353.269831+

381.301491+

413.271181+

495.388021+

593.343401+

693.577241+

817.629121+

F-score-1-b_000002.d: +MS

244.08081321.24280

1+

413.270721+

553.397781+

615.325521+

693.575661+

817.627041+

873.59822

SF-1-b_000002.d: +MS

234.150221+

357.281961+

439.227361+

593.340571+

663.463911+

785.60641855.60818

1+

SPE-LM-2-b_000003.d: +MS

381.300091+

437.198091+

553.397041+ 637.31479

1+

675.270571+

719.440141+

BD-Top-1-b_000002.d: +MS

234.150281+

300.292011+

381.300991+ 439.22740

1+

517.290482+

557.450192+

637.314091+

677.512901+

787.553461+

835.448572+

962.758231+

BD-Bot-2-b_000002.d: +MS

0.0

0.5

1.0

1.5

2.0

9x10

Intens.

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100 200 300 400 500 600 700 800 900 m/z

Optimized Metabolite Extraction from Fecal Material for

Biomarker AnalysisEmily R. Sekera, Heather L. Rudolph, and Troy D. Wood

Conclusions

• Although the Bligh-Dyer extraction proved to be promising to

lessen matrix suppression, the appearance of labelled stercobilin

in the bottom layer would alter the calculations of concentrations

as both labelled and unlabeled compounds were not effected

equally.

• The use of a PA-2 column did separate bilins and other

components to enhance signal and lessen suppression from

other compounds but requires further evaluation as the intensity

provided is not a proper evaluation technique for percent loss.

• Due to the ability of solid phase extraction to be tailored

specifically for the extraction of bilins meanwhile lessening matrix

suppression, it has been chosen for further analysis.

• Solid phase extraction also proved to have acceptable

reproducibility with the least percent loss reported.

Figure A: neque

dignissim, and in

aliquet nisl et umis.

Department of Chemistry

State University of New York at Buffalo

http://woodgroupms.wixsite.com/twood

OverviewAutism Spectrum Disorder (ASD) is a general term for a group of

complex neurodevelopmental disorders that lacks a clinical biomarker.

Emerging evidence shows that the intestinal microbiome in ASD subjects

can be distinguished from controls, suggesting metabolite differences

may be observable due to the action of intestinal microbes. Our lab has

previously studied the metabolites stercobilin and stercobilinogen as

putative biomarkers for ASD wherein a 45% depletion of stercobilin was

observed with a p < 0.001. In this study, we investigate the utilization of

extraction techniques for the analysis of metabolites within the feces of a

murine model of ASD. Through the utilization of HRMS, we examine the

use of syringe filters, solid phase and Bligh-Dyer extractions, as well as

in-line columns to further identify metabolites of interest within fecal

material as potential biomarkers.1,2

IntroductionWhat is autism?

• Autism spectrum disorder (ASD) and autism are both general terms

for a group of complex disorders of brain development.2

• ASD can be associated with intellectual disability, difficulties in motor

coordination and attention, and physical health issues such as sleep

and gastrointestinal disturbances 3

What Mouse Model is Used?

• Timothy Syndrome (TS) mice have been shown to be accurate

behavioral animal models for autism. 4

• TS is a rare autosomal dominant disorder in which 80% of patients

tend to develop ASD. 4

• There are 3 strains of TS in the colony: TS1-neo (inverted neomycin

resistance cassette), TS2-neo, and TS1no-neo.

What is Stercobilin?

• Stercobilin (C33H46N4O6,

MW: 594.34 g/mol) is a member of

the group of mammalian metabolites

known as bilin tetrapyrroles.5

• Stercobilin is a product of heme

metabolism formed from the

degradation of bilirubin.5

InstrumentationAll analysis was done by a Bruker Daltonics 12T Solarix FT-ICR MS or

a Thermo Scientific Orbitrap Q Exactive Focus using electrospray

ionization in positive ion mode.

References1. E. R. Sekera, H.L. Rudolph, et.al.. Depletion of Stercobilin in Fecal Matter from

a Mouse Model of Autism Spectrum Disorders. Metabolomics. 2017, 11,132.

2. T.D. Wood, A. F. Charlebois, E.R. Sekera, et.al. Autism Spectrum Disorders-

Advances at the End of the Second Decade of the 21st Century. InTech Open.

