ASMS
ASILOMAR 2017Mark J. Sartain1, Ariel R. Brumbaugh2, andMichael A. Fischbach2

Tools to Understand the Gut Microbiome:
A Refined LC-MS/MS Method to Profile Bacteria-derived Bile Acids

1Agilent Technologies, Inc., Santa Clara, CA, USA

POSTER #Fri/Sat 3

2Department of Bioengineering, Stanford University, CA

Introduction

Experimental

Results and Discussion

Secondary bile acids represent the most abundant and variablemicrobiota-derived metabolites and have been shown to affect hostphysiology. Yet much remains to be discovered regarding theircompositional differences across individuals, their biologicalactivities, and the bacterial strains and genes that produce them.1

Compound MS/MS Optimization
Similar to Wegner et al.2, we found that for many bile acids MRMbased scans of (M+NH4)+ precursors were superior to SIM-based
analyses of (M-H)- ions. Although detection limits were comparable
in neat solvent, the additional analytical selectivity of MRM helped toreduce interference from matrix components in biological samples.

Robustness
To assess robustness, 200 injections of a mouse cecal extract
spiked with standards were analyzed over a >4-day period withoutinterruption. The unique LC/MS solvent system enabled excellent
retention time reproducibility even with minimal sample preparation.

ABStandards

7-oxo-CA(-) SIM 405.3  405.3(+) MRM 424.3  335.2(+) MRM 424.3  353.2(+) MRM 424.3  371.2(+) MRM 424.3  389.2

6.0E+45.0E+4(-) SIM 405.3  405.3(+) MRM 424.3  335.2(+) MRM 424.3  353.2(+) MRM 424.3  371.2(+) MRM 424.3  389.2

Mouse Fecal
Extract7-oxo-CA

Chromatography
Multiple stationary phases and mobile phase compositions wereevaluated. Acetone was found to possess superior lipid eluotropic
properties in comparison to acetonitrile, fully eluting problematic
phospholipids and triacylglycerols at the end of each analytical run.
AAcetonitrile Mobile Phase B
Acetone Mobile Phase B

2000 V (+), 0 V (-)

Scan type

Dynamic MRM (dMRM)

Cycle time

750 msTotal number of MRMs

110

Min/max dwell time

17 / 748 ms

LCA

Nozzle voltage

Allo LCA

4500 V (+), 3000 V (-)

α-MCA

3.4%

0.11%

LCA

3.3%

0.06%

-MCA

2.0%

0.10%

TUDCA

2.8%

0.21%

GHDCA

3.0%

0.12%

Average

2.7%

0.15%

Quantitation
Dilutions of selected standards were prepared in neat solvent andevaluated at 16 standard concentrations ranging from the LOD at
100 pM to the upper limit of quantitation at 10 µM. Correlation
coefficients (R2) for calibration curves were higher than 0.99 over upto 4.0 orders of dynamic range.
LLOD (nM)

LLOQ (nM)

CA

0.2

1

GCA

0.5

1

α-MCA

0.5

1LCA

25

-MCA

55GHDCA

0.513-oxo-CA

0.1

0.2

Alpha-Murocholic Acid: 1nM – 10µM

R2 = 0.996

Cecal
ConventionalConclusions
A targeted LC-MS/MS method for bile acid profiling and quantitation
has been newly developed that provides:• Enhanced selectivity in biological matrices• Increased robustness due to unique LC mobile phases

DCA

Capillary voltage

0.12%• Excellent analytical sensitivity

CDCA

40 psi

HDCA

Nebulizer gas (nitrogen)

2.1%• Minimal sample preparation requirements
GDCA

10 L/min, 200 ºC

7-Keto-LCA

Sheath gas (nitrogen), Temp

CA12 L/min, 200 ºC

UDCA

Drying gas (nitrogen), Temp

GCDCA

Positive and Negative Switching

Muro-CAPolarity

The water-acetone based mobile phase maintained excellent
separation of bile acid isomers. The gradient was optimized toachieve baseline separation of 24 of the 26 compounds and partial
separation of early-eluting Tauro α- and β-MCA.
3-oxo-CA

Agilent Jet Stream ESI

GCA

Fecal

Gamma MCA

Ion source

0.10%

Cecal
Gnotobiotic

Comparison of PC/SM Elution (A) and TAG elution (B) from a liver total lipid
extract with acetonitrile or acetone as mobile phase B. Ammonium formate
was infused post-column to enable ionization of TAGs.

