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INTRODUCTION

'Steroidomics' is the qualitative and quantitative study of steroid-type molecules found within the metabolome. Bile acids for example, are classified as acidic sterols that are synthesised mainly by the liver from cholesterol and aid digestion and fat solubilisation. The presence of

multiple isomeric bile acids poses a great challenge for steroidomic research. Ion mobility -mass spectrometry (IM-MS) was combined with molecular modelling for the separation and configurational analysis of thirteen medically relevant bile acids. The usefulness of the rotationally averaged collision cross-section (CCS) information derived from the experiment-tally derived IM measurements of relevant bile acids may be used to enhance specificity and augment steroidomic-type research and aid the diagnosis, prognosis and management of disease.

METHODS

Mass Spectrometry

MS: Vion IMS Q-ToF and Synapt G2-Si Mode: ESI and MALDI (-VE)

Capillary voltage: 2kV Cone: 40V

Source temperature: 110°C Scan rate: 1 spectrum/s

ESI

The bile acids were infused at a concentration of 0.1ng/µL (MeOH) and the signal attenuated with the DRE lens

MALDI-Imaging

Isomeric bile acid mixtures were spotted on a 30 µm mouse

brain section mounted on a glass slide. The slide was spray coated with matrix using the SunChrom SunCollect Sprayer.

30 coats were applied at a flow rate of 20 µL/min. MALDI

images were processed using High Definition Imaging software v1.3. Matrix: 9-aminoacridine (0.5 mg/mL in 4:1 EtOH:H2O).

Ion Mobility

Mobility bath gas: N2 (Vion) N2 (G2-Si) CO2 (G2-Si only)

Ion mobility cell: ~3.0mbar ~3.0mbar ~3.0mbar

IMS Wave velocity: 850 m/s 900 m/s 900 m/s Trap Wave Height: 40-60V 40V 40V

Workflow

ESI-MS was used to measure ion drift-times upon a hybrid ion

mobility/ quadrupole / oa-ToF MS (Vion IMS Q-ToF), ESI and MALDI-imaging was also used upon a hybrid quadrupole / ion

mobility / oa-ToF MS (Synapt G2-Si). N2 was used as the

mobility gas in both instruments and CO2 in the Synapt G2-Si only.

THE ANALYSIS OF BILE ACIDS: ENHANCEMENT OF SPECIFICITY USING AN ION MOBILITY-TOFMS BASED APPROACH

Jonathan P Williams1, Martin Palmer1, Jonas Abdel-Khalik2, Yuqin Wang2, Sarah M Stow3, Mark Towers1, Giuseppe Astarita1, James Langridge1 and William J Griffiths2 1 Waters Corporation, Wilmslow, Manchester UK; 2 College of Medicine, Swansea University UK; 3 Laboratory for Structural Mass Spectrometry, Vanderbilt University, TN, USA

RESULTS

New and improved methods were sought for the identification, quantification, and characterization of bile acids, oxysterols, and other sterols and steroids. The involvement of these molecules in

neurogenesis and immunity is investigated. The use of IM as an analytical tool to aid direct infusion ESI, DESI and

MALDI shotgun steroidomic-type analysis was investigated. Bile acids present themselves in biological type samples as complex mixtures. Structural information may be obtained using MS/MS but in the absence

of a chromatographic step, unambiguous characterisation using MS/MS can be challenging since the selected precursor ion can be composed of chemical isomers and interfering isobaric ions.

The individual rotationally averaged CCS measured experimentally using direct infusion ESI-T-Wave ion mobility are shown in Table 1. The results

represent an average of three measurements. Isomeric bile acids are highlighted in grey.

CONCLUSIONS

Good correlation was achieved between the two T-

Wave ion mobility instruments used in this study.

In combination with accurate mass measurement,

the additional molecular descriptor of CCS can aid bile

acid ion identification

The results indicate that the addition of CCS

measurements to searchable databases within a ‘steroidomic-type’ workflow increases the specificity

and selectivity of bile acid analysis, improving the confidence in identification compared to traditional

analytical approaches

Structures of the bile acids.

IMS comprises a travelling wave RF ion guide, which incorpo-

rates a repeating sequence of transient DC pulses to propel ions through the guide in the presence of N2 bath gas. Upon

exiting the IMS cell, ions can be selected with the quadrupole and undergo CID for structural elucidation prior to detection

with the TOFMS. The T-Wave mobility device was calibrated for estimated rotationally averaged TWCCSN2 measurements using

drift tube obtained DTCCSN2 measurements of ions produced from polyalanine.

