Metabolomics Creative Proteomics

Bile Acids Analysis with Absolute Quantitation & Isomer Resolution

Bile acids are structurally complex and exist in diverse forms, making accurate measurement challenging. Creative Proteomics offers targeted, isomer-aware analysis delivering absolute quantitation across primary, secondary, conjugated, sulfated, glucuronidated, and muricholic acids—optimized for your matrix and research goals.

Key advantages

  • Isomer resolution to prevent data misinterpretation
  • Ultra-sensitive detection in complex matrices
  • Comprehensive coverage in a single run
  • Validated methods with isotope-labeled standards
  • Decision-ready results and full QC
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What Are Bile Acids?

Bile acids are cholesterol-derived amphipathic molecules that act as detergents, modulators of nutrient absorption, and potent signaling ligands (e.g., via FXR and TGR5). Their chemistry is intricate: primary and secondary species exist as free acids and as glycine/taurine conjugates, with additional sulfated and glucuronidated forms and numerous positional isomers. Because biological matrices contain wide concentration ranges and strong matrix effects, a purpose-built, isomer-aware LC-MS/MS method is essential for robust quantification.

Creative Proteomics provides a customer-oriented bile acids analysis solution that delivers absolute quantitation and isomer-level resolution across human and preclinical matrices, optimized for discovery, mechanism-of-action studies, biomarker research, microbiome–host interaction projects, nutrition science, toxicology, and pharmaceutical R&D.

What Problems We Solve for Our Clients

  • Differentiate challenging isomers such as CA vs CDCA or DCA vs UDCA, avoiding data misinterpretation.
  • Quantify at ultra-low concentrations in complex matrices like feces, plasma, or bile.
  • Minimize matrix effects using optimized extraction and isotope-labeled internal standards.
  • Measure a broad spectrum of bile acid species (primary, secondary, conjugated, sulfated, glucuronidated, and muricholic acids) in a single run.
  • Deliver decision-ready results with full traceability, validation, and documentation.

Service Scope — Creative Proteomics Bile Acids Analysis

Bile Acid Synthesis & Metabolism Assay

Quantitative profiling of unconjugated, glycine- and taurine-conjugated bile acids, plus key biosynthetic intermediates, using LC-MS/MS with isomer resolution.

Matrices: Serum, Plasma, Urine, Feces, Tissue, Cells

Bile Acid Conjugates Profiling

Comprehensive detection of glycine-, taurine-, sulfated-, and glucuronidated bile acids in a single targeted panel.

Matrices: Serum, Plasma, Bile, Feces

Rodent-Specific Muricholic Acids Panel

Targeted quantitation of α-, β-, and ω-muricholic acids and conjugates for preclinical models.

Matrices: Mouse/Rat serum, feces, tissue

Bile Acid Precursors Analysis

Measurement of C4 and cholestenoic acid series to study biosynthetic flux and pathway regulation.

Matrices: Serum, Plasma, Liver

Custom Bile Acid Panel Development

Fit-for-purpose assay design for rare or novel bile acids, tailored to your research needs.

