copy2015 Waters Corporation 1

Foodomics Applications Improving consumer well being health and

knowledge using the latest -omic techniques

copy2015 Waters Corporation 2

Overview

Introduction to foodomics and nutrimetabolomics

ndash Influence of diet on health (eg Dietary flavonoids)

Metabolomic approaches for molecular characterisation of foods

ndash Characterisation of nutritionally valuable natural products (Passiflora example)

ndash Investigation of flavonoid markers of dietary intake

Rapid Evaporative Ionisation Mass Spectrometry (REIMS)

ndash Direct analysis for rapid profiling

Summary

Acknowledgements

copy2015 Waters Corporation 2

Overview

Introduction to foodomics and nutrimetabolomics

ndash Influence of diet on health (eg Dietary flavonoids)

Metabolomic approaches for molecular characterisation of foods

ndash Characterisation of nutritionally valuable natural products (Passiflora example)

ndash Investigation of flavonoid markers of dietary intake

Rapid Evaporative Ionisation Mass Spectrometry (REIMS)

ndash Direct analysis for rapid profiling

Summary

Acknowledgements

copy2015 Waters Corporation 3

Foodomics

ndash Refers to the metabolite profiling of

foods prior to consumption

(biomarkers of consumption)

Nutritional metabolomics has

emerged with two major goals

ndash (1) to determine the effects of

dietary compounds on host

metabolism after consumption

(biomarkers of effect)

ndash (2) identify metabolic disease that is

influenced by nutrients amp to develop

targeted diet-based treatments

(disease risk biomarkers)

Foodomics and nutrimetabolomics

Heart disease

Alheizmers

Cancer

Asthma

Diabetes

copy2015 Waters Corporation 5

Links between diet and healthhellip

copy2015 Waters Corporation 5

Links between diet and healthhellip

copy2015 Waters Corporation 6

Functional foods

Flavonoids are one of the largest widespread class of plant secondary

metabolites with diverse biological amp pharmacological properties

Highly bioactive and play a wide variety of different roles in health of

pants animals and human health

Many flavonoid containing plants are utilized as functional foods amp

phytomedicines

Functional foods represent one of the fastest growing markets with

interest in the systematic characterization of flavonoids in plant crops

Flavonoids

Reduce blood

pressure

Antioxidants

Anti-inflammator

y action

Improve endothelial

function

Reduce platelet activity

Enzymatic Modulation

copy2015 Waters Corporation 7

Characterisation of functional foods

Functional foods amp phytomedicines

ndash Natural product profiling - flavonoids

ndash Analytical challenges

Technology overview

ndash Introduction to CCS

ndash HRAMS with ion mobility

ndash Reducing sample complexity (spectral cleanup)

ndash Increased selectivity with ion mobility

Profiling of Passiflora species (marker flavonoids) using ion

mobility with UNIFI processing

copy2015 Waters Corporation 9

Natural product profiling Analytical challengeshellip

Sample complexity

Crude (non-selective) sample preparation

Generic chromatography conditions (no chromatography)

ndash gt30k features

Spectral interpretation

Isomeric compounds

ndash different pharmacological affects

Structural elucidation of ldquounknownsrdquo

Identification of authentic product

ndash Identification of active components

ndash Quantitation of active components

copy2015 Waters Corporation 10

IMS-MS for natural product profiling

IMS-MS can help overcome the challenge of matrix complexity

IMS is a rapid separation approach orthogonal to UPLC that provides

increased peak capacity

Separation can help spectral decongestion for complex samples

improving selectivity amp specificity

Can separate isomeric species and provide structural confirmatory

information

Spectral clarity improves confidence in identification and aids in

structural elucidation of unknowns

copy2015 Waters Corporation 11

Introduction to ion mobility amp Synapt G2-Si

technology

copy2015 Waters Corporation 12

Ion mobility separation

Separation of ionic species as they drift through a gas under the influence of an electric field

Rate of drift is dependent on ionrsquos mobility in the gas

Leads to separation based on 3D molecular conformation size amp charge

Isobaric ions

IMS

-9 -8 -7 -6

TOF MS LC

-5 -4 -3 -2 -1 0 1 2 3 4 n

10n seconds

copy2015 Waters Corporation 13

SYNAPT G2-Si High Definition MS (HDMS) Orthogonal acceleration QToF

1 Increased sensitivity

2 Ion mobility 3 Accurate mass

measurement

15mm

5mm

~11mm

CID amp ETD

copy2015 Waters Corporation 14

HDMSE Unlimited Product Ion Acquisition

Co-eluting precursor ions

Mobility separation

Drift time aligned precursors and products

Drift time

mz

Drift time

mz

copy2015 Waters Corporation 16

MSE ndash (no mobility separation)

Low Energy

High Energy

copy2015 Waters Corporation 17

HDMSE ndash MSE with a mobility separation

Low Energy

High Energy

copy2015 Waters Corporation 18

Collision Cross Section (CCS)

Measured drift time can be converted into CCS

CCS is an important distinguishing characteristic of an ion which is related to

ndash chemical structure

ndash 3-dimensional conformation

ndash Charge

CCS is a physicochemical property of an ion

copy2015 Waters Corporation 19

Combining analytical discrimination

Analytical measurement

rt

mz

dt

Property of molecule

KD

mass

CCS

UPLC

Mass spectrometer

Ion mobility

cell

Peak capacity

copy2015 Waters Corporation 20

Profiling analysis of Passifloraceae spp

Also known as passion flower or passion vine

~ 500 species of flowering plants

Passiflora species used in production of food products amp dietary supplements ndash Passion fruit juices teas and confectionery

ndash Herbal remedies amp supplements

Sedative properties of some species used to alleviate nervous anxiety and insomnia

Collaboration between Waters Janete Harumi Yariwake and Cintia Matiucci

copy2015 Waters Corporation 11

Introduction to ion mobility amp Synapt G2-Si

technology

copy2015 Waters Corporation 12

Ion mobility separation

Separation of ionic species as they drift through a gas under the influence of an electric field

Rate of drift is dependent on ionrsquos mobility in the gas

Leads to separation based on 3D molecular conformation size amp charge

Isobaric ions

IMS

-9 -8 -7 -6

TOF MS LC

-5 -4 -3 -2 -1 0 1 2 3 4 n

10n seconds

copy2015 Waters Corporation 13

SYNAPT G2-Si High Definition MS (HDMS) Orthogonal acceleration QToF

1 Increased sensitivity

2 Ion mobility 3 Accurate mass

measurement

15mm

5mm

~11mm

CID amp ETD

copy2015 Waters Corporation 14

HDMSE Unlimited Product Ion Acquisition

Co-eluting precursor ions

Mobility separation

Drift time aligned precursors and products

Drift time

mz

Drift time

mz

copy2015 Waters Corporation 16

MSE ndash (no mobility separation)

Low Energy

High Energy

copy2015 Waters Corporation 17

HDMSE ndash MSE with a mobility separation

Low Energy

High Energy

copy2015 Waters Corporation 18

Collision Cross Section (CCS)

Measured drift time can be converted into CCS

CCS is an important distinguishing characteristic of an ion which is related to

ndash chemical structure

ndash 3-dimensional conformation

ndash Charge

CCS is a physicochemical property of an ion

copy2015 Waters Corporation 19

Combining analytical discrimination

Analytical measurement

rt

mz

dt

Property of molecule

KD

mass

CCS

UPLC

Mass spectrometer

Ion mobility

cell

Peak capacity

copy2015 Waters Corporation 20

Profiling analysis of Passifloraceae spp

Also known as passion flower or passion vine

~ 500 species of flowering plants

Passiflora species used in production of food products amp dietary supplements ndash Passion fruit juices teas and confectionery

ndash Herbal remedies amp supplements

Sedative properties of some species used to alleviate nervous anxiety and insomnia

