3
A Fast and Robust HPLC Separation of Bile Acids, and their
Conjugates, with Ascentis® Express C18
Clare Glicksman1, Michael Wright2, Dave Bell3 and Anders Fridström3
1
Biochemistry department, King’s College Hospital, London, UK
2
Biochemistry department, Cambridge University Hospital Trust, Cambridge, UK
3
Supelco®, Sigma-Aldrich
Introduction
Bile acids are increasingly recognised as playing an important signalling
role in the control of immune response and energy metabolism.
Recently, there has been interest in measuring bile acid concentrations
in plasma and their impact on hormone concentrations, for example
as affected by bariatric surgery (Figure 1).
Primary bile acids are synthesised in the liver from cholesterol. The
majority are conjugated, increasing hydrophilicity, with either glycine
or taurine. They are stored in the gall bladder and released into the
small intestine on food consumption. Bacteria in the gut can convert
primary bile acids to secondary bile acids which are then reabsorbed
and taken back to the liver as part of enterohepatic circulation. Here
secondary bile acids can also be glycine or taurine conjugated. Spillover from this system results in bile acids in the peripheral circulation.
There are potentially 15 species which can be measured in a peripheral
plasma sample (Figure 2).
Methodology
The quantitation of plasma bile acids by tandem mass spectrometry
MS/MS presents an analytical challenge. When employing collisioninduced 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).
Figure 1. Roux-en-Y gastric bypass and gastric banding are two possible
options for reducing dietary food intake. Both procedures create a small
pouch at the start of the stomach by either restricting the stomach with
a gastric band or by surgically creating a separate pouch from the
stomach and then attaching it to the remainder of the GI tract.
Pouch
Small Stomach
Pouch
Roux-en-Y Gastric Bypass
Bypassed Portion
of the Stomach
Duodenum
Gastric Band
Long Roux-Limb
Larger
Stomach
Portion
Port
Colon
Common Limb
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, several
orthogonal HPLC stationary phases were screened for selectivity. The
best separation was achieved with an Ascentis® Express C18 analytical
column (150 x 4.6 mm, 2.7 µm), based on Fused-Core® technology.
Figure 2. Structural overview of bile acids and conjugated salts with corresponding m/z values
Primary
Cholic acid (CA)
Primary
Chenodeoxycholic acid (CDC)
Secondary Deoxycholic acid (DC)
Secondary Lithocholic acid (LC)
Secondary Ursodexoycholic acid (UDC)
Bile Acid(s)
m/z transition
DC, CDC, UDC
391.2/391.2
CA407.1/407.1
LC375.2/375.2
GDC, GCDC, GUDC
448.2/74.1
GCA464.2/74.1
GLC432.1/74.1
TDC, TCDC, TUDC
498.2/80.0
TCA514.0/80.0
TLC482.2/80.0
d4-DC395.2/395.2
d4-GDC452.1/74.1
d4-TDC502.2/80.0
R1R2R3
OH
OH
OH
OH
OH
H
OH
H
OH
OH
H
H
OH
OH
H
Unconjugated
Tauro (T)
Glyco (G)
X =OH
X = NHCH2CH2SO3H
X = NHCH2COOH
O
R3
X
12
3
R1
7
R2
(continued on page 4)
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HPLC/LC
Helping to Elucidate the Role of Plasma Bile Acid Signalling in Humans
4
Figure 3. A typical chromatogram showing all analytes and internal standards with full resolution of all derivatives with similar m/z values
1.10 e4
XIC of – MRM (12paits): 391.2/391.2 amu from Sample 4 (Std. 1uM) of 2009_07_13 liver patient dil.wiff (Turbo Spray)
Max. 4930.0 cps.
1.05 e4
D4-DC
1.00 e4
9500.00
9000.00
8500.00
HPLC/LC
critical pairs with same mass
TUDC
8000.00
7500.00
7000.00
Intensity, cps
6500.00
6000.00
UDC
5500.00
GCDC
5000.00
11.63
TDC
4500.00
D4-GDC
CDC
D4-TDC
4000.00
TCDC
3500.00
DC
11.30
TLC
7.62
3000.00
TCA
2500.00
2000.00
CA
GUDC
GDC
1500.00
LC
GLC
GCA
1000.00
500.00
0.00
12.48
1
2
3
4
5
6
7
Time, min
8
9
10
11
12
13
Sample preparation
Conclusion
Plasma proteins were removed with addition of 900 µL of ACN, 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.
10 µL (corresponding to 8.57 µL of plasma) was injected into the HPLC.
A fast robust LC-MS/MS method was developed on a C18 Ascentis
Express Fused-Core® Particle Column, allowing the 15 species to be
measured individually rather than the alternative measurement of a
total concentration by colourimetric kinetic enzyme assays. This is
particularly useful in research into the role of individual bile acids as
signalling molecules. The method is suitable for clinical laboratories
and is currently being used to investigate potential mechanisms
linked to gut hormone profiles and glycaemic control.
Chromatography
Column: Ascentis® Express C18 (150 x 4.6 mm, 2.7 μm)
Solvent A: Methanol (5 mM ammonium acetate and 0.012% formic acid)
Solvent B: Water (5 mM ammonium acetate and 0.012% formic acid)
Gradient
Time (min.)
Solvent A (%)
Solvent B (%)
0
7030
10
955
14
955
References
1. Glicksman, C. Investigation of Fractionated Plasma Bile Acids in Lean and
Obese Subjects by LC-MSMS. Diploma work , MSc Clinical Biochemistry,
University College, London, February 2010.
2. Glicksman, C.; Pournaras, D.J.; Wright, M.; Roberts, R.; Mahon, D.; Welbourn, R.;
Sherwood, R.; Alaghband-Zadeh, J.; le Roux, C.W. Ann Clin Biochem 2010;
47: 482 – 484.
Temperature
40 °C
3. Watanabe, M.; Houten, S.M.; Mataki, C. et al. Bile acids induce energy
expenditure by promoting intracellular thyroid hormone activation.
Nature 2006 439, 484 – 489.
Mass Spectrometry
Negative ESI with m/z showed in Figure 2
4. Katsuma, S.; Hirasawa, A.; Tsujimoto, G. Bile acids promote glucagon-like
peptide-1 secretion through TGR5 in a murine enteroendocrine cell line
STC-1. Biochem. Biophys. Res. Commun. 2005 329, 386 –390.
Results
See Figure 3.
sigma-aldrich.com/express
5. Maruyama, T.; Tanaka, K.; Suzuki, J. et al. Targeted disruption of
G protein-coupled bile acid receptor 1 (Gpbar1-M-Bar) in mice.
J Endocrinol. 2006 191, 197– 205.
6. Tagliacozzi, D.; Mozzi, A.; Casetta, B. et al. Quantitative Analysis of bile acids
in human plasma by liquid chromatography-electrospray tandem mass
spectrometry: A simple and rapid one-step method. Clinical Chemistry and
Laboratory Medicine 2003 41, 1633 –1641.