Matrices: Client-specified

Bile Acids and Related Metabolites – Target Panel Overview

Category Representative Compounds Notes
Primary Bile Acids Cholic acid (CA), Chenodeoxycholic acid (CDCA) Synthesized in liver from cholesterol; key in lipid digestion
Secondary Bile Acids Deoxycholic acid (DCA), Lithocholic acid (LCA), Ursodeoxycholic acid (UDCA), Hyodeoxycholic acid (HDCA), Isodeoxycholic acid (IDCA), Allocholic acid (ACA), Alloisolithocholic acid (AILCA) Formed via gut microbial metabolism of primary bile acids
Glycine-Conjugated Bile Acids Glycocholic acid (GCA), Glycochenodeoxycholic acid (GCDCA), Glycodeoxycholic acid (GDCA), Glycolithocholic acid (GLCA), Glycoursodeoxycholic acid (GUDCA), Glycohyodeoxycholic acid (GHDCA), Glycohyocholic acid (GHCA), Glycodehydrocholic acid (GDHCA), Glycoallocholic acid (GACA) Increased solubility; facilitates reabsorption in enterohepatic circulation
Taurine-Conjugated Bile Acids Taurocholic acid (TCA), Taurochenodeoxycholic acid (TCDCA), Taurodeoxycholic acid (TDCA), Taurolithocholic acid (TLCA), Tauroursodeoxycholic acid (TUDCA), Taurohyodeoxycholic acid (THDCA), Taurohyocholic acid (THCA), Taurodehydrocholic acid (TDHCA) Increased solubility; important in bile acid transport
Sulfated Bile Acids Lithocholic acid-3-sulfate (LCA-3S), Deoxycholic acid-3-sulfate (DCA-3S), Cholic acid-3-sulfate (CA-3S), Glycolithocholic acid-3-sulfate (GLCA-3S), Glycodeoxycholic acid-3-sulfate (GDCA-3S), Taurolithocholic acid-3-sulfate (TLCA-3S), Taurodeoxycholic acid-3-sulfate (TDCA-3S), Tauroursodeoxycholic acid-3-sulfate (TUDCA-3S) Phase II metabolism products; more water-soluble, excretory forms
Glucuronidated Bile Acids Deoxycholic acid-3-glucuronide (DCA-3G), Lithocholic acid-3-glucuronide (LCA-3G), Chenodeoxycholic acid-3-glucuronide (CDCA-3G), Chenodeoxycholic acid-24-β-D-glucuronide (CDCA-24G), Lithocholic acid-24-glucuronide (LCA-24G) Detoxification products; aid excretion via urine/bile
Rodent-Specific Bile Acids Alpha-muricholic acid (α-MCA), Beta-muricholic acid (β-MCA), Omega-muricholic acid (ω-MCA), Lambda-muricholic acid (λ-MCA, Hyocholic acid), Tauro-alpha-muricholic acid (TαMCA), Tauro-beta-muricholic acid (TβMCA), Tauro-omega-muricholic acid (TωMCA), Tauro-lambda-muricholic acid (TλMCA, Taurohyocholic acid) Predominant in mouse/rat bile acid profiles
Bile Acid Precursors & Intermediates 7α-hydroxy-4-cholesten-3-one (C4), 3α,7α-dihydroxycholestenoic acid (DHCA), 3α,7α,12α-trihydroxycholestenoic acid (THCA), 3β-hydroxycholestenoic acid (3βHCA), 3β,7α-dihydroxycholestenoic acid (3β,7αDHCA), 7α-hydroxy-3-oxo-4-cholestenoic acid (7αOH-3O-4CA), Norcholic acid (NorCA), Nordeoxycholic acid (NorDCA), Norursodeoxycholic acid (NorUDCA), Ursocholanic acid (UCA) Key biosynthetic intermediates in cholesterol-to-bile acid pathway

Don't see a metabolite? We can extend the panel with additional standards or develop a fit-for-purpose assay.

Why Choose Our Bile Acids Analysis Service: Key Advantages

  • Targeted coverage, isomer aware — Chromatographic methods resolve critical isobaric pairs, protecting data integrity.
  • Absolute quantitation with stable-isotope internal standards — Matrix-matched calibration and isotope correction for reliable quantification.
  • Dual analytical modes — UPLC-QQQ MRM for sensitivity and throughput; UPLC-HRMS for structural confirmation.
  • Method validation data included — R² ≥ 0.99, accuracy/precision within ±15% (±20% at LLOQ).
  • Cross-matrix expertise — Harmonized workflows for serum, plasma, feces, bile, liver, cells, and culture media.
  • Decision-ready deliverables — Quantitative tables, QC summaries, chromatograms, and pathway analysis.