Collaboration between Waters Janete Harumi Yariwake and Cintia Matiucci

copy2015 Waters Corporation 12

Ion mobility separation

Separation of ionic species as they drift through a gas under the influence of an electric field

Rate of drift is dependent on ionrsquos mobility in the gas

Leads to separation based on 3D molecular conformation size amp charge

Isobaric ions

IMS

-9 -8 -7 -6

TOF MS LC

-5 -4 -3 -2 -1 0 1 2 3 4 n

10n seconds

copy2015 Waters Corporation 13

SYNAPT G2-Si High Definition MS (HDMS) Orthogonal acceleration QToF

1 Increased sensitivity

2 Ion mobility 3 Accurate mass

measurement

15mm

5mm

~11mm

CID amp ETD

copy2015 Waters Corporation 14

HDMSE Unlimited Product Ion Acquisition

Co-eluting precursor ions

Mobility separation

Drift time aligned precursors and products

Drift time

mz

Drift time

mz

copy2015 Waters Corporation 16

MSE ndash (no mobility separation)

Low Energy

High Energy

copy2015 Waters Corporation 17

HDMSE ndash MSE with a mobility separation

Low Energy

High Energy

copy2015 Waters Corporation 18

Collision Cross Section (CCS)

Measured drift time can be converted into CCS

CCS is an important distinguishing characteristic of an ion which is related to

ndash chemical structure

ndash 3-dimensional conformation

ndash Charge

CCS is a physicochemical property of an ion

copy2015 Waters Corporation 19

Combining analytical discrimination

Analytical measurement

rt

mz

dt

Property of molecule

KD

mass

CCS

UPLC

Mass spectrometer

Ion mobility

cell

Peak capacity

copy2015 Waters Corporation 20

Profiling analysis of Passifloraceae spp

Also known as passion flower or passion vine

~ 500 species of flowering plants

Passiflora species used in production of food products amp dietary supplements ndash Passion fruit juices teas and confectionery

ndash Herbal remedies amp supplements

Sedative properties of some species used to alleviate nervous anxiety and insomnia

Collaboration between Waters Janete Harumi Yariwake and Cintia Matiucci

copy2015 Waters Corporation 13

SYNAPT G2-Si High Definition MS (HDMS) Orthogonal acceleration QToF

1 Increased sensitivity

2 Ion mobility 3 Accurate mass

measurement

15mm

5mm

~11mm

CID amp ETD

copy2015 Waters Corporation 14

HDMSE Unlimited Product Ion Acquisition

Co-eluting precursor ions

Mobility separation

Drift time aligned precursors and products

Drift time

mz

Drift time

mz

copy2015 Waters Corporation 16

MSE ndash (no mobility separation)

Low Energy

High Energy

copy2015 Waters Corporation 17

HDMSE ndash MSE with a mobility separation

Low Energy

High Energy

copy2015 Waters Corporation 18

Collision Cross Section (CCS)

Measured drift time can be converted into CCS

CCS is an important distinguishing characteristic of an ion which is related to

ndash chemical structure

ndash 3-dimensional conformation

ndash Charge

CCS is a physicochemical property of an ion

copy2015 Waters Corporation 19

Combining analytical discrimination

Analytical measurement

rt

mz

dt

Property of molecule

KD

mass

CCS

UPLC

Mass spectrometer

Ion mobility

cell

Peak capacity

copy2015 Waters Corporation 20

Profiling analysis of Passifloraceae spp

Also known as passion flower or passion vine

~ 500 species of flowering plants

Passiflora species used in production of food products amp dietary supplements ndash Passion fruit juices teas and confectionery

ndash Herbal remedies amp supplements

Sedative properties of some species used to alleviate nervous anxiety and insomnia

Collaboration between Waters Janete Harumi Yariwake and Cintia Matiucci

copy2015 Waters Corporation 14

HDMSE Unlimited Product Ion Acquisition

Co-eluting precursor ions

Mobility separation

Drift time aligned precursors and products

Drift time

mz

Drift time

mz

copy2015 Waters Corporation 16

MSE ndash (no mobility separation)

Low Energy

High Energy

copy2015 Waters Corporation 17

HDMSE ndash MSE with a mobility separation

Low Energy

High Energy

copy2015 Waters Corporation 18

Collision Cross Section (CCS)

Measured drift time can be converted into CCS

CCS is an important distinguishing characteristic of an ion which is related to

ndash chemical structure

ndash 3-dimensional conformation

ndash Charge

CCS is a physicochemical property of an ion

copy2015 Waters Corporation 19

Combining analytical discrimination

Analytical measurement

rt

mz

dt

Property of molecule

KD

mass

CCS

UPLC

Mass spectrometer

Ion mobility

cell

Peak capacity

copy2015 Waters Corporation 20

Profiling analysis of Passifloraceae spp

Also known as passion flower or passion vine

~ 500 species of flowering plants

Passiflora species used in production of food products amp dietary supplements ndash Passion fruit juices teas and confectionery

ndash Herbal remedies amp supplements

Sedative properties of some species used to alleviate nervous anxiety and insomnia

Collaboration between Waters Janete Harumi Yariwake and Cintia Matiucci

copy2015 Waters Corporation 16

MSE ndash (no mobility separation)

Low Energy

High Energy

copy2015 Waters Corporation 17

HDMSE ndash MSE with a mobility separation

Low Energy

High Energy

copy2015 Waters Corporation 18

Collision Cross Section (CCS)

Measured drift time can be converted into CCS

CCS is an important distinguishing characteristic of an ion which is related to

ndash chemical structure

ndash 3-dimensional conformation

ndash Charge

CCS is a physicochemical property of an ion

copy2015 Waters Corporation 19

Combining analytical discrimination

Analytical measurement

rt

mz

dt

Property of molecule

KD

mass

CCS

UPLC

Mass spectrometer

Ion mobility

cell

Peak capacity

copy2015 Waters Corporation 20

Profiling analysis of Passifloraceae spp

Also known as passion flower or passion vine

~ 500 species of flowering plants

Passiflora species used in production of food products amp dietary supplements ndash Passion fruit juices teas and confectionery

ndash Herbal remedies amp supplements

Sedative properties of some species used to alleviate nervous anxiety and insomnia

Collaboration between Waters Janete Harumi Yariwake and Cintia Matiucci

copy2015 Waters Corporation 17

HDMSE ndash MSE with a mobility separation

Low Energy

High Energy

copy2015 Waters Corporation 18

Collision Cross Section (CCS)

Measured drift time can be converted into CCS

CCS is an important distinguishing characteristic of an ion which is related to

ndash chemical structure

ndash 3-dimensional conformation

ndash Charge

CCS is a physicochemical property of an ion

copy2015 Waters Corporation 19

Combining analytical discrimination

Analytical measurement

rt

mz

dt

Property of molecule

KD

mass

CCS

UPLC

Mass spectrometer

Ion mobility

cell

Peak capacity

copy2015 Waters Corporation 20

Profiling analysis of Passifloraceae spp

Also known as passion flower or passion vine

~ 500 species of flowering plants

Passiflora species used in production of food products amp dietary supplements ndash Passion fruit juices teas and confectionery

ndash Herbal remedies amp supplements

Sedative properties of some species used to alleviate nervous anxiety and insomnia

Collaboration between Waters Janete Harumi Yariwake and Cintia Matiucci

copy2015 Waters Corporation 18

Collision Cross Section (CCS)

Measured drift time can be converted into CCS

CCS is an important distinguishing characteristic of an ion which is related to

ndash chemical structure

ndash 3-dimensional conformation

ndash Charge

CCS is a physicochemical property of an ion

copy2015 Waters Corporation 19

Combining analytical discrimination

Analytical measurement

rt

mz

dt

Property of molecule

KD

mass

CCS

UPLC

Mass spectrometer

Ion mobility

cell

Peak capacity

copy2015 Waters Corporation 20

Profiling analysis of Passifloraceae spp

Also known as passion flower or passion vine

~ 500 species of flowering plants

Passiflora species used in production of food products amp dietary supplements ndash Passion fruit juices teas and confectionery

ndash Herbal remedies amp supplements

Sedative properties of some species used to alleviate nervous anxiety and insomnia

