 10 minutes, providing reliable services for the precise analysis of the bile acid metabolic network. Figure 11 shows the TIC chromatogram of some bile acids.

Figure 11. Analysis chromatogram of selected bile acids

WuXi AppTec DMPK's development of targeted quantitative analysis service capabilities in lipidomics is not limited to the types mentioned above. Systematic analytical methods have been established for various lipids, including triglycerides (glycerolipids), ceramides, and sphingosine-1-phosphate (sphingolipids), erythromycin (polyketides), lipid IVA, and DSMP (saccharolipids), among others.

As an emerging discipline, lipidomics provides powerful tools for uncovering the mysteries of lipid molecules. Targeted quantitative analysis of lipids is not only significant in basic research but also shows broad application prospects in disease diagnosis, precision medicine, bioenergy, and other fields. In the future, with continuous technological advancements, lipidomics will continue to make important contributions to human health and sustainable development.

Final Words

Leveraging a comprehensive mass spectrometry technology platform, WuXi AppTec DMPK has established targeted lipidomics analysis service capabilities based on triple quadrupole and high-resolution mass spectrometers, enabling precise quantitative analysis of lipids. We are progressively exploring and establishing untargeted lipidomics analysis capabilities based on high-resolution mass spectrometers such as ThermoScientific Orbitrap Eclipse Tribrid MS System, Waters Vion IMS Q-Tof, and Xevo G2S Q-Tof. We have cultivated an efficient, high-quality, and professional analysis team. Based on analytical experience and lipid databases, we provide one-stop solutions from sample processing and multi-omics data integration to biomarker discovery, supporting drug research and development, disease mechanism exploration, and accelerating innovative drug development and the implementation of precision medicine.

Figure 12. WuXi AppTec DMPK lipidomics analysis service platforms

Authors: Wuyun Gong, Xianchun Zhang, Qian Chen, Shuo Zhang, Hongmei Wang, Zhiyu Li, Lili Xing

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