lipid%mediator%metabololipidomicslc2ms2ms%spectra%book2014%serhanlab.bwh.harvard.edu/wp-content/uploads/2018/11/spectra-boo… ·...

Lipid Mediator Metabololipidomics LC-‐MS-‐MS Spectra Book 2014

Jonathan FITZGERALD, Romain COLAS, Masakazu SHINOHARA, Jesmond DALLI and Charles SERHAN

Center for Experimental TherapeuFcs and Reperfusion Injury, Harvard InsFtute of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MassachuseOs 02115 U.S.A.

Correspondence should be addressed to: Prof. Charles N. Serhan Harvard InsFtute of Medicine 77 Avenue Louis Pasteur, HIM 829 Boston, MA 02115.

Email: [email protected]; Tel: 617-‐525-‐5001; Fax: 617-‐525-‐5017

HARVARD MEDICAL SCHOOL

BRIGHAM AND WOMEN’S HOSPITAL

Version: 02142014 1/104

Table of Contents

Lipid Mediator Structures & Stereochemistry 3 Arachidonic Acid Metabolome 4 Eicosapentaenoic Acid Metabolome 9 Docosahexaenoic Acid Metabolome 13 Aspirin-‐Triggered Specialized Pro-‐resolving Mediators 18 Deuterium Labeled Internal Standards 22

Methods 25 OperaEonalizaEon of lipid mediator metabololipidomics 26 High Pressure Liquid Chromatography 27 Mass Spectrometry 27 Methods validaEon 30

Linearity and aHributed internal standard 30 Recovery, matrix interference and limit of detecEon 31 Repeatability and reproducibility 32 Chemical stability of selected endogenous lipid mediators idenEfied in human serum 36

MulEple ReacEon Monitoring TransiEons 37 Lipid Mediators 38 Aspirin-‐Triggered Lipid Mediators 38 Further Metabolites of Lipid Mediators 39

Lipid Mediator BiosyntheEc Pathways 40 Prostaglandins, Leukotrienes and Lipoxins 41 E-‐Series Resolvins 42 D-‐Series Resolvins 43 Aspirin-‐Triggered D-‐Series Resolvins 44 ProtecEns 45 Maresins 46

LC-‐MS/MS Spectra 47 Arachidonic Acid Metabolome 48

Prostaglandins & Thromboxane 48 Leukotrienes 53 Lipoxins 57 MonoHydroxyETE Products 61

Eicosapentaenoic Acid Metabolome 65 Lipoxins 65 E-‐Series Resolvins 68 MonoHydroxyEPE Products 72

Docosahexaenoic Acid Metabolome 77 D-‐Series Resolvins 77 Maresins 83 ProtecEns 86 MonoHydroxyDHA Products 88

Aspirin-‐Triggered Specialized Pro-‐resolving Mediators 93 Deuterium Labeled Internal Standards 99

2/104

Prof. Charles N. SERHAN Laboratory Brigham & Women’s Hospital Center for Experimental TherapeuFcs and Reperfusion Injury Harvard Medical School


© 2014 Serhan et al.