Collaboration between Waters Janete Harumi Yariwake and Cintia Matiucci

copy2015 Waters Corporation 19

Combining analytical discrimination

Analytical measurement

rt

mz

dt

Property of molecule

KD

mass

CCS

UPLC

Mass spectrometer

Ion mobility

cell

Peak capacity

copy2015 Waters Corporation 20

Profiling analysis of Passifloraceae spp

Also known as passion flower or passion vine

~ 500 species of flowering plants

Passiflora species used in production of food products amp dietary supplements ndash Passion fruit juices teas and confectionery

ndash Herbal remedies amp supplements

Sedative properties of some species used to alleviate nervous anxiety and insomnia

Collaboration between Waters Janete Harumi Yariwake and Cintia Matiucci

copy2015 Waters Corporation 20

Profiling analysis of Passifloraceae spp

Also known as passion flower or passion vine

~ 500 species of flowering plants

Passiflora species used in production of food products amp dietary supplements ndash Passion fruit juices teas and confectionery

ndash Herbal remedies amp supplements

Sedative properties of some species used to alleviate nervous anxiety and insomnia

Collaboration between Waters Janete Harumi Yariwake and Cintia Matiucci

copy2015 Waters Corporation 21

Product profiling - analytical strategy

Component detection

Determine chemical structures

Assign identifications for knownunknowns look for unknowns

Resolve analytes chromatographically OR

direct analysis

Component identification

isobaric analytes isomers different charge states resolve co-eluting analytes

UPLC separation for sensitivity and resolution

Infusion ASAP

Synapt G2-Si ESI [pos amp neg]

HDMSE unbiased data acquisition

UNIFI 18 Exact mass precursor amp fragments isotope

pattern rtime drift time (CCS)

Chemical elucidation toolset Elemental Composition

Mass Fragment for structure assignment amp confirmation Chemspider library searches

copy2015 Waters Corporation 22

Answering the challenge of natural product

characterisation

Sample complexity

copy2015 Waters Corporation 23

UPLC separation Passiflora edularis

gt10 000 components

detected

copy2015 Waters Corporation 24

Ion mobility separation Increased peak capacity

Conventional UPLC separation

copy2015 Waters Corporation 25

Answering the challenge of natural product

characterisation

Spectral interpretation

copy2015 Waters Corporation 26

MSE multi component precursor and fragment ion spectra Vitexin (mz 431) retention time 8395 mins

Conventional HRMS Vitexin

Low energy

High energy

Vitexin

copy2015 Waters Corporation 27

HDMSE single component precursor and ion mobility product ion spectra

Vitexin (mz 431) retention time 8395 mins drift time 427 ms

Resolved from chromatographically coeluting components using ion mobility

Ion mobility spectral clean-up Vitexin

Vitexin

Low energy

High energy

copy2015 Waters Corporation 28

Answering the challenge of natural product

characterisation

Isomers

copy2015 Waters Corporation 29

Active marker flavonoid structures

OH

OH

O

OH

O

O

OH

OH

OH

OH

OH

O

OH

O

OH

O

OH

OH

OH

OH

OH

OH

OH

O

OH

O

O

OH

OH

OH

OH

OH

OH

O

OH

O

OH

O

OH

OH

OH

OH

Isoorientin Orientin

Isovitexin Vitexin

6C glycocides 8C glycocides

6

8

6

8

Luteolin-8-C-glucoside C21H20O11

Luteolin-6-C-glucoside C21H20O11

Apigenin-6-C-glucoside C21H20O10

Apigenin-8-C-glucoside C21H20O10

copy2015 Waters Corporation 30

Orientinisoorientin isomer separation amp CCS values

Isoorientin

19768Ǻ2

Orientin

18765Ǻ2

Identical twins with shared retention time amp accurate mass BUT different DRIFT times

C21H20O11

448377

∆ =10 Ǻ2

copy2015 Waters Corporation 22

Answering the challenge of natural product

characterisation

Sample complexity

copy2015 Waters Corporation 23

UPLC separation Passiflora edularis

gt10 000 components

detected

copy2015 Waters Corporation 24

Ion mobility separation Increased peak capacity

Conventional UPLC separation

copy2015 Waters Corporation 25

Answering the challenge of natural product

characterisation

Spectral interpretation

copy2015 Waters Corporation 26

MSE multi component precursor and fragment ion spectra Vitexin (mz 431) retention time 8395 mins

Conventional HRMS Vitexin

Low energy

High energy

Vitexin

copy2015 Waters Corporation 27

HDMSE single component precursor and ion mobility product ion spectra

Vitexin (mz 431) retention time 8395 mins drift time 427 ms

Resolved from chromatographically coeluting components using ion mobility

Ion mobility spectral clean-up Vitexin

Vitexin

Low energy

High energy

copy2015 Waters Corporation 28

Answering the challenge of natural product

characterisation

Isomers

copy2015 Waters Corporation 29

Active marker flavonoid structures

OH

OH

O

OH

O

O

OH

OH

OH

OH

OH

O

OH

O

OH

O

OH

OH

OH

OH

OH

OH

OH

O

OH

O

O

OH

OH

OH

OH

OH

OH

O

OH

O

OH

O

OH

OH

OH

OH

Isoorientin Orientin

Isovitexin Vitexin

6C glycocides 8C glycocides

6

8

6

8

Luteolin-8-C-glucoside C21H20O11

Luteolin-6-C-glucoside C21H20O11

Apigenin-6-C-glucoside C21H20O10

Apigenin-8-C-glucoside C21H20O10

copy2015 Waters Corporation 30

Orientinisoorientin isomer separation amp CCS values

Isoorientin

19768Ǻ2

Orientin

18765Ǻ2

Identical twins with shared retention time amp accurate mass BUT different DRIFT times

C21H20O11

448377

∆ =10 Ǻ2

copy2015 Waters Corporation 23

UPLC separation Passiflora edularis

gt10 000 components

detected

copy2015 Waters Corporation 24

Ion mobility separation Increased peak capacity

Conventional UPLC separation

copy2015 Waters Corporation 25

Answering the challenge of natural product

characterisation

Spectral interpretation

copy2015 Waters Corporation 26

MSE multi component precursor and fragment ion spectra Vitexin (mz 431) retention time 8395 mins

Conventional HRMS Vitexin

Low energy

High energy

Vitexin

copy2015 Waters Corporation 27

HDMSE single component precursor and ion mobility product ion spectra

Vitexin (mz 431) retention time 8395 mins drift time 427 ms

Resolved from chromatographically coeluting components using ion mobility

Ion mobility spectral clean-up Vitexin

Vitexin

Low energy

High energy

copy2015 Waters Corporation 28

Answering the challenge of natural product

characterisation

Isomers

copy2015 Waters Corporation 29

Active marker flavonoid structures

OH

OH

O

OH

O

O

OH

OH

OH

OH

OH

O

OH

O

OH

O

OH

OH

OH

OH

OH

OH

OH

O

OH

O

O

OH

OH

OH

OH

OH

OH

O

OH

O

OH

O

OH

OH

OH

OH

Isoorientin Orientin

Isovitexin Vitexin

6C glycocides 8C glycocides

6

8

6

8

Luteolin-8-C-glucoside C21H20O11

Luteolin-6-C-glucoside C21H20O11

Apigenin-6-C-glucoside C21H20O10

Apigenin-8-C-glucoside C21H20O10

copy2015 Waters Corporation 30

Orientinisoorientin isomer separation amp CCS values

Isoorientin

19768Ǻ2

Orientin

18765Ǻ2

Identical twins with shared retention time amp accurate mass BUT different DRIFT times

