Simultaneous Determination of Bile Acids Utilizing an Immobilized Enzyme Column

February 4, 2021


Bile acids have a common hydroxyl group located at the 3α position of their steroidal backbone. 3α-HSD (3α-Hydroxysteroid Dehydrogenase) is an enzyme that causes this hydroxyl group to be selectively oxidized in the presence of the co-enzyme NAD (Nicotinamide Adenine Dinucleotide). In this reaction, when a molecule of bile acid is oxidized to 3-ketosteroid one NADH molecule (the reduced form of NAD) is generated and it has intense fluorescence (Ex=340 nm, Em=470 nm). In this method a standard mixture of bile acids was measured using post-column derivatization for detecting NADH generated by continuously mixing the reaction solution containing NAD with the column eluent and passing this mixed solution through a 3α-HSD enzyme-immobilized column.


LC-4000 HPLC system


Experimental Condition
Column: Bilepak-II (4.6 mmI.D. x 125 mmL, 5 µm)
Enzyme Column: Enzymepak 3α-HSD (4.0 mmI.D. x 20 mmL)
Eluent A: 30 mM Ammonium acetate buffer (pH 6.8)/Acetonitrile/Methanol (60/20/20)
Eluent B: 30 mM Ammonium acetate buffer (pH 6.8)/Acetonitrile/Methanol (40/30/30)
Gradient Condition: (A/B), 0 min (100/0) -> 32 min (0/100) -> 60 min (0/100) -> 60.1 min (100/0) 1 cycle; 80 min
Flow Rate: 1.0 mL/min
Reagent: 0.3 mM NAD, 1 mM EDTA-2Na, 0.05% 2-mercaptoethanol, 10 mM potassium dihydrogenphosphate, pH 7.8 (adjusted with potassium hydroxide)
Reagent Flow Rate: 1.0 mL/min
Column Temp.: 25 ºC
Wavelength: Ex. 345 nm, Em. 470 nm, Gain 100x
Injection Volume: 10 µL
Standard Sample: 15 Bile acids (50 µmol/mL each)

Figure 1 shows the enzyme reaction for the oxidation of bile acids and reduction of NAD.

Figure 1. Mechanism of Enzyme Reaction

Figure 2 shows a flow diagram for the system used to analyze the bile acids.

Fig. 2. Flow Diagram

No.1: Eluent, No.2: Quarternary pump, No.3: Cooled Autosampler, No.4: Column oven, No.5: Column (Bilepak II), No.6: Enzyme column (Enzymepak 3a-HSD ), No.7: Reagent, No.8: Reagent pump, No.9: Fluorescence detector, No.10: Backpressure coil


Bile acids, NAD, NADH, Enzymepak 3α-HSD, Bilepak-II, Fluorescence detector


Figure 3 shows the chromatogram of a standard mixture of 15 bile acids and an internal standard (I.S.), which were well separated in under 50 minutes.

Figure 3. Chromatogram of the Standard Sample of Mixed Bile Acids

1: Glycoursodeoxycholic acid (GUDCA), 2: Tauroursodeoxycholic acid (TUDCA), 3: Ursodeoxycholic acid  (UDCA), 4: Glycocholic acid (GCA), 5: Taurocholic acid (TCA), 6: Cholic acid (CA), 7: Glycochenodeoxycholic  acid (GCDCA), 8: Taurochenodeoxycholic acid (TCDCA), 9: Glycodeoxycholic acid (GDCA), 10: Taurodeoxycholic acid (TDCA), 11: Chenodeoxycholic acid (CDCA), 12: Deoxycholic acid (DCA),  13: Glycolithocholic acid (GLCA), 14: Taurolithocholic acid (TLCA), I.S.: 5β-pregnan-3α,17α,20α-triol,  15: Lithocholic acid (LCA)

Table 1 shows the retention time and peak area reproducibility of each bile acid when a 0.5 nmol standard mixture of bile acids (injection volume: 10 µL) (n=10). The %RSD of retention time and peak area for each component obtained was 0.2 %~ 0.34% and 0.8% ~ 2.13% respectively.

Table 1. Reproducibility (n = 10)

Bile acid %RSD
Retention time Peak area
GUDCA 0.34 1.43
TUDCA 0.33 1.25
UDCA 0.28 1.47
GCA 0.33 1.36
TCA 0.33 1.16
CA 0.26 1.5
GCDCA 0.24 2.04
TCDCA 0.23 2.13
GDCA 0.24 1.8
TDCA 0.22 1.17
CDCA 0.2 2.11
DCA 0.2 1.16
GLCA 0.22 0.8
TLCA 0.23 0.89
LCA 0.29 1.38

Table 2 shows the limit of detection (S/N = 3) of each bile acid. Limit of detection is 3 – 7.5 pmol, and it is expected that this method will be used to measure small volume of bile acids in  biological fluid (such as blood) with high-sensitivity and selectability.

Bile acid amount (pmol)
UDCA 3.0
GCA 4.5
TCA 4.5
CA 4.5
GDCA 4.5
TDCA 3.0
CDCA 4.5
DCA 3.0
GLCA 4.5
TLCA 6.0
LCA 7.5

About the Author

Chromatography Group