Lipid Mediator Structures & Stereochemistry

3/104




Arachidonic Acid Metabolome

4/104




Complete

Stereo

chem

istry

5Z,8Z,11

Z,14

Z-‐Eicosatetraeno

ic acid

11-‐oxo-‐

9α,15S-‐

dihydroxy-‐

5Z,13E-‐

prostadien

oic acid

9-‐oxo-‐

11α,15S-‐

dihydroxy-‐

5Z,13E-‐

prostadien

oic acid

9α,11α

,15S-‐

trihydroxy-‐

5Z,13E-‐

prostadien

oic acid

Trivial N

ame

Arachidon

ic acid

Prostaglandin D2

Prostaglandin E 2

Prostaglandin F 2

α

Chem

ical Nam

e

Eicosatetraeno

ic

acid

11-‐dehydro-‐

prostaglandin F 2

α

9-‐de

hydro-‐

prostaglandin F 2

α

9α,11α

,15S-‐

trihydroxy-‐

prostaglandin F 2

α

Form

ula

C 20H

32O2

C 20H

32O5

C 20H

32O5

C 20H

34O5

Structure

Abb

reviaE

on

AA

PGD2

PGE 2

PGF 2

α

COOH

OH

HO O

COOH

OH

O

HO

COOH

OH

HO

HO

COOH

5/104

Complete

Stereo

chem

istry

9α,�11,�15

S-‐�

trihydroxy-‐

5Z,�13E-‐

thromba�dien

�oic acid

5S,�12R

-‐� dihydroxy-‐�

6Z,�8E,�10

E,�14

Z-‐�

eicosatetraeno

ic acid

5S,�12R

,20-‐�

trihydroxy-‐�

6Z,�8E,�10

E,�14

Z-‐�

eicosatetraeno

ic acid

5S,�12R

-‐� dihydroxy-‐

�6Z,�8E,�10

E,�14

Z-‐�

20-‐carbo

xy-‐

eicosatetraene

dioic acid

Trivial N

ame

Thrombo

xane

B2

Leukotrien

e B 4

20-‐hydroxy-‐

Leukotrien

e B 4

20-‐carbo

xy-‐

Leukotrien

e B 4

Chem

ical Nam

e

9α,�11,�15

S-‐�

trihydroxy-‐

thrombo

xane

B2

5S,12R

-‐ dihydroxy-‐�

LTB 4

5S,12R

,20-‐

trihydroxy-‐�

LTB 4

5S,12R


20-‐carbo

xy-‐

LTB 4

Form

ula

C 20H

34O6

C 20H

32O4

C 20H

32O5

C 20H

30O6

Structure

Abb

reviaE

on

TxB 2

LTB 4

20-‐OH-‐LTB

4

20-‐COOH-‐LTB

4

COOH

OH

OHO

OH

COOH

OH

OH

COOH

OH

OH

OH

6/104

COOH

COOH

OH

OH

Complete

Stereo

chem

istry

5S,6R,15

S-‐

trihydroxy-‐

7E,9E,11

Z,13

E-‐eicosatetraeno

ic acid

5S,14R

,15S-‐

trihydroxy-‐

6E,8Z,10

E,12

E-‐

eicosatetraeno

ic acid

5S,�15S-‐

�dihydroxy-‐

�6E,�8Z,�11

Z,�13

E-‐�

eicosatetraeno

ic acid

5-‐�

hydroxy-‐�

6E,�8Z,�11

Z,�14

Z-‐�

eicosatetraeno

ic acid

Trivial N

ame

Lipo

xin A4

Lipo

xin B 4

5S,15S-‐

dihydroxy-‐

eicosatetraeno

ic

acid

5-‐

hydroxy-‐

eicosatetraeno

ic

acid

Chem

ical Nam

e

5S,6R,15

S-‐

trihydroxy-‐

LXA4

5S,14R

,15S-‐

trihydroxy-‐

LXB 4

5S,15S-‐

dihydroxy-‐

ETE

5-‐

hydroxy-‐

ETE

Form

ula

C 20H

32O5

C 20H

32O5

C 20H

32O4

C 20H

32O3

Structure

Abb

reviaE

on

LXA4

LXB 4

5S,15S-‐diHETE

5-‐HETE

COOH

OH

HO

OH

COOH

OH

HO

OH

COOH

OH

COOH

OH

OH

7/104

Complete

Stereo

chem

istry

12-‐�

hydroxy-‐�

5Z,�8Z,�10

E,�14

Z-‐�

eicosatetraeno

ic acid

15-‐�

hydroxy-‐�

5Z,�8Z,�11

Z,�13

E-‐�

eicosatetraeno

ic acid

Trivial N

ame

12-‐

hydroxy-‐

eicosatetraeno

ic

acid

15-‐

hydroxy-‐

eicosatetraeno

ic

acid

Chem

ical Nam

e

12-‐

hydroxy-‐

ETE

15-‐

hydroxy-‐

ETE

Form

ula

C 20H

32O3

C 20H

32O3

Structure

Abb

reviaE

on

12-‐HETE

15-‐HETE

COOH

OH

COOH

OH

8/104

Eicosapentaenoic Acid Metabolome

9/104




Complete

Stereo

chem

istry

5Z,8Z,11

Z,14

Z,17

Z-‐eicosape

ntaeno

ic acid

5S,�6R,�15

S-‐�

trihydroxy-‐

�7E,�9E,�11

Z,�13

E,�17

Z-‐�

eicosape

ntaeno

ic acid

5S,14R

,15S-‐

trihydroxy-‐

6E,8Z,10

E,12

E,17

Z-‐

eicosape

ntaeno

ic acid

5S,�15S-‐�

dihydroxy-‐

�6E,�8Z,�11

Z,�13

E,17

Z-‐�

eicosape

ntaeno

ic acid

Trivial N

ame

Eicosape

ntaeno

ic

acid

Lipo

xin A5

Lipo

xin B 5

5S,15S-‐

dihydroxy-‐

eicosape

ntaeno

ic

acid

Chem

ical Nam

e

Eicosape

ntaeno

ic

acid

5S,6R,15

S-‐

trihydroxy-‐

LXA5

5S,14R

,15S-‐

trihydroxy-‐

LXB 5