C21H20O11

448377

∆ =10 Ǻ2

copy2015 Waters Corporation 24

Ion mobility separation Increased peak capacity

Conventional UPLC separation

copy2015 Waters Corporation 25

Answering the challenge of natural product

characterisation

Spectral interpretation

copy2015 Waters Corporation 26

MSE multi component precursor and fragment ion spectra Vitexin (mz 431) retention time 8395 mins

Conventional HRMS Vitexin

Low energy

High energy

Vitexin

copy2015 Waters Corporation 27

HDMSE single component precursor and ion mobility product ion spectra

Vitexin (mz 431) retention time 8395 mins drift time 427 ms

Resolved from chromatographically coeluting components using ion mobility

Ion mobility spectral clean-up Vitexin

Vitexin

Low energy

High energy

copy2015 Waters Corporation 28

Answering the challenge of natural product

characterisation

Isomers

copy2015 Waters Corporation 29

Active marker flavonoid structures

OH

OH

O

OH

O

O

OH

OH

OH

OH

OH

O

OH

O

OH

O

OH

OH

OH

OH

OH

OH

OH

O

OH

O

O

OH

OH

OH

OH

OH

OH

O

OH

O

OH

O

OH

OH

OH

OH

Isoorientin Orientin

Isovitexin Vitexin

6C glycocides 8C glycocides

6

8

6

8

Luteolin-8-C-glucoside C21H20O11

Luteolin-6-C-glucoside C21H20O11

Apigenin-6-C-glucoside C21H20O10

Apigenin-8-C-glucoside C21H20O10

copy2015 Waters Corporation 30

Orientinisoorientin isomer separation amp CCS values

Isoorientin

19768Ǻ2

Orientin

18765Ǻ2

Identical twins with shared retention time amp accurate mass BUT different DRIFT times

C21H20O11

448377

∆ =10 Ǻ2

copy2015 Waters Corporation 25

Answering the challenge of natural product

characterisation

Spectral interpretation

copy2015 Waters Corporation 26

MSE multi component precursor and fragment ion spectra Vitexin (mz 431) retention time 8395 mins

Conventional HRMS Vitexin

Low energy

High energy

Vitexin

copy2015 Waters Corporation 27

HDMSE single component precursor and ion mobility product ion spectra

Vitexin (mz 431) retention time 8395 mins drift time 427 ms

Resolved from chromatographically coeluting components using ion mobility

Ion mobility spectral clean-up Vitexin

Vitexin

Low energy

High energy

copy2015 Waters Corporation 28

Answering the challenge of natural product

characterisation

Isomers

copy2015 Waters Corporation 29

Active marker flavonoid structures

OH

OH

O

OH

O

O

OH

OH

OH

OH

OH

O

OH

O

OH

O

OH

OH

OH

OH

OH

OH

OH

O

OH

O

O

OH

OH

OH

OH

OH

OH

O

OH

O

OH

O

OH

OH

OH

OH

Isoorientin Orientin

Isovitexin Vitexin

6C glycocides 8C glycocides

6

8

6

8

Luteolin-8-C-glucoside C21H20O11

Luteolin-6-C-glucoside C21H20O11

Apigenin-6-C-glucoside C21H20O10

Apigenin-8-C-glucoside C21H20O10

copy2015 Waters Corporation 30

Orientinisoorientin isomer separation amp CCS values

Isoorientin

19768Ǻ2

Orientin

18765Ǻ2

Identical twins with shared retention time amp accurate mass BUT different DRIFT times

C21H20O11

448377

∆ =10 Ǻ2

copy2015 Waters Corporation 26

MSE multi component precursor and fragment ion spectra Vitexin (mz 431) retention time 8395 mins

Conventional HRMS Vitexin

Low energy

High energy

Vitexin

copy2015 Waters Corporation 27

HDMSE single component precursor and ion mobility product ion spectra

Vitexin (mz 431) retention time 8395 mins drift time 427 ms

Resolved from chromatographically coeluting components using ion mobility

Ion mobility spectral clean-up Vitexin

Vitexin

Low energy

High energy

copy2015 Waters Corporation 28

Answering the challenge of natural product

characterisation

Isomers

copy2015 Waters Corporation 29

Active marker flavonoid structures

OH

OH

O

OH

O

O

OH

OH

OH

OH

OH

O

OH

O

OH

O

OH

OH

OH

OH

OH

OH

OH

O

OH

O

O

OH

OH

OH

OH

OH

OH

O

OH

O

OH

O

OH

OH

OH

OH

Isoorientin Orientin

Isovitexin Vitexin

6C glycocides 8C glycocides

6

8

6

8

Luteolin-8-C-glucoside C21H20O11

Luteolin-6-C-glucoside C21H20O11

Apigenin-6-C-glucoside C21H20O10

Apigenin-8-C-glucoside C21H20O10

copy2015 Waters Corporation 30

Orientinisoorientin isomer separation amp CCS values

Isoorientin

19768Ǻ2

Orientin

18765Ǻ2

Identical twins with shared retention time amp accurate mass BUT different DRIFT times

C21H20O11

448377

∆ =10 Ǻ2

copy2015 Waters Corporation 27

HDMSE single component precursor and ion mobility product ion spectra

Vitexin (mz 431) retention time 8395 mins drift time 427 ms

Resolved from chromatographically coeluting components using ion mobility

Ion mobility spectral clean-up Vitexin

Vitexin

Low energy

High energy

copy2015 Waters Corporation 28

Answering the challenge of natural product

characterisation

Isomers

copy2015 Waters Corporation 29

Active marker flavonoid structures

OH

OH

O

OH

O

O

OH

OH

OH

OH

OH

O

OH

O

OH

O

OH

OH

OH

OH

OH

OH

OH

O

OH

O

O

OH

OH

OH

OH

OH

OH

O

OH

O

OH

O

OH

OH

OH

OH

Isoorientin Orientin

Isovitexin Vitexin

6C glycocides 8C glycocides

6

8

6

8

Luteolin-8-C-glucoside C21H20O11

Luteolin-6-C-glucoside C21H20O11

Apigenin-6-C-glucoside C21H20O10

Apigenin-8-C-glucoside C21H20O10

copy2015 Waters Corporation 30

Orientinisoorientin isomer separation amp CCS values

Isoorientin

19768Ǻ2

Orientin

18765Ǻ2

Identical twins with shared retention time amp accurate mass BUT different DRIFT times

C21H20O11

448377

∆ =10 Ǻ2

copy2015 Waters Corporation 28

Answering the challenge of natural product

characterisation

Isomers

copy2015 Waters Corporation 29

Active marker flavonoid structures

OH

OH

O

OH

O

O

OH

OH

OH

OH

OH

O

OH

O

OH

O

OH

OH

OH

OH

OH

OH

OH

O

OH

O

O

OH

OH

OH

OH

OH

OH

O

OH

O

OH

O

OH

OH

OH

OH

Isoorientin Orientin

Isovitexin Vitexin

6C glycocides 8C glycocides

6

8

6

8

Luteolin-8-C-glucoside C21H20O11

Luteolin-6-C-glucoside C21H20O11

Apigenin-6-C-glucoside C21H20O10

Apigenin-8-C-glucoside C21H20O10

copy2015 Waters Corporation 30

Orientinisoorientin isomer separation amp CCS values

Isoorientin

19768Ǻ2

Orientin

18765Ǻ2

Identical twins with shared retention time amp accurate mass BUT different DRIFT times