London. In Press.

3. Autism Speaks. What Is Autism? https://www.autismspeaks.org/what-autism.

4. P. L. Bader, M. Faizi, et. al.. Mouse model of Timothy syndrome recapitulates

triad of autistic traits. Proc. Natl. Acad. Sci. U. S. A., Early Ed. 2011, 108, 15432.

5. E.Kuntz, H.-D. Kuntz. Hepatology: Textbook and Atlas. Springer, Wetzlar, 2008.

Acknowledgements

We gratefully acknowledge the financial support of the NIH through the

National Center for Research Resources (Grant # S10-RR029517-01) for

providing funding used to obtain the FT-ICR instrument. We would also like

to thank the Mark Diamond Research Fund from the University at Buffalo

and the California Scottish Rite for financial support for materials used in

this research. We are also grateful to MSACL for providing an MSACL

Young Investigator Grant.

Future Work

• Further evaluation of percent loss utilizing PA-2 Column.

• Viability of chosen extraction technique with urine samples.

• Determination of the validity of depletion stercobilin and

stercobilinogen across multiple mouse models of autism.

• Further testing of samples for stercobilinogen depletion.

• Testing our putative biomarker in human urine samples.

Figure 1: Structure of Stercobilin

MethodsTimothy Syndrome Mice

• Fecal samples were collected from mice and stored at -20°C until use.

• Murine fecal samples from 12 TS mice and controls are chosen at random to create a pooled fecal slurry to determine the validity and reproducibility of each extraction technique.

Figure 2: Control and TS2 mice.

Extraction of Bilins from Excrement

• Bilins need to be extracted from fecal material to be properly analyzed by mass spectrometry.

• 25 mg of fecal slurry were used for each extraction procedure.

• Each extraction and sample were analyzed in triplicate

• Polypropylene syringe filters, Oasis MAX SPE Cartridges, Bligh-Dyer Extractions, C18 zip tips, and a Polar Advantage II (PA2) liquid chromatography column were all utilized.

• Prior to extraction isotopically labelled stercobilin is added to allow for the examination of percent recovery of the compounds of interest. After extraction, a standard of quinapril is added to determine percent recovery of both the standard and labelled stercobilin.

Results

Sample Collection

Fecal Pooling

Syringe Filter

Solid Phase Extraction

Bligh-Dyer Extraction

Sample for HRMS Analysis

PA-2 Column

C18 Zip Tip

(Top Layer)

Figure 3: Outline of Extraction Protocol.

Figure 5: Comparison of representative mass spectra by multiple extraction

techniques.

No Extraction

Syringe Filter

Solid Phase Extraction

BD Top Layer

BD Bottom Layer

SampleAverage

Percent Loss

Standard

Deviation

Coefficient of

Variance

Syringe Filter 47.1 % 5.1 % 4 - 9 %

Solid Phase

Extraction30.8 % 4.1 % 1 – 8%

Bligh-Dyer

Top Layer76.5 % 10.2 % 0.6 – 4 %

Bligh Dyer-

Bottom Layer99.9 % 0.1 % 0.01 – 0.2 %

Bligh-Dyer Top

Layer with Zip Tip86.2 % 5.6 % 3 – 8 %

PA-2 Column 90.6 % 1.1 % 1 %

Figure 6: Analysis of different extraction techniques.

Separation by LC-MS on Orbitrap

• Through the use of a decreased flow rate (0.180 mL/min) coupled

to the addition of 0.1% formic acid as a charging agent, unlabeled

and labeled stercobilin are able to be resolved from one another.

Quinapril also showed as a separate elution for use in quantitation.

• The utilization of intensity affected the calculated average percent

loss negatively. The utilization of quanbrowser in the future with a

calibration curve of quinapril could more accurately determine the

efficiency of the technique.

Figure 4: TIC of a syringe filter sample on the PA2 Column with representative

mass spectra of the peaks with unlabeled bilins and quinapril.

Direct Injection on FT-ICR

Summary of Results

• The resolving power of the FT-ICR allows for the viewing of more

convoluted samples with increased mass accuracy. This can be

essential in non-targeted analysis.

• As a targeted analysis, solid phase extraction proved to lessen

matrix suppression the best of the techniques.