Bile acid analysis

Step into the intricate world of bile acids and witness the revolution in their quantitative analysis and structural characterization, all thanks to the latest advancements in mass spectrometry.

Analytical challenges of bile acid analysis

Studying bile acids using LC-MS/MS presents several analytical challenges.

Bile acids are a complex class of molecules with diverse structures and properties, making their analysis intricate. They can exist in various forms, including free bile acids, conjugated bile acids (with glycine or taurine) and sulfated or glucuronidated bile acids. This structural diversity requires careful method development to ensure comprehensive detection and quantitation.

Important considerations include the following:

  • Many bile acids are structural isomers, differing only in the position of hydroxyl groups on the steroid nucleus. Differentiating these isomers requires high-resolution separation techniques and accurate mass spectrometry, which can be technically demanding.
  • Bile acids can be present at very low concentrations in some samples, requiring highly sensitive detection methods. Additionally, the dynamic range of bile acid concentrations can be broad, requiring methods that can accurately quantify both low and high concentrations.
  • Biological samples often contain numerous other compounds that can interfere with bile acid detection, causing matrix effects that suppress or enhance ion signals. Careful method validation and the use of internal standards can help mitigate these effects.
  • Bile acids can show varying ionization efficiencies in the mass spectrometer, which can affect sensitivity and quantitation. Optimizing ionization conditions—for example, using appropriate solvents, additives and ionization techniques such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI)—is crucial.
  • Accurate quantitation requires the use of suitable calibration standards and internal standards. Isotopically labeled bile acids are often used as internal standards to correct for variability in sample preparation and analysis.
  • To address these challenges, researchers often employ a combination of advanced chromatographic techniques, high-resolution mass spectrometry, robust sample preparation methods and thorough method validation protocols.

What is a bile acid?

Primary bile acids are cholesterol-derived molecules synthesized in the liver and collected and pre-concentrated in bile contained in the gall bladder. They share a common steroid nucleus with 4 interconnected rings. They have hydroxyl (–OH) groups attached at various positions on the steroid nucleus, which can vary in number and location, leading to different bile acids. They possess a carboxylic acid group attached to a side chain extending from the steroid nucleus.

In humans, 2 primary bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA), undergo intestinal bacterial-mediated bioconversion into chemically distinct secondary bile acids, deoxycholic acid (DCA) and lithocholic acid (LCA). Primary bile acids are often conjugated with the amino acids taurine or glycine (as taurocholic acid and glycocholic acid, for example) before being secreted into the bile. Gut microbiota can further metabolize primary bile acids to form many secondary bile acids.

While the biochemistry of these molecules is poorly understood, that is starting to change. The emerging importance of the microbiome in the gut-brain axis has driven considerable interest and effort into the study of these novel compounds.

The importance of bile acids

Bile acids have several important biological functions:

  • They emulsify dietary fats, increasing their surface area for the action of pancreatic lipases. This process is essential for the efficient digestion and absorption of fats and fat-soluble vitamins (A, D, E and K).
  • Bile acids aid in the excretion of cholesterol from the body. The conversion of cholesterol to bile acids in the liver is a major pathway for the removal of cholesterol.
  • Bile acids activate various signaling pathways by binding to specific receptors, such as the farnesoid X receptor (FXR) and the G-protein-coupled bile acid receptor (TGR5). These signaling pathways regulate metabolism, inflammation and energy homeostasis.
  • Bile acids possess antimicrobial properties, helping to maintain the balance of gut microbiota by inhibiting the growth of certain pathogenic microorganisms.

Although bile acids are often associated with gut health and microbiome research, they play multifaceted roles in human physiology, health and disease. The wider impact of these diverse molecules in cancer research, drug development, cardiovascular health, nutrition, obesity and exposomics make them a focus of ongoing research. The broad interest in bile acids stems from their central role in a variety of biological processes and their potential as therapeutic targets and biomarkers. The interdisciplinary nature of bile acid research underscores their importance, driving collaborative efforts across multiple scientific domains.

Bile acid analysis by LC-MS/MS

There are 2 different targeted strategies for comprehensive detection and quantitation of bile acids in human plasma.

A triple-quadrupole MS-based approach can provide sensitive quantitation of bile acids using highly resolved chromatographic separation and an internal standard strategy. Thomas Horvath, Baylor College of Medicine and Texas Children's Hospital Microbiome Center, developed a high-throughput method that provides baseline separation of a panel of bile acids, including several isobaric species. The use of individual bile acid isomers was required to fully optimize method parameters and compound ionization of this technically demanding assay. The validated method details can be found here.

A high-resolution mass spectrometry (HRMS)-based approach can also provide sufficient sensitivity for quantitation of endogenous bile acids with more comprehensive identification by:

  • Generating a high-resolution full product ion spectrum for each targeted bile acid
  • Using a narrow mass-to-charge (m/z) extraction window for fragment ions to reduce background chemical interferences and improve the signal-to-noise (S/N) of the assay

The addition of novel fragmentation strategies, such as electron activated dissociation (EAD), can help distinguish bile acid isomers in human plasma, removing the need for extensive chromatographic separations and laborious method development.

Understanding bile metabolism through accurate quantitation of bile acid isomers is paving the way for unprecedented metabolomics insights, with implications spanning from metabolic disorders to new therapeutic development.

Webinar

Novel approaches in bile acid analysis by LC-MS/MS

 In this webinar, Thomas D. Horvath, Baylor College of Medicine and Texas Children's Hospital Microbiome Center and Paul Baker, SCIEX, discuss innovative methodologies for enhancing bile acid quantitation using high-resolution mass spectrometry (HRMS) and triple-quadrupole MS (TQMS) systems.

Technical note

Quantitative analysis and structural characterization of bile acids using the ZenoTOF 7600 system

Get technical insight into how the SCIEX 7500 system and the ZenoTOF 7600 system can be used to quantify the bile acid content of human plasma sample extracts.

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