5S,15S-‐

dihydroxy-‐

EPE

Form

ula

C 20H

30O2

C 20H

30O5

C 20H

30O5

C 20H

30O4

Structure

Abb

reviaE

on

EPA

LXA5

LXB 5

5S,15S-‐diHEP

E

COOH

OH

HO

OH

COOH

OH

HO

OH

COOH

OH

OH

COOH

10/104

Complete

Stereo

chem

istry

5S,12R

,18R

-‐ trihydroxy-‐

6Z,8E,10

E,14

Z,16

E-‐eicosape

ntaeno

ic acid

5S,18R

-‐ dihydroxy-‐

6E,8Z,11

Z,14

Z,16

E-‐

eicosape

ntaeno

ic acid

17R,18

R-‐

dihydroxy-‐

5Z,8Z,11

Z,13

E,15

E-‐eicosape

ntaeno

ic acid

5-‐

�hydroxy-‐�

6E,�8Z,�11

Z,�14

Z,�17

Z-‐�

eicosape

ntaeno

ic acid

Trivial N

ame

Resolvin E1

Resolvin E2

Resolvin E3

5-‐

hydroxy-‐

eicosape

ntaeno

ic

acid

Chem

ical Nam

e

5S,12R

,18R

-‐trihydroxy-‐

EPE

5S,18R

-‐ dihydroxy-‐

EPE

17R,18

R-‐

dihydroxy-‐

EPE

5-‐

hydroxy-‐

EPE

Form

ula

C 20H

30O5

C 20H

30O4

C 20H

30O4

C 20H

30O3

Structure

Abb

reviaE

on

RvE1

RvE2

RvE3

5-‐HEP

E

COOH

OH

HO

COOH

OH

11/104

COOH

HO

OH

COOH

HO

OH

OH

Complete

Stereo

chem

istry

12-‐�

hydroxy-‐

�5Z,�8Z,�10

E,�14

Z,�17

Z-‐�

eicosape

ntaeno

ic acid

15-‐

�hydroxy-‐�

5Z,�8Z,�11

Z,�13

E,�17

Z-‐�

eicosape

ntaeno

ic acid

18-‐�

hydroxy-‐�

5Z,�8Z,�11

Z,�14

Z,�16

E-‐�

eicosape

ntaeno

ic acid

Trivial N

ame

12-‐

hydroxy-‐

eicosape

ntaeno

ic

acid

15-‐

hydroxy-‐

eicosape

ntaeno

ic

acid

18-‐

hydroxy-‐

eicosape

ntaeno

ic

acid

Chem

ical Nam

e

12-‐

hydroxy-‐

EPE

15-‐

hydroxy-‐

EPE

18-‐

hydroxy-‐

EPE

Form

ula

C 20H

30O3

C 20H

30O3

C 20H

30O3

Structure

Abb

reviaE

on

12-‐HEP

E

15-‐HEP

E

18-‐HEP

E

COOH

OH

COOH

OH

COOH

OH

12/104

Docosahexaenoic Acid Metabolome

13/104




Complete

Stereo

chem

istry

4Z,7Z,10

Z,13

Z,16

Z,19

Z-‐do

cosahexaen

oic acid

7S,8R,17

S-‐

trihydroxy-‐

4Z,9E,11

E,13

Z,15

E,19

Z-‐

docosahexaen

oic acid

7S,16R

,17S-‐

trihydroxy-‐

4Z,8E,10

Z,12

E,14

E,19

Z-‐

docosahexaen

oic acid

4S,11R

,17S-‐

trihydroxy-‐

5Z,7E,9E,13Z,15E,19Z-‐

docosahexaen

oic acid

Trivial N

ame

Docosahexaeno

ic

acid

Resolvin D1

Resolvin D2

Resolvin D3

Chem

ical Nam

e

Docosahexaeno

ic

acid

7S,8R,17

S-‐trihydroxy-‐

DHA

7S,16R

,17S-‐

trihydroxy-‐

DHA

4S,11R

,17S-‐

trihydroxy-‐

DHA

Form

ula

C 22H

32O2

C 22H

32O5

C 22H

32O5

C 22H

32O5

Structure

Abb

reviaE

on

DHA

RvD1

RvD2

RvD3

COOH

OH

HO

HO

COOH

14/104

COOH

HO

OH

OH

OH

HO

COOH

HO

Complete

Stereo

chem

istry

4S,5,17S-‐

trihydroxy-‐

6E,8E,10

Z,13

Z,15

E,19

Z-‐

docosahexaen

oic acid

7S,17S-‐

dihydroxy-‐

4Z,8E,10

Z,13

Z,15

E,19

Z-‐

docosahexaen

oic acid

4S,17S-‐

dihydroxy-‐

5E,7Z,10

Z,13

Z,15

E,19

Z-‐

docosahexaen

oic acid

7R,14S-‐

dihydroxy-‐

4Z,8E,10

E,12

Z,16

Z,19

Z-‐do

cosahexaen

oic acid

Trivial N

ame

Resolvin D4

Resolvin D5

Resolvin D6

Maresin 1

Chem

ical Nam

e

4S,5R,17

S-‐

trihydroxy-‐

DHA

7S,17S-‐

dihydroxy-‐

DHA

4S,17S-‐

dihydroxy-‐

DHA

7R,14S-‐

dihydroxy-‐

DHA

Form

ula

C 22H

32O5

C 22H

32O4

C 22H

32O4

C 22H

32O4

Structure

Abb

reviaE

on

RvD4

RvD5

RvD6

MaR

1

COOH

HO

HO

15/104

OH

OH

COOH

OH

OH

OH

HOOC

OH

OH

HOOC

Complete

Stereo

chem

istry

4S,14S-‐

dihydroxy-‐

5E,7Z,10

Z,12

E,16

Z,19

Z-‐do

cosahexaen

oic acid

10R,17

S-‐

dihydroxy-‐

4Z,7Z,11

E,13

E,15

Z,19

Z-‐

docosahexaen

oic acid

4-‐�hydroxy-‐�

5E,�7Z,�10

Z,�13

Z,�16

Z,�19

Z-‐�

docosahexaen

oic acid


4Z,�8E,�10

Z,�13

Z,�16

Z,�19

Z-‐�

docosahexaen

oic acid

Trivial N

ame

4,14-‐diHDHA

[Neu

ro]