Bile Acids Assay Technical Details & Coverage

Creative Proteomics quantifies and profiles bile acids using UPLC–MS/MS platforms, including Waters ACQUITY UPLC™ + Sciex Triple Quad™ 5500+/6500+ and Agilent 1290 Infinity II UHPLC + QTRAP® 6500. For high-resolution confirmation, we employ Thermo Scientific™ Q Exactive™ Orbitrap.

Chromatography: Reversed-phase UPLC (C18), gradient elution optimized for bile acid families; isomer-separation conditions applied where needed.

Ionization & detection: Negative-mode ESI for most species; APCI available where advantageous. Triple-quadrupole MRM transitions are established per analyte (quantifier + qualifier).

Calibration: 6–8 matrix-matched levels; R² ≥ 0.99; isotope-labeled internal standards (e.g., D₄-CA, D₄-CDCA).

QC: Pooled-matrix QC at low/mid/high levels, spike-recovery, and matrix-effect checks.

Sensitivity & range (typical): LLOQs in low ng/mL (serum/plasma) and low ng/mg (feces/tissue); dynamic range spanning 3–4 orders of magnitude (analyte- and matrix-dependent).

Acceptance targets: Accuracy and precision within ±15% for QCs (±20% at LLOQ); back-calculated calibrators within predefined limits; retention-time and ion-ratio tolerances monitored.

SCIEX Triple Quad™ 6500+

SCIEX Triple Quad™ 6500+ (Figure from Sciex)

Waters ACQUITY UPLC System

Waters ACQUITY UPLC System (Figure from Waters)

Agilent 1260 Infinity II HPLC

Agilent 1260 Infinity II HPLC (Figure from Agilent)

Thermo Scientific Q Exactive Orbitrap

Q Exactive™ Orbitrap (Figure from Thermo Scientific)

Flexible Service Configurations for Targeted Bile Acid Studies

  • Standard Targeted Panel (absolute quantitation)
    Primary/secondary bile acids and glycine/taurine conjugates quantified with stable-isotope internal standards where available; optional add-ons for sulfates/glucuronides and muricholic acids.
  • Extended Panel (with precursors & intermediates)
    Adds C4 and cholestenoic acids to study biosynthetic flux and pathway regulation.
  • Hybrid Identification Mode (targeted + HRMS)
    For projects requiring additional structural confidence, high-resolution MS/MS libraries and retention-time matching can be enabled.
  • Data Analysis Add-Ons
    Group stats, volcano plots, enrichment analysis (bile acid pathways), microbiome-linked interpretation with fecal/serum ratioing, and publication-ready visualizations.

How Our Bile Acids Analysis Works — Step-by-Step Process

1

Project Consultation — Define analyte panel, matrix types, sensitivity targets, and any custom requirements.

2

Sample Receipt & Verification — Confirm integrity, amount, and compliance with handling guidelines.

3

Sample Preparation — Matrix-specific extraction (protein precipitation, SPE, homogenization) to enrich bile acids.

4

Chromatographic Separation — Reversed-phase UPLC with gradient elution; isomer resolution where required.

5

Mass Spectrometry Detection — Negative-mode ESI LC-MS/MS with validated MRM transitions, qualifier ions, and ion-ratio checks.

6

Calibration & QC — Multi-point calibration, isotope-labeled standards, and interspersed QC samples.

7

Data Processing & Review — Automated quantitation with expert review to confirm compliance with acceptance criteria.

Bile Acids Assay Workflow

How to Prepare Your Samples for Bile Acids Quantification

Matrix / Species Minimum Amount Container & Handling Storage & Shipping
Serum / Plasma ≥50 µL PP or glass vials; EDTA or heparin plasma acceptable Freeze promptly; ship on dry ice
Urine ≥50 µL No additives/detergents Freeze; ship on dry ice
Feces ≥20 mg (homogenized preferred) Sterile, screw-cap tube Freeze; ship on dry ice
Bile ≥50 µL Low-bind tubes Freeze; ship on dry ice
Liver / Tissue ≥10 mg Pre-weighed cryovials or foil Snap-freeze; ship on dry ice
Cells ≥2×10⁶ (pellet) PBS rinse; remove media Snap-freeze pellet; ship on dry ice
Culture Supernatant ≥200 µL Clarify by centrifugation Freeze; ship on dry ice
Dried Blood Spot 2–3 punches (3–6 mm) Card in gas-permeable pouch with desiccant Keep dry & cool; cold-chain preferred

Applications of Bile Acids Profiling & Quantification

Microbiome–Host Interaction

Profiling fecal and systemic bile acids to assess gut microbial metabolism and host response.

