HPLC Method for Separation of  Bile acids (Methyl cholate, Cholic acid, Deoxycholic acid, Chenodeoxycholic acid) on Primesep B Column

HPLC Method for Analysis of Methyl cholate, Cholic acid, Deoxycholic acid, Chenodeoxycholic acid on Primesep B by SIELC Technologies

HPLC Method for Analysis of  Methyl cholate, Cholic acid, Deoxycholic acid, Chenodeoxycholic acid on Primesep B Column by SIELC Technologies
HPLC Method for Analysis of Methyl cholate, Cholic acid, Deoxycholic acid, Chenodeoxycholic acid on Primesep B Column by SIELC Technologies

Bile acids are a class of amphipathic molecules derived from cholesterol that play a crucial role in the digestion and absorption of dietary fats. They are synthesized in the liver and released into the small intestine during digestion.

Cholic Acid is a primary bile acid with the chemical formula C24H40O5. It is synthesized in the liver from cholesterol and contributes to the emulsification and digestion of fats in the small intestine. As a medication, it is used to treat bile acid synthesis disorder, that is caused by defects of the single enzyme, as well as Zellweger syndrome.

Deoxycholic Acid is a secondary bile acid with the chemical formula C24H40O4. It is formed by bacterial action in the colon on cholic acid. Deoxycholic acid aids in the digestion and absorption of fats. In some countries it is even licensed to be used as an emulsifier in the food industry. It has found a lot of use in research focused on immunology, cancer, and nanotechnology.

Chenodeoxycholic Acid, also known as chenocholic acid, is a primary bile acid with the chemical formula C24H40O4. It is synthesized in the liver. It participates in the emulsification of fats. It has been used medically to dissolve gallstones, treat cerebrotendinous xanthomatosis, and constipation.

Methyl cholate is a compound with the chemical formula C25H42O5. Methyl cholate is derived from cholic acid, one of the primary bile acids. The addition of a methyl group to cholic acid results in the formation of methyl cholate. Bile acids, including methyl cholate, contribute to the solubilization of lipids.

These bile acids are part of the bile acid pool, which undergoes enterohepatic circulation—being released into the small intestine, reabsorbed in the terminal ileum, and returned to the liver. Bile acids also play a role in cholesterol metabolism and act as signaling molecules.

Their chemical structures, amphipathic nature, and interactions with lipids are essential for their physiological functions in the digestive process. The balance of bile acids in the body is crucial for proper digestion and absorption of dietary fats.

Methyl cholate, Cholic acid, Deoxycholic acid, Chenodeoxycholic acid can be retained, separated, and analyzed using a Primesep B mixed-mode stationary phase column. The analysis utilizes an isocratic method with a simple mobile phase consisting of water, acetonitrile (MeCN), and sulfuric acid as a buffer. Detection is achieved using UV at 192 nm

ColumnPrimesep B, 4.6 x 150 mm, 5 µm, 100 A, dual ended
Mobile PhaseMeCN/H2O – 60/40%
BufferH3PO4 -0.2%
Flow Rate1.0 ml/min
DetectionUV 192 nm
Samples2 mg/mL in MeCN/H2O – 50/50%
Injection volume10 µl
LOD*0.2 ppm
* LOD was determined for this combination of instrument, method, and analyte, and it can vary from one laboratory to another even when the same general type of analysis is being performed.
Class of Compounds
bile acids
Analyzing CompoundsMethyl cholate, Cholic acid, Deoxycholic acid, Chenodeoxycholic acid

Application Column

Primesep B

Column Diameter: 4.6 mm
Column Length: 150 mm
Particle Size: 5 µm
Pore Size: 100 A
Column options: dual ended

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Application Analytes:
Chenodeoxycholic acid
Cholic acid
Deoxycholic acid
Methyl cholate

Application Detection:
UV Detection
SIELC Technologies usually develops more than one method for each compound. Therefore, this particular method may not be the best available method from our portfolio for your specific application. Before you decide to implement this method in your research, please send us an email to research@sielc.com so we can ensure you get optimal results for your compound/s of interest.