C21H20O11

448377

∆ =10 Ǻ2

copy2015 Waters Corporation 29

Active marker flavonoid structures

OH

OH

O

OH

O

O

OH

OH

OH

OH

OH

O

OH

O

OH

O

OH

OH

OH

OH

OH

OH

OH

O

OH

O

O

OH

OH

OH

OH

OH

OH

O

OH

O

OH

O

OH

OH

OH

OH

Isoorientin Orientin

Isovitexin Vitexin

6C glycocides 8C glycocides

6

8

6

8

Luteolin-8-C-glucoside C21H20O11

Luteolin-6-C-glucoside C21H20O11

Apigenin-6-C-glucoside C21H20O10

Apigenin-8-C-glucoside C21H20O10

copy2015 Waters Corporation 30

Orientinisoorientin isomer separation amp CCS values

Isoorientin

19768Ǻ2

Orientin

18765Ǻ2

Identical twins with shared retention time amp accurate mass BUT different DRIFT times

C21H20O11

448377

∆ =10 Ǻ2

copy2015 Waters Corporation 30

Orientinisoorientin isomer separation amp CCS values

Isoorientin

19768Ǻ2

Orientin

18765Ǻ2

Identical twins with shared retention time amp accurate mass BUT different DRIFT times

C21H20O11

448377

∆ =10 Ǻ2

copy2015 Waters Corporation 31

Answering the challenge of natural product

characterisation

Authenticity amp active compounds

copy2015 Waters Corporation 32

Identification of active compounds

CAERULEA EDULIS

INCARNATA ALATA

Which species has the most potent sedative effects

copy2015 Waters Corporation 32

Identification of active compounds

CAERULEA EDULIS

INCARNATA ALATA

Which species has the most potent sedative effects

copy2015 Waters Corporation 33

Comparative analysis tools Pedulis and Palata

Region of interest isoorientin amp orientin

Region of interest isovitexin amp vitexin

Mirror plots

Species comparison Passiflora edulis (reference) Passiflora alata (control)

copy2015 Waters Corporation 34

Passiflora edulis +++

Passiflora alata +

Vitexin

Isovitexin

Chromatographic view shows intensity differences for active compounds between species

Comparative analysis tools Pedulis and Palata

copy2015 Waters Corporation 35

PE unique region of

interest mz 577

Comparative analysis tools Pedulis and Palata

Drift time

mz

copy2015 Waters Corporation 36

Passiflora Edulis

Passiflora Alata

Different profiles for the markers at mz 577 Candidates for further investigation

Comparative analysis tools Pedulis and Palata

copy2015 Waters Corporation 37

Summary

Natural product profiling is challenged by complex samples with many isomeric

compounds

Combined with UPLC and HRMS ion mobility offers advantages for the

characterisation of these samples

ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak

capacity

ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers

ndash Drift times are measured for all precursor ions

ndash Simultaneous fragmentation data is obtained for all components

ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate

single component fragment ion spectra for all precursors

UNIFI software provides all the required informatics for interpretation of these

comprehensive datasets

ndash UNIFI automatically generates collision cross sections (CCS) for all components

ndash HDMS data viewer and comparison tools allows drift time mapping

copy2015 Waters Corporation 38

Markers of dietary intake

Monitoring a flavanol-rich diet

Dr Vanessa Garcia Larsen

copy2015 Waters Corporation 39

Project background

Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease

Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous

More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures

Aims ndash Can an accurate method be developed to estimate flavonoid markers

of dietary intake

o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)

o Can any biomarkers metabolites of flavonoid intake in vivo be identified

o Is it possible to develop a method of analysis that can be used routinely in studies in humans

copy2015 Waters Corporation 40

Experimental Approach

Dietary assessment of flavonoid intake

ndash Food Frequency Questionnaire (FFQ)

ndash Covered 12 months of dietary intake

ndash High and low dietary intake

Sampling

ndash Serum

Analytical set-up

ndash ACQUITY I-Class

ndash SYNAPT G2-Si HDMS

ndash Progenesis QI

copy2015 Waters Corporation 41

Dietary flavonoid subclasses

ndash Flavanones (eriodictyol hesperidin

and naringenin)

ndash Anthocyanins (cyanidin delphinidin

malvidin pelargononidin petunidin

peonidin)

ndash Flavan-3-ols (catechins epicatechin)

ndash Flavonols (quercetin kaemferol

myricetin isohamnetin)

ndash Flavones (luteolin apigenin)

ndash Flavonoid polymers

(proanthocyanidins theaflavins

thearubigins)

Flavonoids amp their polymers

constitute a large class of food

constituents amp alter metabolic

process amp have a positive impact

on health

Specific groups of foods are rich

sources of 1 or more subclasses

Proposed compound classes Polyphenolics

copy2015 Waters Corporation 34

Passiflora edulis +++

Passiflora alata +

Vitexin

Isovitexin

Chromatographic view shows intensity differences for active compounds between species

Comparative analysis tools Pedulis and Palata

copy2015 Waters Corporation 35

PE unique region of

interest mz 577

Comparative analysis tools Pedulis and Palata

Drift time

mz

copy2015 Waters Corporation 36

Passiflora Edulis

Passiflora Alata

Different profiles for the markers at mz 577 Candidates for further investigation

Comparative analysis tools Pedulis and Palata

copy2015 Waters Corporation 37

Summary

Natural product profiling is challenged by complex samples with many isomeric

compounds

Combined with UPLC and HRMS ion mobility offers advantages for the

characterisation of these samples

ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak

capacity

ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers

ndash Drift times are measured for all precursor ions

ndash Simultaneous fragmentation data is obtained for all components

ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate

single component fragment ion spectra for all precursors

UNIFI software provides all the required informatics for interpretation of these

comprehensive datasets

ndash UNIFI automatically generates collision cross sections (CCS) for all components

ndash HDMS data viewer and comparison tools allows drift time mapping

copy2015 Waters Corporation 38

Markers of dietary intake

Monitoring a flavanol-rich diet

Dr Vanessa Garcia Larsen

copy2015 Waters Corporation 39

Project background

Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease

Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous

More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures

Aims ndash Can an accurate method be developed to estimate flavonoid markers

of dietary intake

o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)

o Can any biomarkers metabolites of flavonoid intake in vivo be identified

o Is it possible to develop a method of analysis that can be used routinely in studies in humans

copy2015 Waters Corporation 40

Experimental Approach

Dietary assessment of flavonoid intake

ndash Food Frequency Questionnaire (FFQ)

ndash Covered 12 months of dietary intake

ndash High and low dietary intake

Sampling

ndash Serum

Analytical set-up

ndash ACQUITY I-Class

ndash SYNAPT G2-Si HDMS

ndash Progenesis QI

copy2015 Waters Corporation 41

Dietary flavonoid subclasses

ndash Flavanones (eriodictyol hesperidin

and naringenin)

ndash Anthocyanins (cyanidin delphinidin

malvidin pelargononidin petunidin

peonidin)

ndash Flavan-3-ols (catechins epicatechin)

ndash Flavonols (quercetin kaemferol

myricetin isohamnetin)

ndash Flavones (luteolin apigenin)

ndash Flavonoid polymers

(proanthocyanidins theaflavins

thearubigins)

Flavonoids amp their polymers

constitute a large class of food

constituents amp alter metabolic

process amp have a positive impact

on health

Specific groups of foods are rich

sources of 1 or more subclasses

Proposed compound classes Polyphenolics

copy2015 Waters Corporation 35

PE unique region of

interest mz 577

Comparative analysis tools Pedulis and Palata

Drift time

mz

copy2015 Waters Corporation 36

Passiflora Edulis

Passiflora Alata

Different profiles for the markers at mz 577 Candidates for further investigation