Metabolic Pathway Studies

Mapping bile acid biosynthesis, conversion, and transport in liver–gut axis research.

Nutritional Science

Evaluating diet, probiotics, or functional foods on bile acid composition and metabolism.

Preclinical & Translational Research

Monitoring bile acid perturbations in disease models and therapeutic interventions.

Toxicology & Safety Assessment

Detecting bile acid accumulation or imbalance linked to compound exposure.

Biomarker Discovery

Identifying bile acid signatures associated with physiological or pathological states.

Deliverables: What You Get from Our Bile Acids Analysis Service

Comprehensive Concentration Tables — Absolute values in ng/mL, nmol/L, or ng/mg as appropriate.

QC Performance Summary — Calibration curve metrics, QC recoveries, and precision data.

Annotated Chromatograms — Demonstrating analyte resolution and peak quality.

Method Sheet — Key parameters for transparency and reproducibility.

Optional Data Analysis — Statistics, fold-change, volcano plots, pathway enrichment, and microbiome-related interpretation.

Publication-Ready Visuals — Box plots, heatmaps, and bile acid pathway diagrams.

EIC of GDCA and GDCA-d4 internal standard with labeled retention times and MRM transitions.

Extracted ion chromatogram (EIC) of glycodeoxycholic acid (GDCA, m/z 448.3→74.0) and isotopically labeled internal standard (GDCA-d4), showing baseline separation and retention time alignment.

Product ion spectrum of taurocholic acid showing taurine-specific fragment peaks at m/z 80, 124, and 107.

Negative-mode ESI–MS/MS product ion spectrum of taurocholic acid (TCA, m/z 514.3), highlighting diagnostic fragment ions at m/z 80.0, 124.0, and 107.0 characteristic of taurine conjugation.

Calibration curve of DCA showing linearity from 0.5 to 500 ng/mL with regression equation and R-squared value.

Matrix-matched calibration curve for deoxycholic acid (DCA, m/z 391.3→345.3) over 0.5–500 ng/mL with 1/x² weighting, R² = 0.998.

Comprehensive Bile Acids Profiling in a High-Fat Diet Rodent Model


Journal: Communications Biology

Published: 2023

DOI: https://doi.org/10.1038/s42003-023-04739-9


Objective

A pharmaceutical research team sought to evaluate the impact of a high-fat diet (HFD) on bile acid composition in a murine model, aiming to understand gut–liver axis alterations and potential metabolic dysregulation. The study required isomer-level resolution, quantitation across multiple biological matrices, and comparative analysis against control animals.


Approach

Creative Proteomics applied its extended panel bile acids analysis using UPLC–Triple Quad™ 6500+ MS/MS. Sample matrices included serum, feces, and liver extracts from HFD-fed and control mice.

  • Sample Preparation: Matrix-specific extraction with protein precipitation and SPE clean-up to minimize matrix effects.
  • Chromatographic Separation: Reversed-phase C18 UPLC with optimized gradient to resolve critical isomers such as α-, β-, and ω-muricholic acids.
  • Detection & Quantitation: Negative-mode ESI with MRM transitions validated for each analyte; stable-isotope-labeled internal standards for accuracy.
  • Data Analysis: Concentration normalization per matrix type, fold-change calculations, and bile acid pathway enrichment mapping.