ProtecFn

D1

4-‐hydroxy-‐

DHA

7-‐hydroxy-‐

DHA

Chem

ical Nam

e

4S,14S-‐

dihydroxy-‐

DHA

10R,17

S-‐

dihydroxy-‐

DHA

4-‐hydroxy-‐

DHA

7-‐hydroxy-‐

DHA

Form

ula

C 22H

32O4

C 22H

32O4

C 22H

32O3

C 22H

32O3

Structure

Abb

reviaE

on

4S,14S-‐diHDHA

PD1

4-‐HDHA

7-‐HDHA

COOH

OH

OH

COOH

OH

OH

COOH

OH

COOH

OH

16/104

Complete

Stereo

chem

istry


4Z,�7Z,�10

Z,�12

E,�16

Z,�19

Z-‐�

docosahexaen

oic acid


4Z,�7Z,�10

Z,�13

Z,�15

E,�19

Z-‐�

docosahexaen

oic acid

Trivial N

ame

14-‐hydroxy-‐

DHA

17-‐hydroxy-‐

DHA

Chem

ical Nam

e

14-‐hydroxy-‐

DHA

17-‐hydroxy-‐

DHA

Form

ula

C 22H

32O3

C 22H

32O3

Structure

Abb

reviaE

on

14-‐HDHA

17-‐HDHA

OH

COOH

COOH

OH

17/104

Aspirin-‐Triggered Specialized Pro-‐resolving Mediators

18/104




Complete

Stereo

chem

istry

5S,6R,15

R-‐

trihydroxy-‐

7E,9E,11

Z,13

E-‐eicosatetraeno

ic acid

5S,14R

,15R

-‐ trihydroxy-‐

6E,8Z,10

E,12

E-‐

eicosatetraeno

ic acid

7S,8R,17

R-‐

trihydroxy-‐

4Z,9E,11

E,13

Z,15

E,19

Z-‐

docosahexaen

oic acid

7S,16R

,17R

-‐ trihydroxy-‐

4Z,8E,10

Z,12

E,14

E,19

Z-‐do

cosahexaen

oic acid

Trivial N

ame

15-‐epi-‐Lipoxin A

4

15-‐epi-‐Lipoxin B

4

Aspirin-‐triggered

Re

solvin D1


Re

solvin D2

Chem

ical Nam

e

5S,6R,15

R-‐

trihydroxy-‐

LXA4

5S,14R

,15R

-‐ trihydroxy-‐

LXB 4

7S,8R,17

R-‐trihydroxy-‐

DHA

7S,16R

,17R

-‐trihydroxy-‐

DHA

Form

ula

C 20H

32O5

C 20H

32O5

C 22H

32O5

C 22H

32O5

Structure

Abb

reviaE

on

AT-‐LXA

4

AT-‐LXB

4

AT-‐RvD

1

AT-‐RvD

2

COOH

OH

HO

OH

COOH

OH

HO

OH

COOH

OH

HO

HO

19/104

COOH

OH

HO

OH

Complete

Stereo

chem

istry

4S,11R

,17R

-‐ trihydroxy-‐

5Z,7E,9E,13Z,15E,19Z-‐

docosahexaen

oic acid

4S,5,17R

-‐ trihydroxy-‐

6E,8E,10

Z,13

Z,15

E,19

Z-‐

docosahexaen

oic acid

7S,17R

-‐ dihydroxy-‐

4Z,8E,10

Z,13

Z,15

E,19

Z-‐

docosahexaen

oic acid

4S,17R

-‐ dihydroxy-‐

5E,7Z,10

Z,13

Z,15

E,19

Z-‐

docosahexaen

oic acid

Trivial N

ame


Re

solvin D3


Re

solvin D4


Re

solvin D5


Re

solvin D6

Chem

ical Nam

e

4S,11R

,17R

-‐trihydroxy-‐

DHA

4S,5,17R

-‐ trihydroxy-‐

DHA

7S,17R

-‐ dihydroxy-‐

DHA

4S,17R

-‐ dihydroxy-‐

DHA

Form

ula

C 22H

32O5

C 22H

32O5

C 22H

32O4

C 22H

32O4

Structure

Abb

reviaE

on

AT-‐RvD

3

AT-‐RvD

4

AT-‐RvD

5

AT-‐RvD

6

HOOC

OH

OH

OH

COOH

HO

HO

20/104

OH

HO

COOH

HO

OH

OH

HOOC

Complete

Stereo

chem

istry

10R,17

R-‐

dihydroxy-‐

4Z,7Z,11

E,13

E,15

Z,19

Z-‐

docosahexaen

oic acid

Trivial N

ame


[Neu

ro]

ProtecFn

D1

Chem

ical Nam

e

10R,17

R-‐

dihydroxy-‐

DHA

Form

ula

C 22H

32O4

Structure

Abb

reviaE

on

AT-‐PD1

COOH

OH

OH

21/104

Deuterium Labeled Internal Standards

22/104




Complete

Stereo

chem

istry

9-‐oxo-‐

11α,15S-‐

dihydroxy-‐

5Z,13E-‐

3,3,4,4-‐d 4-‐

prostadien

oic acid

5S,6R,15

S-‐

trihydroxy-‐

7E,9E,11

Z,13

E-‐

19,�19,�20,�20,�20-‐�d

5-‐

eicosatetraeno

ic acid

5S,�12R

-‐� dihydroxy-‐�

6Z,�8E,�10

E,�14

Z-‐�

6,�7,�14,�15-‐�d 4-‐

eicosatetraeno

ic acid

5S-‐�

hydroxy-‐�

6E,�8Z,�11

Z,�14

Z-‐�

5,6,8,9,11,12,14,15-‐�d 8-‐

eicosatetraeno

ic acid

Trivial N

ame

d 4-‐Prostagland

in E

2

d 5-‐Lipoxin A

4

d 4-‐Leu

kotriene

B4

d 8-‐5S-‐

hydroxy-‐

eicosatetraeno

ic

acid

Chem

ical Nam

e

9-‐de

hydro-‐

prostaglandin F 2

α-‐d 4

5S,6R,15

S-‐

trihydroxy-‐

LXA4-‐d 5

5S,12R


LTB 4-‐d

4

5S-‐

hydroxy-‐

ETE-‐d 8

Form

ula

C 20H

28D4O

5

C 20H

27D5O

5

C 20H

28D4O

4

C 20H

24D8O

3

Structure

Abb

reviaE

on

d 4-‐PGE 2

d 5-‐LXA

4

d 4-‐LTB

4

d 8-‐5S-‐HETE

COOH

OH

OH

DD

D

D

COOH

OH

O

HO

DD

DD

COOH

OH

HO

OH

D

D

DD

D

COOH

OH

DD

DD

DD

DD

23/104

Complete

Stereo

chem

istry

7S,16R

,17S-‐

trihydroxy-‐

4Z,8E,10

Z,12

E,14

E,19

Z-‐19

,19,20

,20,20

,20-‐d 5-‐

docosahexaen

oic acid

Trivial N

ame

d 5-‐Resolvin D2

Chem

ical Nam

e

7S,16R

,17S-‐

trihydroxy-‐

DHA -‐d

5

Form

ula

C 22H

27D5O

5

Structure

Abb

reviaE

on

d 5-‐RvD

2

24/104

COOH

OH

HO

OH D D

D

DD

Methods

25/104




26/104




Auto-‐extracFon (Solid Phase Extrac6on)

Samples

LC-‐MS-‐MS

Targeted MRM and EPI

Data dependent scan/EMS/MSn

IdenFficaFon

Matching to LM database (RT+ ≥ 6 diagnos6c ions)

QuanFtaFon

Deuterated internal standards

Synthesis of authenFc and syntheFc deuterated

standards

ConstrucFon of authenFc and syntheFc SPM and eicosanoids

database

MSn

Tissue validaFon and reference human samples

(i.e. SRM1950)

ComputaFonal data analysis

Cluster analysis

Principal component analysis

Cells, Fssues, organs and individual profiles

LM profiles (>55) Targeted bioacFve metabolomes

OperaEonalizaEon of lipid mediator metabololipidomics

IdenFficaFon and signature profiles for pro-‐resolving and inflammatory lipid mediators in human Fssue Colas RA, Shinohara M, Dalli J, Chiang N, Serhan CN. Am J Physiol Cell Physiol. 2014

Table 1: High Performance Liquid Chromatography sesngs.

