Bile Acids Kit for LC-MS

A Rapid and Reliable Kit for Bile Acids Research

Bile Acids Research Significance

Bile Acids (BA) are synthesized in the liver and play important roles in cholesterol homeostasis, absorption of vitamins and lipids, and various key metabolic processes. BA analysis is an important area of study in various settings including clinical laboratories and ADME and Toxicology studies. Abnormalities in BA biosynthesis and accumulation are linked to multiple human diseases and mechanisms causing hepatotoxicity including drug-induced cholestasis.

ADME/Tox Liver Model for Bile Acids Research

HepaRG (5F Clone) control cells (Cat. No. MTOX1010-1VL) is a highly active human liver cell line for bile acid research.

Methodology

The quantitation of bile acids from plasma, cells or media by tandem mass spectrometry (MS/MS) presents an analytical challenge. When employing collision-induced dissociation, bile acids will either form no useful fragments for quantitation (unconjugated), or produce a charged fragment originating from a conjugation group (conjugated taurine and glycine salts).

As three of the bile acids have the same molecular weight and elemental composition, they cannot be differentiated by MS/MS alone and require chromatographic separation of the group in both their unconjugated and conjugated forms. To enable separation, an Ascentis® Express C18 analytical column based on Fused-Core® technology is used.

Bile Acids Kit Products

Description Cat. No.
HPLC Column  
Ascentis Express C18, 15 cm x 4.6 mm I.D., 2.7 µm particle size 53829-U
Standards  
Conjugated bile acids kit (contains GCDC, GCA, TCDC, TCA, TDC) CBA-1KT
Chenodeoxycholic acid (CDC) C9377
Cholic acid (CA) C1129
Deoxycholic acid (DC) D2510
Glycochenodeoxycholic acid (GCDC) G0759
Glycocholic acid (GCA) G2878
Glycoursodeoxycholic acid (GUDC) 06863
Lithocholic acid (LC) L6250
Taurochenodeoxycholic acid (TCDC) T6260
Taurocholic acid (TCA) T4009
Taurodeoxycholic acid (TDC) T0875
Taurolithocholic acid (TLC) T7515
Tauroursodeoxycholic acid (TUDC) T0266
Ursodeoxycholic acid (UDC) U5127

 

Structural Overview of Bile Acids and Conjugated Salts with Corresponding m/z Values

 

Unconjugated X = OH
Tauro (T) X = NHCH2CH2SO3H
Glyco (G) X = NHCH2COOH

 

    R1 R2 R3
Primary Cholic acid (CA) OH OH OH
Primary Chenodeoxycholic acid (CDC) OH OH H
Secondary Deoxycholic acid (DC) OH H OH
Secondary Lithocholic acid (LC) OH H H
Secondary Ursodeoxycholic acid (UDC) OH OH H

 

Bile Acid(s) m/z transition
DC,CD,UDC 391.2/391.2
C 407.1/407.1
LC 375.2/375.2
GDC,GCDC,GUDC 448.2/74.1
GCA 464.2/74.1
GLC 432.1/74.1
TDC, TCDC, TUDC 498.2/80.0
TCA 514.0/80.0
TLC 482.2/80.0
d4-DC 395.2/395.2
d4-GDC 452.1/74.1
d4-TDC 502.2/80.0

 

Sample Preparation

Plasma proteins are removed with addition of 900 µL of acetonitrile containing deuterated internal standards, to 250 µL of human EDTA plasma. The mixture was vortexed, centrifuged, and the supernatant evaporated before being reconstituted in a 50:50 solution of methanol and water. A 10 µL aliquot (corresponding to 8.57 µL of plasma) is injected into the HPLC.

Results

A chromatogram showing the fifteen bile acid species separated on an Ascentis® Express C18 Fused-Core® column is shown below. The method is fast and robust and allowed the compounds to be measured individually rather than the alternative measurement of a total concentration by colorimetric kinetic enzyme assays. This is particularly useful in research into the role of individual bile acids as signaling molecules. The kit is suitable for ADME/Tox and clinical laboratories.

Results