How to qualitatively and quantitatively detect bile acid composition and content in feces of C57 neonatal mice?

To perform qualitative and quantitative analysis of the bile acid profile in the feces of newborn C57 mice, the following key technical points need to be addressed: complex sample matrix, numerous types of bile acids with significant polarity differences, and potentially very low content. Below is the recommended experimental process and method:

 

I. Sample Preparation

1. Feces Collection

(1) Collection Time: Ensure timely collection after the first intestinal excretion of newborn mice to avoid contamination with maternal components.

(2) Storage Method: Immediately freeze in liquid nitrogen and store long-term at -80°C to prevent bile acid degradation.

 

2. Bile Acid Extraction

(1) Sample Weighing: Accurately weigh 10–50 mg of freeze-dried feces.

(2) Extraction Solvent: Add pre-cooled 70% methanol (containing 0.1% formic acid) or 75% acetonitrile/water at a volume ratio of 1:20~1:40.

(3) Internal Standard Addition: Add stable isotope-labeled bile acid internal standards (such as d4-CA, d4-CDCA, d4-DCA) for subsequent quantification.

(4) Ultrasonic Vibration + Vortex Mixing: Ice bath sonication for 15–30 minutes, followed by centrifugation at 4°C.

(5) Supernatant Filtration: Treat with a 0.22 μm filter membrane and transfer to a sample vial.

 

II. Chromatography-Mass Spectrometry Platform Selection

It is recommended to use the UPLC-MS/MS platform combined with the MRM mode for high-sensitivity quantitative analysis.

1. Chromatographic Conditions (Recommended)

(1) Column: C18 reversed-phase column (such as Waters Acquity UPLC BEH C18, 1.7 μm, 2.1×100 mm)

(2) Mobile Phase:

• A: 0.1% formic acid aqueous solution

• B: 0.1% formic acid acetonitrile

(3) Gradient Elution: Set a gradient suitable for bile acid separation (e.g., increase from 20% B to 95% B within 30 minutes)

(4) Flow Rate: 0.3 mL/min

(5) Column Temperature: 40°C

(6) Injection Volume: 5 μL

 

2. Mass Spectrometry Conditions (Taking Triple Quadrupole as an Example)

(1) Ionization Mode: Negative ion mode (ESI-)

(2) Monitoring Mode: MRM (Multiple Reaction Monitoring)

(3) Parameter Optimization: Optimize fragmentation voltage and collision energy for each bile acid (including conjugated and free types)

 

III. Data Processing and Quantification Method

1. Standard Quantification

• Use commercially available bile acid standards (such as Sigma or Cayman) to establish a concentration gradient standard curve.

• Each bile acid should have an independent quantification channel, combining internal standard to correct for matrix effects and losses.

 

2. Data Analysis

• Use software like MassLynx, Analyst, or Xcalibur to extract MRM peak areas.

• Compare unknown sample peak areas with the standard curve to calculate concentration (units can be ng/mg fecal dry weight).

 

IV. Analyzable Target Compounds

Commonly detected bile acids include:

• Primary bile acids: CA (cholic acid), CDCA (chenodeoxycholic acid)

• Secondary bile acids: DCA (deoxycholic acid), LCA (lithocholic acid), UDCA (ursodeoxycholic acid)

• Conjugated bile acids: TCA, GCA, TCDCA, GCDCA, etc.

 

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