Table 2: Mass Spectrometer sesngs for MulFple ReacFon Monitoring and Enhance Product Ion for AB Sciex 5500 Q TRAP.

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Shimadzu LC20AD HPLC Sesngs

Solvent A H2O (0.01% aceFc acid)

Solvent B Methanol (0.01% aceFc acid)

Flow rate 0.4mL/min

Column Eclipse plus C18, 1.8 µm, 4.6 x 100 mm, AGILENT

Column Heater ThermaSphere TS-‐130, 50°C, PHENOMENEX

Gradient

Time (min) Solvent (%B) 0.0 55 2.0 55 10.0 85 18.0 98 20.0 98 20.1 55 24.5 End

AB Sciex 5500 Q TRAP MS sesng for MRM and EPI

MRM EPI Curtain Gas 25 25 Collision Gas Medium Medium Ion Spray Voltage -‐4000 -‐4000 Temperature 500 °C 500 °C Ion Source Gas 1 40 40 Ion Source Gas 2 40 40




Table 3: Mass Spectrometer sesngs for MulFple ReacFon Monitoring and Enhance Product Ion for AB Sciex 6500 Q TRAP.

AB Sciex 6500 Q TRAP MS sesng for MRM and EPI

MRM EPI Curtain Gas 30 30 Collision Gas Medium Medium Ion Spray Voltage -‐4500 -‐4500 Temperature 580 °C 580 °C Ion Source Gas 1 80 80 Ion Source Gas 2 70 70

Table 4: Mass Spectrometer sesngs for MulFple ReacFon Monitoring forAB Sciex 5500 Q TRAP. DP=Declustering PotenFal, EP=Entrance potenFal, CE=Collision Energy, CXP=Collision Cell Exit PotenFal

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AB Sciex 5500 Q TRAP Individual lipid mediator, pathway marker, and further metabolite sesng for MRM (V)

Compound DP EP CE CXP AA -‐90 -‐10 -‐41 -‐22 PGD2 -‐80 -‐10 -‐30 -‐15 PGE2 -‐80 -‐10 -‐30 -‐15 PGF2α -‐80 -‐10 -‐30 -‐15 TxB2 -‐80 -‐10 -‐30 -‐15 LTB4 -‐110 -‐10 -‐22 -‐17 20-‐OH-‐LTB4 -‐100 -‐10 -‐26 -‐17 20-‐COOH-‐LTB4 -‐90 -‐10 -‐26 -‐17 LXA4 -‐95 -‐10 -‐22 -‐11 LXB4 -‐90 -‐10 -‐30 -‐21 5,15-‐diHETE -‐110 -‐10 -‐22 -‐17 5-‐HETE -‐90 -‐10 -‐21 -‐22 12-‐HETE -‐70 -‐10 -‐21 -‐15 15-‐HETE -‐65 -‐10 -‐19 -‐18

EPA -‐90 -‐10 -‐41 -‐22 LXA5 -‐95 -‐10 -‐22 -‐11 LXB5 -‐90 -‐10 -‐30 -‐21 5,15-‐diHEPE -‐110 -‐10 -‐22 -‐17 RvE1 -‐80 -‐10 -‐24 -‐16 RvE2 -‐40 -‐10 -‐22 -‐17 RvE3 -‐40 -‐10 -‐22 -‐17 5-‐HEPE -‐60 -‐10 -‐22 -‐11 12-‐HEPE -‐70 -‐10 -‐21 -‐15 15-‐HEPE -‐65 -‐10 -‐19 -‐8 18-‐HEPE -‐65 -‐10 -‐18 -‐23

DHA -‐90 -‐10 -‐41 -‐22 RvD1 -‐90 -‐10 -‐22 -‐13 RvD2 -‐90 -‐10 -‐24 -‐13 RvD3 -‐80 -‐10 -‐28 -‐19 RvD5 -‐40 -‐10 -‐22 -‐17 RvD6 -‐40 -‐10 -‐22 -‐17 MaR1 -‐40 -‐10 -‐22 -‐17 4,14-‐diHDHA -‐40 -‐10 -‐22 -‐17 PD1 -‐40 -‐10 -‐22 -‐17 4-‐HDHA -‐40 -‐10 -‐18 -‐13 7-‐HDHA -‐40 -‐10 -‐18 -‐13 14-‐HDHA -‐70 -‐10 -‐21 -‐15 17-‐HDHA -‐80 -‐10 -‐20 -‐18

d4-‐PGE2 -‐80 -‐10 -‐30 -‐11 d4-‐LXA4 -‐135 -‐10 -‐24 -‐17 d4-‐LTB4 -‐135 -‐10 -‐24 -‐17 d8-‐5S-‐HETE -‐100 -‐10 -‐22 -‐11

d5-‐RvD2 -‐90 -‐10 -‐24 -‐13

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AB Sciex 6500 Q TRAP Individual lipid mediator, pathway marker, and further metabolite sesng for MRM (V)