Comparative analysis tools Pedulis and Palata

copy2015 Waters Corporation 37

Summary

Natural product profiling is challenged by complex samples with many isomeric

compounds

Combined with UPLC and HRMS ion mobility offers advantages for the

characterisation of these samples

ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak

capacity

ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers

ndash Drift times are measured for all precursor ions

ndash Simultaneous fragmentation data is obtained for all components

ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate

single component fragment ion spectra for all precursors

UNIFI software provides all the required informatics for interpretation of these

comprehensive datasets

ndash UNIFI automatically generates collision cross sections (CCS) for all components

ndash HDMS data viewer and comparison tools allows drift time mapping

copy2015 Waters Corporation 38

Markers of dietary intake

Monitoring a flavanol-rich diet

Dr Vanessa Garcia Larsen

copy2015 Waters Corporation 39

Project background

Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease

Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous

More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures

Aims ndash Can an accurate method be developed to estimate flavonoid markers

of dietary intake

o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)

o Can any biomarkers metabolites of flavonoid intake in vivo be identified

o Is it possible to develop a method of analysis that can be used routinely in studies in humans

copy2015 Waters Corporation 40

Experimental Approach

Dietary assessment of flavonoid intake

ndash Food Frequency Questionnaire (FFQ)

ndash Covered 12 months of dietary intake

ndash High and low dietary intake

Sampling

ndash Serum

Analytical set-up

ndash ACQUITY I-Class

ndash SYNAPT G2-Si HDMS

ndash Progenesis QI

copy2015 Waters Corporation 41

Dietary flavonoid subclasses

ndash Flavanones (eriodictyol hesperidin

and naringenin)

ndash Anthocyanins (cyanidin delphinidin

malvidin pelargononidin petunidin

peonidin)

ndash Flavan-3-ols (catechins epicatechin)

ndash Flavonols (quercetin kaemferol

myricetin isohamnetin)

ndash Flavones (luteolin apigenin)

ndash Flavonoid polymers

(proanthocyanidins theaflavins

thearubigins)

Flavonoids amp their polymers

constitute a large class of food

constituents amp alter metabolic

process amp have a positive impact

on health

Specific groups of foods are rich

sources of 1 or more subclasses

Proposed compound classes Polyphenolics

copy2015 Waters Corporation 36

Passiflora Edulis

Passiflora Alata

Different profiles for the markers at mz 577 Candidates for further investigation

Comparative analysis tools Pedulis and Palata

copy2015 Waters Corporation 37

Summary

Natural product profiling is challenged by complex samples with many isomeric

compounds

Combined with UPLC and HRMS ion mobility offers advantages for the

characterisation of these samples

ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak

capacity

ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers

ndash Drift times are measured for all precursor ions

ndash Simultaneous fragmentation data is obtained for all components

ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate

single component fragment ion spectra for all precursors

UNIFI software provides all the required informatics for interpretation of these

comprehensive datasets

ndash UNIFI automatically generates collision cross sections (CCS) for all components

ndash HDMS data viewer and comparison tools allows drift time mapping

copy2015 Waters Corporation 38

Markers of dietary intake

Monitoring a flavanol-rich diet

Dr Vanessa Garcia Larsen

copy2015 Waters Corporation 39

Project background

Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease

Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous

More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures

Aims ndash Can an accurate method be developed to estimate flavonoid markers

of dietary intake

o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)

o Can any biomarkers metabolites of flavonoid intake in vivo be identified

o Is it possible to develop a method of analysis that can be used routinely in studies in humans

copy2015 Waters Corporation 40

Experimental Approach

Dietary assessment of flavonoid intake

ndash Food Frequency Questionnaire (FFQ)

ndash Covered 12 months of dietary intake

ndash High and low dietary intake

Sampling

ndash Serum

Analytical set-up

ndash ACQUITY I-Class

ndash SYNAPT G2-Si HDMS

ndash Progenesis QI

copy2015 Waters Corporation 41

Dietary flavonoid subclasses

ndash Flavanones (eriodictyol hesperidin

and naringenin)

ndash Anthocyanins (cyanidin delphinidin

malvidin pelargononidin petunidin

peonidin)

ndash Flavan-3-ols (catechins epicatechin)

ndash Flavonols (quercetin kaemferol

myricetin isohamnetin)

ndash Flavones (luteolin apigenin)

ndash Flavonoid polymers

(proanthocyanidins theaflavins

thearubigins)

Flavonoids amp their polymers

constitute a large class of food

constituents amp alter metabolic

process amp have a positive impact

on health

Specific groups of foods are rich

sources of 1 or more subclasses

Proposed compound classes Polyphenolics

copy2015 Waters Corporation 37

Summary

Natural product profiling is challenged by complex samples with many isomeric

compounds

Combined with UPLC and HRMS ion mobility offers advantages for the

characterisation of these samples

ndash Ion mobility separation is orthogonal to chromatography providing enhanced peak

capacity

ndash The combination of UPLC and IMS helps resolve coeluting compounds and even isomers

ndash Drift times are measured for all precursor ions

ndash Simultaneous fragmentation data is obtained for all components

ndash The added dimension of ion mobility enables spectral cleanup and the ability to generate

single component fragment ion spectra for all precursors

UNIFI software provides all the required informatics for interpretation of these

comprehensive datasets

ndash UNIFI automatically generates collision cross sections (CCS) for all components

ndash HDMS data viewer and comparison tools allows drift time mapping

copy2015 Waters Corporation 38

Markers of dietary intake

Monitoring a flavanol-rich diet

Dr Vanessa Garcia Larsen

copy2015 Waters Corporation 39

Project background

Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease

Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous

More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures

Aims ndash Can an accurate method be developed to estimate flavonoid markers

of dietary intake

o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)

o Can any biomarkers metabolites of flavonoid intake in vivo be identified

o Is it possible to develop a method of analysis that can be used routinely in studies in humans

copy2015 Waters Corporation 40

Experimental Approach

Dietary assessment of flavonoid intake

ndash Food Frequency Questionnaire (FFQ)

ndash Covered 12 months of dietary intake

ndash High and low dietary intake

Sampling

ndash Serum

Analytical set-up

ndash ACQUITY I-Class

ndash SYNAPT G2-Si HDMS

ndash Progenesis QI

copy2015 Waters Corporation 41

Dietary flavonoid subclasses

ndash Flavanones (eriodictyol hesperidin

and naringenin)

ndash Anthocyanins (cyanidin delphinidin

malvidin pelargononidin petunidin

peonidin)

ndash Flavan-3-ols (catechins epicatechin)

ndash Flavonols (quercetin kaemferol

myricetin isohamnetin)

ndash Flavones (luteolin apigenin)

ndash Flavonoid polymers

(proanthocyanidins theaflavins

thearubigins)

Flavonoids amp their polymers

constitute a large class of food

constituents amp alter metabolic

process amp have a positive impact

on health

Specific groups of foods are rich

sources of 1 or more subclasses

Proposed compound classes Polyphenolics

copy2015 Waters Corporation 38

Markers of dietary intake

Monitoring a flavanol-rich diet

Dr Vanessa Garcia Larsen

copy2015 Waters Corporation 39

Project background

Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease

Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous

More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures

Aims ndash Can an accurate method be developed to estimate flavonoid markers

of dietary intake

o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)

o Can any biomarkers metabolites of flavonoid intake in vivo be identified

o Is it possible to develop a method of analysis that can be used routinely in studies in humans

copy2015 Waters Corporation 40

Experimental Approach

Dietary assessment of flavonoid intake

ndash Food Frequency Questionnaire (FFQ)

ndash Covered 12 months of dietary intake

ndash High and low dietary intake

Sampling

ndash Serum

Analytical set-up

ndash ACQUITY I-Class

ndash SYNAPT G2-Si HDMS

ndash Progenesis QI

copy2015 Waters Corporation 41

Dietary flavonoid subclasses

ndash Flavanones (eriodictyol hesperidin

and naringenin)

ndash Anthocyanins (cyanidin delphinidin

malvidin pelargononidin petunidin

peonidin)

ndash Flavan-3-ols (catechins epicatechin)

ndash Flavonols (quercetin kaemferol

myricetin isohamnetin)

ndash Flavones (luteolin apigenin)

ndash Flavonoid polymers

(proanthocyanidins theaflavins

thearubigins)