Key Findings

  • Significant Elevation of taurine-conjugated muricholic acids (TαMCA, TβMCA) in feces of HFD-fed mice, indicating altered bile acid metabolism and reduced microbial deconjugation activity.
  • Reduced Levels of secondary bile acids such as deoxycholic acid (DCA) and lithocholic acid (LCA), suggesting microbiota compositional shifts.
  • Pathway Analysis revealed downregulation of bile salt hydrolase–related metabolism and altered FXR/TGR5 signaling potential.

Deliverables to Client

  • Absolute quantitation tables for >40 bile acid species across all matrices.
  • QC performance summary (R² ≥ 0.99, precision within ±15%).
  • Extracted ion chromatograms and representative product ion spectra for key compounds.
  • Pathway enrichment diagrams highlighting differences between HFD and control groups.

Reference

  1. Liu, Jui-Tung, et al. "A human iPSC-derived hepatocyte screen identifies compounds that inhibit production of Apolipoprotein B." Communications Biology 6.1 (2023): 452.

Can your targeted LC-MS/MS bile acids analysis resolve isomers accurately?

Yes. Our bile acid profiling workflow uses reversed-phase UPLC with isomer-specific separation conditions, combined with targeted LC-MS/MS multiple reaction monitoring (MRM) to distinguish challenging isobaric and positional isomers such as cholic acid (CA) vs. chenodeoxycholic acid (CDCA), and α-muricholic acid vs. β-muricholic acid.

How do you ensure accurate bile acids quantification across diverse biological matrices?

We apply matrix-matched calibration curves and stable-isotope labeled internal standards for absolute quantitation. Quality control (QC) samples at multiple concentration levels are analyzed alongside study samples to ensure reproducibility in plasma, serum, feces, bile, urine, tissue, and cell extracts.

Can you analyze conjugated and modified bile acids in a single run?

Yes. Our targeted bile acids analysis panel can quantify primary, secondary, glycine-conjugated, taurine-conjugated, sulfated, and glucuronidated bile acids in one LC-MS/MS run, minimizing sample consumption while maximizing analyte coverage.

Do you offer bile acids analysis for fecal samples or complex matrices?

Yes. We have validated extraction protocols for high-matrix-content samples such as feces, intestinal contents, and bile, enabling sensitive quantitation of bile acids despite strong matrix effects.

Can bile acids analysis be integrated with microbiome or metabolomics studies?

Absolutely. We can align bile acid quantification data with microbiome sequencing or untargeted metabolomics results to study microbiome–host co-metabolism, bile acid transformation pathways, and their potential role in health or disease models.

How do you deal with bile acids with low ionization efficiency?

We optimize ionization mode selection (negative ESI or APCI) and MS/MS transition parameters for each compound. Where necessary, we apply derivatization or alternative chromatographic strategies to enhance sensitivity for difficult analytes.

Can I request a custom bile acids panel for specific targets?

Yes. We can design fit-for-purpose panels, from a single bile acid target to an extended library including muricholic acids, bile acid precursors (C4, cholestenoic acids), and rare metabolites, tailored to your study requirements.

Quantifying forms and functions of intestinal bile acid pools in mice

Sudo, K., Delmas-Eliason, A., Soucy, S., Barrack, K. E., Liu, J., Balasubramanian, A., … & Sundrud, M. S.

Journal: bioRxiv

Year: 2024

A human iPSC-derived hepatocyte screen identifies compounds that inhibit production of Apolipoprotein B

Liu, J. T., Doueiry, C., Jiang, Y. L., Blaszkiewicz, J., Lamprecht, M. P., Heslop, J. A., … & Duncan, S. A.

Journal: Communications Biology

Year: 2023

Choleoeimeria pogonae Alters the Bile Acid Composition of the Central Bearded Dragon (Pogona vitticeps)

Johnston, Andrea N., Stöhr, Anke C., et al.

Journal: Journal of Herpetological Medicine and Surgery

Year: 2021

For Research Use Only. Not for use in diagnostic procedures.
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