Compound DP EP CE CXP AA -‐10.0 -‐10.0 -‐10.0 -‐12.0 PGD2 -‐30.0 -‐10.0 -‐22.0 -‐8.0 PGE2 -‐30.0 -‐10.0 -‐22.0 -‐8.0 PGF2a -‐60.0 -‐10.0 -‐33.3 -‐12.0 TxB2 -‐70.0 -‐10.0 -‐25.0 -‐22.0 LTB4 -‐45.0 -‐10.0 -‐21.0 -‐10.8 20-‐OH-‐LTB4 -‐60.0 -‐10.0 -‐22.0 -‐8.0 20-‐COOH-‐LTB4 -‐60.0 -‐10.0 -‐22.0 -‐8.0 LXA4 -‐30.0 -‐10.0 -‐18.2 -‐10.0 LXB4 -‐45.0 -‐10.0 -‐21.5 -‐12.0 5,15-‐diHETE -‐35.0 -‐10.0 -‐18.0 -‐12.0 5-‐HETE -‐30.0 -‐10.0 -‐18.0 -‐9.5 12-‐HETE -‐30.0 -‐10.0 -‐18.0 -‐9.5 15-‐HETE -‐30.0 -‐10.0 -‐18.0 -‐9.5

EPA -‐10.0 -‐10.0 -‐10.0 -‐12.0 LXA5 -‐30.0 -‐10.0 -‐18.6 -‐10.0 LXB5 -‐30.0 -‐10.0 -‐21.2 -‐10.0 5,15-‐diHEPE -‐30.0 -‐12.0 -‐17.0 -‐12.0 RvE1 -‐50.0 -‐10.0 -‐21.1 -‐10.0 RvE2 -‐30.0 -‐12.0 -‐18.6 -‐10.0 RvE3 -‐35.0 -‐10.0 -‐20.5 -‐14.0 5-‐HEPE -‐30.0 -‐10.0 -‐18.0 -‐9.5 12-‐HEPE -‐30.0 -‐10.0 -‐18.0 -‐9.5 15-‐HEPE -‐30.0 -‐10.0 -‐18.0 -‐9.5 18-‐HEPE -‐30.0 -‐10.0 -‐18.0 -‐9.5

DHA -‐10.0 -‐10.0 -‐10.0 -‐12.0 RvD1 -‐40.0 -‐10.0 -‐19.0 -‐9.0 RvD2 -‐40.0 -‐10.0 -‐27.8 -‐10.0 RvD3 -‐55.0 -‐10.0 -‐24.6 -‐9.5 RvD5 -‐42.0 -‐10.0 -‐18.0 -‐11.0 RvD6 -‐50.0 -‐10.0 -‐20.0 -‐11.0 MaR1 -‐38.0 -‐10.0 -‐19.6 -‐11.0 4,14-‐diHDHA -‐50.0 -‐10.0 -‐20.0 -‐11.0 PD1 -‐45.0 -‐10.0 -‐20.8 -‐9.5 4-‐HDHA -‐30.0 -‐10.0 -‐18.0 -‐9.5 7-‐HDHA -‐30.0 -‐10.0 -‐18.0 -‐9.5 14-‐HDHA -‐30.0 -‐10.0 -‐18.0 -‐9.5 17-‐HDHA -‐30.0 -‐10.0 -‐18.0 -‐9.5

d4-‐PGE2 -‐30.0 -‐10.0 -‐26.0 -‐14.0 d4-‐LXA4 -‐40.0 -‐10.0 -‐17.5 -‐7.5 d4-‐LTB4 -‐55.0 -‐10.0 -‐21.7 -‐10.0 d8-‐5S-‐HETE -‐40.0 -‐10.0 -‐24.0 -‐12.0 d5-‐RvD2 -‐40.0 -‐10.0 -‐20.3 -‐13.0




Table 5: Mass Spectrometer sesngs for MulFple ReacFon Monitoring for AB Sciex 5500 Q TRAP. DP=Declustering PotenFal, EP=Entrance potenFal, CE=Collision Energy, CXP=Collision Cell Exit PotenFal

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Compound name Linear equaEon r2 Internal standard RvD1 y=0.00002081x 0.999 d5-‐RvD2 RvD2 y=0.00009118x 0.997 d5-‐RvD2 RvD3 y=0.000008163x 0.990 d5-‐RvD2 RvD5 y=0.00001137x 0.993 d4-‐LTB4 PD1 y=0.00001425x 0.995 d4-‐LTB4 MaR1 y=0.00007534x 0.999 d4-‐LTB4

17-‐HDHA y=0.00001132x 0.996 d8-‐5HETE 14-‐HDHA y=0.00001916x 0.999 d8-‐5HETE 7-‐HDHA y=0.00001096x 0.996 d8-‐5HETE 4-‐HDHA y=0.00001040x 0.996 d8-‐5HETE LXA5 y=0.00002043x 0.999 d5-‐LXA4 RvE1 y=0.00004358x 0.999 d5-‐LXA4 RvE2 y=0.0001647x 0.996 d4-‐LTB4 RvE3 y=0.0001047x 0.998 d4-‐LTB4

18-‐HEPE y=0.00002131x 0.998 d8-‐5HETE 15-‐HEPE y=0.00002002x 0.999 d8-‐5HETE 12-‐HEPE y=0.00001899x 0.997 d8-‐5HETE 5-‐HEPE y=0.00001301x 0.998 d8-‐5HETE LXA4 y=0.00001607x 0.996 d5-‐LXA4 LXB4 y=0.00007325x 0.995 d5-‐LXA4

5S,15S-‐diHETE y=0.00008011x 0.997 d4-‐LTB4 LTB4 y=0.00001074x 0.991 d4-‐LTB4

20-‐OH-‐LTB4 y=0.00001524x 0.999 d4-‐LTB4 20-‐COOH-‐LTB4 y=0.00007931x 0.999 d4-‐LTB4

PGD2 y=0.00001019x 0.993 d4-‐PGE2 PGE2 y=0.00001698x 0.998 d4-‐PGE2 PGF2α y=0.00001643x 0.997 d4-‐PGE2 TXB2 y=0.00001054x 0.997 d4-‐PGE2

