Bile Acids Analysis with Absolute Quantitation & Isomer Resolution

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

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+ (Figure from Sciex)

Waters ACQUITY UPLC System (Figure from Waters)

Agilent 1260 Infinity II HPLC (Figure from Agilent)

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.

How to Prepare Your Samples for Bile Acids Quantification

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

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.

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.

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

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.