An Improved Reverse Phase LC-MS/MS Method for The Measurement of Bile
Acids in Biological Samples

Experimental
LC/MS Method Development and Sample Analysis
A total of 13 bile acid standards were individually
dissolved in methanol and sequentially injected in an
Agilent 6495C triple quadrupole mass spectrometer(Figure 1) by flow injection to enable automatedoptimization of MRM compound transitions, AJS
source, and iFunnel acquisition parameters with
MassHunter Optimizer. Single bile acid standards
were then pooled together, and the resulting mixture
was utilized to develop a UHPLC method (Table 2) ona high-resolution C-18 column. Details of the MS andLC methods are shown in Table 1 and 2, respectively.

Figure 1. Agilent 6495C Triple Quadrupole Mass
Spectrometer with 3rd Generation Ion Funnel
Technology coupled to an Agilent Infinity II UHPLC pietro.morlacchi@agilent.com

Results and Discussion

Results and Discussion

Optimized LC-MS/MS Method

Table 1. 6495C QQQ MS conditions
MS Conditions
Instrument

Agilent 6495C QQQ withiFunnel Technology

Ionization Source

Agilent Jet Stream ESI

Polarity

NegativeDrying Gas

240 °C; 13 L/min

Nebulizer30 psi

Sheath Gas

400 °C; 10 L/min

Capillary

4000 V

1000 VHigh Pressure
RFiFunnel Parameters 90 V

Agilent Technologies, Inc., MA, USA, 2Augusta University, GA, USA, 3Memorial Sloan
Kettering Cancer Center, NY, USA

The bile acid mixture was chromatographically resolved with excellent baseline peak separation (Figure 2). We
attempted to optimize the MS acquisition parameters in both positive and negative ion mode and found that negative ion
mode (-ESI) favored better ionization thus more intense MRM transitions Three bile acids in the panel, UDCA, CDCA, andLCA did not produce any measurable MRM transition in both polarities. These compounds were measured in SIM modein the final method. (Figure 3).
The retention time (RT) of each bile acid in the panel was acquired by single standard analysis. The RTs were included inthe acquisition method to build the final dynamic MRM (dMRM) method.
We also analyzed the bile acid mixture on an Agilent 6470 QQQ and found that the 6495C with improved iFunnel
technology resulted about 3-5 time more sensitive (Figure 4).
**TUDCA*LC Conditions

50 °C

Mobile Phase

A: water + 0.1% formic acid
B: acetonitrile +0.1% formic acid

Flow Rate

500 μL/min

GradientTime%A %B(min)075 25175 251640 60
1740 60
17.30
298
20.30
298
21.30
75 25
23.00
75 25
Post Time: 1.0 min

Injection Volume
Multisampler
Temperature

2-5 μL
4 °C

TDCA
TCDCA
LCA

TUDCA
TCA
GUDCA
GCA
TCDCA
TDCA
CA
UDCA
GCDCA
GDCA
TLCA
CDCA
LCA

6×10 7

TCA

Dynamic MRM (dMRM)

ColumnTemperature

8×10 7

TLCA

Table 2. 1290 Infinity II LC conditions

ColumnFigure 4. TUDCA calibration curve representative of the bile acids mixture

*UDCA

Low
Pressure RF
60 V

Agilent Zorbax RRHD Eclipse
Plus C18, 100 x 2.1 mm,1.8 μm(p/n 959758-902)

1.0 – 100 ng/mL- ESI

NozzleScan TypeTUDCA

Response

Bile acids are a class of steroidal compounds derived
from cholesterol catabolism in the liver.1 Thesemolecules play important roles in the absorption anddigestion of lipids, maintaining lipids, glucose, andenergy homeostasis, and working as activators ofcell signaling pathways associated with theirbiosynthesis.2 In the intestine, the main secreted bileacids produced in the liver (primary bile acids)
undergo additional modifications by gut microbesthat results in a bouquet of isomeric and isobaric
species (secondary bile acids). Many of thesesecondary bile acid species are difficult to beaccurately measured by LC-MS/MS because of lackof chromatographic baseline separation, poor
fragmentation, and low detection limit due to theirintrinsic low abundance in biological matrixes. Herein
we report a UPLC -MS/MS method for the analysis ofbile acids that partially overcomes these limitations

Experimental

1Bile AcidIntroduction2021
Metabolomics
Society Annual
Meeting
POSTER # 429

Pietro Morlacchi1, Juan Zou2, Ruben Ramos3, Justin Cross3

*

GUDCA

4×10 72×10 7

GDCA
Figure 2. Final LC-MS/MS (-ESI) acquisition method depicting the MRM
and SIM chromatograms from the bile acid standard mixture. Asterisks
denote bile acids measured in SIM mode.

Neg
Pos

GCA
GCDCA

01030

4050

R2TCDCA
TUDCA
TDCA
TCA
GUDCA
CA
TLCA
GCA
GDCA
GCDCA
UDCA
CDCA
LCA

0.999
0.999
0.999
0.999
0.996
0.995
0.987
0.993
0.992
0.993
0.995
0.985
0.958

*Response
Figure 3. Signal intensity of the mostabundant MRM transition in positive andnegative ion mode

Figure 4. TIC comparison between 6495C and 6470 QQQ models.
Calibration Curve for Quantitation
Calibration standards were prepared from each bile acid stock solution by serial dilution with neat solvent. The
concentration of the standards ranged from 1.0 ng/mL to 10 μg/mL. As shown in Table VV, the correlation coefficient(R2) for most of the bile acid in the mixture was 0.99 over the specified concentration range (Table 3). LLOQ values arealso reported in Table 3.

LLOQ(pmol/mL)
0.5
0.6
0.6
0.6
0.6
0.6
1.5
1.5
1.6
1.6
18.2
18.2
30.6

Table 4. LLOQ and correlation
coefficient relative to the bile
acids in the mixture

60Injections

CACDCA

20

Bile Acid

Figure 4. Bile acid instrument response across consecutive injection andcorresponding RSD (%)

Bile Acid
TUDCA
TCA
GUDCA
GCA
TCDCA
TDCA
CA
UDCA
GCDCA
GDCA
TLCA
CDCA
LCA

RSD (%)
2.9
2.9
1.5
1.9
2.8
2.8
2.6
2.3
1.9
2.0
4.6
3.2
5.3

Conclusions
The implementation of the new Agile 6495C triple quadrupole mass spectrometer in the development of this analyticalmethod allowed a much higher sensitivity towards bile acid detection compared to previous models. Overall, the method
presented good linearity over a wide range of concentrations, and the use of the dynamic multiple reaction monitoring(dMRM) ensured the acquisition of higher quality data. Although this method requires a more rigorous validation incomplex matrices, it should greatly simplify bile acid quantification and thereby facilitate research and discovery of novel
biomarkers in biological samples.

References
1John Y.L. Chiang, J Lipid Res, 2009 Oct; 50(10): 1955-1966
2Tiara R. Ahmad & Rebecca A. Haeusler, Nature Reviews Endocrinology 2019, 15: 701-712

For Research Use Only. Not for use in diagnostic procedures.

DE44406.296412037