15-‐HETE y=0.00001377x 0.999 d8-‐5HETE 12-‐HETE y=0.00001142x 0.994 d8-‐5HETE 5-‐HETE y=0.00001010x 0.990 d8-‐5HETE

Table 6: Linear equaFon, coefficient of determinaFon (r2) and aOributed internal standard

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Recovery (%) in plasma

Interference (% signal remaining)

LOD 5500 (pg) in

LOD 6500 (pg) in

Internal standards phase plasma phase plasma d5-‐RvD2 73 ± 16 28 ± 9 0.17 0.43 0.04 0.09 d5-‐LXA4 75 ± 2 7 ± 2 0.10 0.25 0.02 0.05 d4-‐LTB4 80 ± 3 18 ± 2 0.10 0.51 0.05 0.22 d4-‐PGE2 63 ± 13 21 ± 6 0.19 0.47 0.09 0.23 d8-‐5S-‐HETE 68 ± 3 28 ± 5 0.10 0.26 0.07 0.15 d5-‐authenEc lipid mediators d5-‐10S,17S-‐diHDHA 88 ± 4 8 ± 2 0.06 0.45 0.03 0.15 d5-‐5S,15S-‐diHEPE 94 ± 6 9 ± 5 0.13 0.95 0.03 0.15 d5-‐15-‐HEPE 63 ± 12 26 ± 4 0.09 0.23 0.10 0.19

Table 7: DeterminaFon of recovery, interference and LOD. Plasma recoveries, interference and limit of detecFon (LOD) of the internal standards and authenFc d5-‐lipid mediators used as representaFve of specific chromatographic regions and physical properFes of compounds. LOD was assessed in phase and in the matrix. Mean ± SEM, n = 6 for recoveries and interference, d = 3 for LOD.

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Compound name

Nominal concentraEon

(pg)

Intraday (repeatability) Interday (reproducibility)

Measured (pg) %RSD Accuracy Measured (pg) %RSD Accuracy RvD1 5 5.0 ± 0.1 1% 99% 4.6 ± 0.1 2% 92%

20 17.2 ± 0.7 7% 86% 23.2 ± 0.7 5% 84% 100 109.8 ± 2.2 4% 90% 118.5 ± 1.8 3% 82% 200 227.8 ± 2.5 2% 86% 233.5 ± 1.8 2% 83%

RvD2 5 5.2 ± 0.1 4% 96% 4.6 ± 0.1 4% 93% 20 16.9 ± 0.7 7% 85% 22.6 ± 0.5 4% 87% 100 113.0 ± 2.3 4% 87% 116.5 ± 1.6 3% 84% 200 227.8 ± 3.1 3% 86% 235.5 ± 2.5 2% 82%

RvD3 5 5.7 ± 0.1 3% 86% 5.4 ± 0.1 4% 91% 20 19.8 ± 0.5 4% 99% 26.0 ± 0.3 2% 70% 100 118.3 ± 1.4 2% 82% 125.3 ± 0.9 2% 75% 200 226.8 ± 2.2 2% 87% 226.8 ± 1.7 1% 87%

RvD5 5 4.8 ± 0.1 5% 97% 4.6 ± 0.04 2% 93% 20 16.7 ± 0.6 6% 83% 24.0 ± 0.3 2% 80% 100 114.3 ± 0.8 1% 86% 117.0 ± 0.0 0% 83% 200 217.0 ± 2.6 2% 92% 217.0 ± 2.4 2% 92%

PD1 5 4.6 ± 0.1 5% 93% 4.4 ± 0.05 2% 88% 20 16.7 ± 0.3 3% 84% 23.7 ± 0.5 4% 82% 100 114.0 ± 0.7 1% 86% 118.5 ± 1.0 2% 82% 200 218.5 ± 1.3 1% 91% 220.0 ± 1.3 1% 90%

MaR1 5 4.6 ± 0.2 9% 91% 4.2 ± 0.02 1% 85% 20 16.1 ± 0.1 1% 81% 22.3 ± 0.5 4% 89% 100 107.0 ± 3.5 7% 93% 110.8 ± 1.4 3% 89% 200 221.3 ± 2.3 2% 89% 229.0 ± 1.8 2% 86%

17-‐HDHA 5 3.5 ± 0.1 4% 69% 4.4 ± 0.05 2% 87% 20 17.8 ± 0.3 3% 89% 23.7 ± 0.8 6% 82% 100 115.0 ± 1.1 2% 85% 120.0 ± 0.6 1% 80% 200 222.0 ± 1.2 1% 89% 227.5 ± 1.6 1% 86%

14-‐HDHA 5 3.2 ± 0.1 5% 64% 4.3 ± 0.02 1% 86% 20 17.3 ± 0.4 4% 86% 22.2 ± 0.4 3% 89% 100 114.8 ± 2.4 4% 85% 109.5 ± 2.3 4% 91% 200 212.3 ± 2.3 2% 94% 233.5 ± 0.9 1% 83%

7-‐HDHA 5 3.5 ± 0.1 4% 70% 4.5 ± 0.1 3% 90% 20 19.7 ± 0.1 1% 99% 25.8 ± 0.4 3% 71% 100 123.8 ± 2.3 4% 76% 128.0 ± 2.5 4% 72% 200 236.5 ± 1.0 1% 82% 240.5 ± 2.4 2% 80%

4-‐HDHA 5 3.6 ± 0.1 6% 73% 4.6 ± 0.02 1% 91% 20 19.8 ± 0.3 2% 99% 26.0 ± 0.3 2% 70% 100 125.8 ± 2.0 3% 74% 128.5 ± 1.6 2% 72% 200 236.3 ± 2.1 2% 82% 240.3 ± 1.4 1% 80%

Table 8 (part 1/4): Precision and accuracy of LM metabololipidomics. %RSD, relaFve standard deviaFon; %RSD = (SEM/mean) x 100; mean ± SEM; n = 4