Flavonoids amp their polymers

constitute a large class of food

constituents amp alter metabolic

process amp have a positive impact

on health

Specific groups of foods are rich

sources of 1 or more subclasses

Proposed compound classes Polyphenolics

copy2015 Waters Corporation 39

Project background

Recent epidermiologic evidence has suggested a protective effect of diets rich in flavonoids against stroke and respiratory disease

Current methods to assess nutrient intake involve the recall of food over a prolonged period of time and a more rapid technique would be advantageous

More accurate methods to estimate markers of dietary intake including flavonoids will improve our understanding on the complex relationship between disease and dietary exposures

Aims ndash Can an accurate method be developed to estimate flavonoid markers

of dietary intake

o Is the use of HR-MS useful for this experiment (unknown markers in humans samples)

o Can any biomarkers metabolites of flavonoid intake in vivo be identified

o Is it possible to develop a method of analysis that can be used routinely in studies in humans

copy2015 Waters Corporation 40

Experimental Approach

Dietary assessment of flavonoid intake

ndash Food Frequency Questionnaire (FFQ)

ndash Covered 12 months of dietary intake

ndash High and low dietary intake

Sampling

ndash Serum

Analytical set-up

ndash ACQUITY I-Class

ndash SYNAPT G2-Si HDMS

ndash Progenesis QI

copy2015 Waters Corporation 41

Dietary flavonoid subclasses

ndash Flavanones (eriodictyol hesperidin

and naringenin)

ndash Anthocyanins (cyanidin delphinidin

malvidin pelargononidin petunidin

peonidin)

ndash Flavan-3-ols (catechins epicatechin)

ndash Flavonols (quercetin kaemferol

myricetin isohamnetin)

ndash Flavones (luteolin apigenin)

ndash Flavonoid polymers

(proanthocyanidins theaflavins

thearubigins)

Flavonoids amp their polymers

constitute a large class of food

constituents amp alter metabolic

process amp have a positive impact

on health

Specific groups of foods are rich

sources of 1 or more subclasses

Proposed compound classes Polyphenolics

copy2015 Waters Corporation 40

Experimental Approach

Dietary assessment of flavonoid intake

ndash Food Frequency Questionnaire (FFQ)

ndash Covered 12 months of dietary intake

ndash High and low dietary intake

Sampling

ndash Serum

Analytical set-up

ndash ACQUITY I-Class

ndash SYNAPT G2-Si HDMS

ndash Progenesis QI

copy2015 Waters Corporation 41

Dietary flavonoid subclasses

ndash Flavanones (eriodictyol hesperidin

and naringenin)

ndash Anthocyanins (cyanidin delphinidin

malvidin pelargononidin petunidin

peonidin)

ndash Flavan-3-ols (catechins epicatechin)

ndash Flavonols (quercetin kaemferol

myricetin isohamnetin)

ndash Flavones (luteolin apigenin)

ndash Flavonoid polymers

(proanthocyanidins theaflavins

thearubigins)

Flavonoids amp their polymers

constitute a large class of food

constituents amp alter metabolic

process amp have a positive impact

on health

Specific groups of foods are rich

sources of 1 or more subclasses

Proposed compound classes Polyphenolics

copy2015 Waters Corporation 41

Dietary flavonoid subclasses

ndash Flavanones (eriodictyol hesperidin

and naringenin)

ndash Anthocyanins (cyanidin delphinidin

malvidin pelargononidin petunidin

peonidin)

ndash Flavan-3-ols (catechins epicatechin)

ndash Flavonols (quercetin kaemferol

myricetin isohamnetin)

ndash Flavones (luteolin apigenin)

ndash Flavonoid polymers

(proanthocyanidins theaflavins

thearubigins)

Flavonoids amp their polymers

constitute a large class of food

constituents amp alter metabolic

process amp have a positive impact

on health

Specific groups of foods are rich

sources of 1 or more subclasses

Proposed compound classes Polyphenolics

copy2015 Waters Corporation 42

UPLC Chromatogram ESI negative trace

Flavanoids Non-polar metabolites

copy2015 Waters Corporation 43

UPLC Chromatogram ESI negative trace

Flavanoids Non-polar metabolites

Gallic acid C7H6O5

copy2015 Waters Corporation 44

Unsupervised PCA analysis Low vs high grape consumption

copy2015 Waters Corporation 43

UPLC Chromatogram ESI negative trace

Flavanoids Non-polar metabolites

Gallic acid C7H6O5

copy2015 Waters Corporation 44

Unsupervised PCA analysis Low vs high grape consumption

copy2015 Waters Corporation 44

Unsupervised PCA analysis Low vs high grape consumption

copy2015 Waters Corporation 45

S-Plot Low vs high grape consumption

Potential biomarkers for the high samples

Potential biomarkers for the low samples

Visualisation of covariance amp correlation between the

metabolites and the class designation

copy2015 Waters Corporation 46

Progenesis QI Marker identification ndash database searching phase

copy2015 Waters Corporation 47

Initial results amp future work

43000 compounds of interest = comprehensive data set to

determine the most appropriate markers for the study

ndash Data interpretation Progenesis and Ezinfo

Further evaluation planned to test the model with a larger study

group

Use the data to construct a routine assay using MS

ndash Three biomarkers have already been identified and will be used as an

objective tool to estimate dietary intake of flavonoids

copy2015 Waters Corporation 48

copy2015 Waters Corporation 49

Sample

REIMS instrument configuration

iKnife and bipolar forceps Hand-held

sampling device(s)

Diathermy generator

Informatics system

Source

REIMS is an emerging technique that allows rapid characterisation of

biological tissues

Xevo G2-XS Qtof and

Synapt G2Si

copy2015 Waters Corporation 50

How does it work

copy2015 Waters Corporation 51

REIMS workflow Direct analysis with chemometric profiling

Xevo G2-XS Qtof or Synapt G2Si

Model Generation

Multivariate statistical analysis

Different tissues

Different species

Different production

Biomarker ID amp verification

Rapid screening of unknowns by similarity to database

entries

copy2015 Waters Corporation 52

Direct analysis for flexible amp rapid screening

Sub-sampling Determination

(5s)

Data analysis (2s)

Report

Sample to report in seconds

Point-of-control analysis

copy2015 Waters Corporation 47

Initial results amp future work

43000 compounds of interest = comprehensive data set to

determine the most appropriate markers for the study

ndash Data interpretation Progenesis and Ezinfo

Further evaluation planned to test the model with a larger study

group

Use the data to construct a routine assay using MS

ndash Three biomarkers have already been identified and will be used as an

objective tool to estimate dietary intake of flavonoids

copy2015 Waters Corporation 48

copy2015 Waters Corporation 49

Sample

REIMS instrument configuration

iKnife and bipolar forceps Hand-held

sampling device(s)

Diathermy generator

Informatics system

Source

REIMS is an emerging technique that allows rapid characterisation of

biological tissues

Xevo G2-XS Qtof and

Synapt G2Si

copy2015 Waters Corporation 50

How does it work

copy2015 Waters Corporation 51

REIMS workflow Direct analysis with chemometric profiling

Xevo G2-XS Qtof or Synapt G2Si

Model Generation

Multivariate statistical analysis

Different tissues

Different species

Different production

Biomarker ID amp verification

Rapid screening of unknowns by similarity to database

entries

copy2015 Waters Corporation 52

Direct analysis for flexible amp rapid screening

Sub-sampling Determination

(5s)

Data analysis (2s)

Report

Sample to report in seconds

Point-of-control analysis

copy2015 Waters Corporation 48

copy2015 Waters Corporation 49

Sample

REIMS instrument configuration

iKnife and bipolar forceps Hand-held

sampling device(s)