α-MCA
β-MCA

Agilent 6470 Triple Quadrupole Mass Spectrometer

1.8%32% B

TDCA

0.20 ml/min, 32% B hold for 6 min, 0.4 ml/min to65% B at 25 min, to 98% B at 25.1 min, followed
by further steps for column cleaning andequilibration. Total time = 32.1 min

200CAPlasma

7-oxo-CA

A = 0.1% Formic Acid
+ 20mM Ammonium Acetate in Water
B = 0.1% Formic Acid in Acetone

TCDCA

Mobile phase

GUDCA
GHDCA
GCA

2-5 µL150

RT CVAcetonitrile Mobile Phase BAcetone Mobile Phase BTCAInjection volume100

Area CV98% B

Sum of 3 Neutral Loss Scans for TAGs

TUDCA

45 ºCB50CompoundProfiling
Agilent Mass Profiler Professional software was used to compare
bile acid compositions from mouse extracts and plasma andrevealed significant differences.

32% B

Tβ-MCA

Column temperature

Analytical gradient

98% B

m/z 184 Precursor Ion Scan for PC/SMs

Agilent Poroshell EC-C18, 2.1x150mm, 2.7 µm
Agilent Poroshell EC-C18, 2.1x5mm, 2.7 µm

1.0E+4CompoundTα-MCA

Guard column

2.0E+4Injection Number

Comparison of 7-oxo-CA MRM vs SIM scans for 100 ppb standard in neatsolvent (A) and endogenous 7-oxo-CA in mouse fecal extract (B). The same
LC/MS method was used for both samples.

Agilent UHPLC 1290 Infinity II System
Column3.0E+40Iso LCA

Method Design
MassHunter Optimizer software was used to optimize MRM
transitions for 26 bile acids. Analysis was carried out with positive
and negative electrospray ionization in dynamic MRM (dMRM) in asingle analytical run. Major UHPLC and MS parameters are as
follows:

4.0E+40.0E+0

Experimental
Reagents and Sample Preparation
Twenty-six authentic chemical standards were purchased fromcommercial vendors. To establish lower limits of quantification(LLOQs), dilutions of analytes were prepared in neat solvent. Fecalpellets and cecal contents from gnotobiotic and conventionallyraised mice were homogenized and extracted in the presence ofmethanol, and particulate was removed after addition of an equalvolume of water. NIST SRM 1950 plasma was protein-precipitatedwith methanol.

Key

7.0E+4

Raw Peak Area

Recently, there has been increased interest in targeted LC-MS
methodologies to profile bile acids in lieu of established GC-MS
approaches that require extensive sample preparation. Achromatographic method was developed with a unique mobile
phase system to efficiently resolve close structural bile acid isomers
while also clearing problematic lipid accumulation, enhancing
method robustness. In conjunction with the LC method a triple
quadrupole mass spectrometer operated with optimized MRM
parameters enabled sensitive and specific detection of bile acids.

8.0E+4

Overlaid MRM and SIM chromatograms from a mixture of bile acid standards
acquired with the final dMRM LC/MS method. Bile acids sharing the same
precursor m/z are indicated by color.

References
1. Devlin, A.S., Fischbach, M.A. Nature Chem Biol. 2015 Sep;11(9):685-90
2. Wegner, K. et al. Anal Bioanal Chem. 2017 Feb;409(5):1231-1245

For Research Use Only. Not for use in diagnostic procedures.
RA44448.4289351852