Modeling and Theoretical CCS determination

Obtain structure from PubChem Remove hydrogen to create deprotonated molecules [M-H]-

of the bile acids Run Gaussian Optimisation for starting structure and partial

charges Run Distance Geometry to generate a set of conformations.

8000 conformation limit was set for the deprotonated mole-cules of the bile acids

Run energy minimization for candidate low energy confor-mations

Theoretical CCS were obtained using the Trajectory Method in MOBCAL and from preliminary N2 parameters using the

Projection Superposition Approximation method

Fig. 1 Schematic of the Vion IMS Q-ToF

Fig.1 shows a schematic of Vion. In brief, the instrument com-

prises an IM separation device, a quadrupole and segmented collision cell prior to the TOFMS. Ions are accumulated in the

trap travelling-wave (T-Wave) and periodically released into the T-Wave IM where they separate according to their mobil-

ity.

Direct infusion-ESI Ion Mobility MS: CCS measurements

of the bile acids investigated

Fig. 2 Overlaid drift times (ms) of DCA, CA, CDA and HA. UA has a

very similar drift time to CDA and is different by only 1 scan (the flight time for the pusher frequency). The inset shows an overlay of

the individual drift times (ms) for UA, CDA and HA using a higher T-Wave ion mobility velocity of 1500 m/s.

Time9.00 10.00

%

0

100

Time1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

%

0

100

1. DCA 2. CA

3. CDA 4. HA

UA

CDA

HA

1

2 3

4

MALDI Imaging Ion Mobility MS measurements of the

isomeric bile acids deoxycholic acid and hyodeoxy-cholic acid

Fig. 3 MALDI-Imaging Synapt G2-Si of DCA and HA. Although

isomeric, the two bile acids, previously been detected in brain. could easily be differentiated using ion mobility and detected

where spotted on to the mouse brain tissue section.

Gas-Phase separation re-optimisation of bile acid isomers;

effect of mobility gas alteration from N2 to CO2

CDA

Time2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50

%

0

100

2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50

%

0

100

2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50

%

0

100

DCA

DCA

DCA

UA

HA

Fig. 4 Gas-phase separation optimisation for the bile acid iso-

mers obtained in N2 upon the Synapt G2-Si.

Time2.00 2.50 3.00 3.50 4.00 4.50 5.00

%

0

100

2.00 2.50 3.00 3.50 4.00 4.50 5.00

%

0

100

2.00 2.50 3.00 3.50 4.00 4.50 5.00

%

0

100

DCA HA

UA

DCA

DCA

CDA

Fig. 5 Gas-phase separation optimization of the bile acid iso-

mers obtained in CO2 upon the Synapt G2-Si

Modeling and Theoretical CCS generation

Fig. 6 Experimental CCS vs. theoretical CCS ranges for CCS

values obtained in N2 drift gas. Theoretical values were ob-tained using the Trajectory Method (green) in MOBCAL and

from the PSA Method (blue). Theoretical conformations were generated with distance geometry.

Fig. 7 Sample conformations for the bile acids obtained from

computational modeling for a) Glycodeoxycholic Acid, b) Taurodeoxycholic Acid, c) Deoxycholic Acid, and d) Chenode-

oxycholic Acid. Bile acids that have more interactions between the carboxylic or sulfonate end group on the tail structure with

the hydroxyl groups on the fused ring system correspond to the bile acids that fall closer to the lower bound on the theo-

retical range.

BILE ACID [M-H]- VionCCSN2 (Å2) G2-SiCCSN2

(Å2)

DEOXYCHOLIC ACID 202.6 198.9

CHOLIC ACID 204.1 200.8

CHENODEOXYCHOLIC ACID 209.0 205.5

LITHOCHOLIC ACID 208.6 204.6

URSODEOXYCHOLIC ACID 208.3 205.5

GLYCODEOXYCHOLIC ACID 199.8 196.9

GLYCOCHOLIC ACID 202.7 200.0

HYODEOXYCHOLIC ACID 209.6 206.5

TAUROCHENODEOXYCHOLIC ACID 208.4 204.9

GLYCOCHENODEOXYCHOLIC ACID 201.9 198.0

TAUROLITHOCHOLIC ACID 208.1 204.1

TAURODEOXYCHOLIC ACID 207.0 203.9

TAUROCHOLIC ACID 208.4 205.7

Table 1 VionCCSN2 and G2-SiCCSN2 of [M-H]- of the Bile Acids investigated

Representative conformations from distance geometry

modeling for the bile acids investigated