Diathermy generator

Informatics system

Source

REIMS is an emerging technique that allows rapid characterisation of

biological tissues

Xevo G2-XS Qtof and

Synapt G2Si

copy2015 Waters Corporation 50

How does it work

copy2015 Waters Corporation 51

REIMS workflow Direct analysis with chemometric profiling

Xevo G2-XS Qtof or Synapt G2Si

Model Generation

Multivariate statistical analysis

Different tissues

Different species

Different production

Biomarker ID amp verification

Rapid screening of unknowns by similarity to database

entries

copy2015 Waters Corporation 52

Direct analysis for flexible amp rapid screening

Sub-sampling Determination

(5s)

Data analysis (2s)

Report

Sample to report in seconds

Point-of-control analysis

copy2015 Waters Corporation 49

Sample

REIMS instrument configuration

iKnife and bipolar forceps Hand-held

sampling device(s)

Diathermy generator

Informatics system

Source

REIMS is an emerging technique that allows rapid characterisation of

biological tissues

Xevo G2-XS Qtof and

Synapt G2Si

copy2015 Waters Corporation 50

How does it work

copy2015 Waters Corporation 51

REIMS workflow Direct analysis with chemometric profiling

Xevo G2-XS Qtof or Synapt G2Si

Model Generation

Multivariate statistical analysis

Different tissues

Different species

Different production

Biomarker ID amp verification

Rapid screening of unknowns by similarity to database

entries

copy2015 Waters Corporation 52

Direct analysis for flexible amp rapid screening

Sub-sampling Determination

(5s)

Data analysis (2s)

Report

Sample to report in seconds

Point-of-control analysis

copy2015 Waters Corporation 50

How does it work

copy2015 Waters Corporation 51

REIMS workflow Direct analysis with chemometric profiling

Xevo G2-XS Qtof or Synapt G2Si

Model Generation

Multivariate statistical analysis

Different tissues

Different species

Different production

Biomarker ID amp verification

Rapid screening of unknowns by similarity to database

entries

copy2015 Waters Corporation 52

Direct analysis for flexible amp rapid screening

Sub-sampling Determination

(5s)

Data analysis (2s)

Report

Sample to report in seconds

Point-of-control analysis

copy2015 Waters Corporation 51

REIMS workflow Direct analysis with chemometric profiling

Xevo G2-XS Qtof or Synapt G2Si

Model Generation

Multivariate statistical analysis

Different tissues

Different species

Different production

Biomarker ID amp verification

Rapid screening of unknowns by similarity to database

entries

copy2015 Waters Corporation 52

Direct analysis for flexible amp rapid screening

Sub-sampling Determination

(5s)

Data analysis (2s)

Report

Sample to report in seconds

Point-of-control analysis

copy2015 Waters Corporation 52

Direct analysis for flexible amp rapid screening

Sub-sampling Determination

(5s)

Data analysis (2s)

Report

Sample to report in seconds

Point-of-control analysis

copy2015 Waters Corporation 53

Genotype influences phenotypic characteristics

Ca 22k genes

20 different amino acids combined to give gt100k

expressed proteins

gtgt post-translational modifications

Metabolites are close to the phenotype

copy2015 Waters Corporation 54

Speciation buffalo vs bovine spectra REIMS Tof MS neg ion IPA 150 ulmin 50-1200 mz

copy2015 Waters Corporation 55

Polyunsaturated fatty acid (PUFA) content

Ca x16

Arachidonic acid Ω6

C20H32O2

Eicosapentaenoic acid Ω3

C20H30O2

Ca x7

Good ratio of Ω36 acids

copy2015 Waters Corporation 56

Multi-species muscle tissue ndash MVA models

2 biological replicates of each species 10 technical replicates per sample

PCA model

Non-ruminants

Ruminants

Buffalo Bovine

Porcine

Ovine

Gallus gallus

copy2015 Waters Corporation 57

PROTOTYPE real-time recogniser software

copy2015 Waters Corporation 57

PROTOTYPE real-time recogniser software

copy2015 Waters Corporation 58

Food Fraud amp authenticity applications

copy2015 Waters Corporation 59

REIMS spectra

Kaempferol

Apigenin

Oleanolic acid ursolic acid

Herbs amp spices

PDO status Belgian butter

Botanical origin of honey

Essential Ω6 fatty acid

Ω9

Galangin Myricetin

Geographical origin of coffee

Caffeine Tanzania

Peru

Kenyan

Colombian

copy2015 Waters Corporation 60

Butter fatty acid profile 220-290 mz Tof MS neg ion

Myristic acid C140

C14H27O2

[M-H]- Δ-06

Palmitoleic acid

C161 (9Z) C16H29O2

[M-H]- Δ 07

Palmitic acid C160

C16H31O2

[M-H]- Δ 06

Linoleic acid C182 (9Z12Z)

C18H31O2

[M-H]- Δ 28

Oleic acid C181 (9Z)

C18H33O2

[M-H]- Δ 08

Stearic acid C180

C18H35O2

[M-H]- Δ 06

Pentadecanoic acid C150

C15H29O2

[M-H]- Δ 02

Heptadecanoic acid C17H33O2

[M-H]- Δ 05

Butterfat is a triglyceride derived from fatty acids such as myristic palmitic and

oleic acids Saturated fatty acids Palmitic acid 31 Myristic acid 12 Stearic acid 11 Lower (at most 12 carbon atoms) saturated fatty acids 11 pentadecanoic acid and heptadecanoic acid traces Unsaturated fatty acids Oleic acid 24 Palmitoleic acid 4 Linoleic acid 3 α-Linolenic acid 1

α-Linoleic acid C183 (9Z12Z 15Z)

C18H29O2

[M-H]- Δ 13

Δ mDa error

copy2015 Waters Corporation 61

Rosmarinus officinalis Tof MS neg ion

Kaempferol C15H10O6

-016ppm

Apigenin C15H10O5

-22ppm

Oleanolic acid ursolic acid

C30H48O3

-184ppm

Quinic acid C7H16O6

123ppm

57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7

-394ppm

Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids

2-hydroxybenzoic acid (salicyclic acid) C7H6O3

146ppm

copy2015 Waters Corporation 61

Rosmarinus officinalis Tof MS neg ion

Kaempferol C15H10O6

-016ppm

Apigenin C15H10O5

-22ppm

Oleanolic acid ursolic acid

C30H48O3

-184ppm

Quinic acid C7H16O6

123ppm

57-Dihydroxy-345-trimethoxyflavone (ayanin) C18H16O7

-394ppm

Flavonoids Polyphenols Terpenes terpene alcohols amp terpenoids

2-hydroxybenzoic acid (salicyclic acid) C7H6O3

146ppm

copy2015 Waters Corporation 62

Rosmarinus officinalis Tof MSMS rosmarinic acid 3591 mz

copy2015 Waters Corporation 63

Summary

There is a link between food and health

ndash Much research is directed to understand the relationship between food intake key metabolites health issues and disease

ndash ldquoDevelopment of new products for functional foods nutraceuticals and natural health products sector is one of the fastest expanding areas of research todayrdquo [CORDIS]

Metabolomic approach using HRMS screening

ndash Allows a holistic view of food composition amp contaminants

ndash Ion mobility separation is orthogonal to chromatography

o Enhanced peak capacity from ion mobility reduces sample complexity

REIMS emerging technique for direct analysis

ndash Non-targeted analysis with real-time chemometric profiling

ndash Point-of-control analysis

copy2015 Waters Corporation 64

Acknowledgements

Prof Clare Mills

Mike McCullagh

Antonietta Wallace

Lee Gethings

Martin Wells

copy2015 Waters Corporation 64

Acknowledgements

Prof Clare Mills

Mike McCullagh

Antonietta Wallace

Lee Gethings

Martin Wells