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Compound name


(pg)


Measured (pg) %RSD Accuracy Measured (pg) %RSD Accuracy LXA5 5 5.6 ± 0.1 2% 87% 5.0 ± 0.1 4% 99%

20 18.0 ± 0.3 3% 90% 21.6 ± 0.7 6% 92% 100 111.0 ± 2.6 5% 89% 110.3 ± 1.9 3% 90% 200 212.3 ± 2.8 3% 94% 240.3 ± 3.3 3% 80%

RvE1 5 5.8 ± 0.1 4% 85% 5.1 ± 0.1 3% 99% 20 17.4 ± 0.1 1% 87% 24.3 ± 0.3 2% 78% 100 113.3 ± 2.3 4% 87% 121.8 ± 1.7 3% 78% 200 242.0 ± 3.7 3% 79% 241.8 ± 1.7 1% 79%

RvE2 5 5.5 ± 0.4 14% 91% 4.7 ± 0.1 3% 93% 20 16.4 ± 0.7 8% 82% 20.7 ± 0.7 6% 97% 100 108.8 ± 2.8 5% 91% 101.2 ± 2.9 6% 99% 200 199.3 ± 3.3 3% 100% 240.0 ± 1.1 1% 80%

RvE3 5 6.1 ± 0.3 8% 78% 5.0 ± 0.4 16% 99% 20 17.8 ± 1.7 17% 89% 23.7 ± 1.6 12% 81% 100 105.0 ± 3.0 6% 95% 116.5 ± 1.0 2% 84% 200 218.5 ± 3.4 3% 91% 227.8 ± 3.1 3% 86%

18-‐HEPE 5 4.1 ± 0.1 3% 82% 4.3 ± 0.03 2% 87% 20 17.6 ± 0.2 2% 88% 23.3 ± 0.5 4% 83% 100 117.5 ± 2.0 3% 83% 121.3 ± 1.5 2% 79% 200 239.0 ± 2.3 2% 81% 241.8 ± 1.5 1% 79%

15-‐HEPE 5 4.3 ± 0.1 3% 87% 4.4 ± 0.04 2% 88% 20 17.6 ± 0.2 2% 88% 22.8 ± 0.5 4% 86% 100 115.5 ± 2.5 4% 85% 122.3 ± 1.1 2% 78% 200 231.3 ± 1.7 1% 84% 239.3 ± 1.7 1% 80%

12-‐HEPE 5 4.6 ± 0.04 2% 93% 4.4 ± 0.1 3% 88% 20 18.1 ± 0.2 1% 91% 22.6 ± 0.4 3% 87% 100 122.5 ± 1.3 2% 78% 117.3 ± 1.7 3% 83% 200 231.3 ± 2.3 2% 84% 227.8 ± 1.3 1% 86%

5-‐HEPE 5 4.6 ± 0.05 2% 91% 4.6 ± 0.04 2% 92% 20 18.1 ± 0.2 2% 91% 23.4 ± 0.5 4% 83% 100 115.3 ± 1.3 2% 85% 119.5 ± 0.5 1% 81% 200 223.5 ± 2.0 2% 88% 233.3 ± 1.0 1% 83%

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Compound name


(pg)


Measured (pg) %RSD Accuracy Measured (pg) %RSD Accuracy LXA4 5 5.5 ± 0.1 2% 90% 4.9 ± 0.1 4% 98%

20 17.7 ± 0.8 8% 88% 23.5 ± 0.1 1% 82% 100 114.0 ± 2.1 4% 86% 119.0 ± 1.1 2% 81% 200 226.3 ± 2.1 2% 87% 233.5 ± 1.5 1% 83%

LXB4 5 6.2 ± 0.1 4% 76% 5.4 ± 0.1 3% 92% 20 18.4 ± 0.6 6% 92% 22.9 ± 0.4 3% 85% 100 116.8 ± 1.7 3% 83% 114.5 ± 1.2 2% 86% 200 220.0 ± 4.5 4% 90% 231.8 ± 1.6 1% 84%

5S,15S-‐diHETE 5 5.1 ± 0.1 4% 98% 5.4 ± 0.1 2% 93% 20 20.3 ± 0.5 4% 98% 26.4 ± 0.2 1% 68% 100 123.3 ± 1.4 2% 77% 129.0 ± 2.0 3% 71% 200 246.5 ± 3.7 3% 77% 251.0 ± 6.1 5% 75%

LTB4 5 5.0 ± 0.03 1% 99% 5.8 ± 0.1 2% 84% 20 22.3 ± 0.3 2% 89% 27.5 ± 0.4 3% 63% 100 125.0 ± 0.6 1% 75% 130.0 ± 1.8 3% 70% 200 231.0 ± 3.2 3% 85% 226.8 ± 2.9 3% 87%

20-‐OH-‐LTB4 5 5.4 ± 0.1 4% 93% 4.9 ± 0.03 1% 98% 20 17.1 ± 0.5 6% 86% 22.6 ± 0.4 3% 87% 100 111.3 ± 2.7 5% 89% 116.0 ± 0.8 1% 84% 200 223.3 ± 1.1 1% 88% 229.0 ± 3.0 3% 86%

20-‐COOH-‐LTB4 5 5.1 ± 0.4 16% 99% 5.2 ± 0.1 2% 97% 20 18.0 ± 0.9 9% 90% 21.4 ± 1.6 13% 93% 100 117.5 ± 1.3 2% 83% 114.0 ± 2.2 4% 86% 200 227.3 ± 2.3 2% 86% 253.8 ± 3.4 3% 73%

PGD2 5 5.7 ± 0.1 2% 87% 5.2 ± 0.1 4% 96% 20 17.1 ± 0.2 2% 86% 22.8 ± 0.3 3% 86% 100 105.5 ± 2.3 4% 95% 115.5 ± 1.0 2% 85% 200 213.3 ± 3.1 3% 93% 218.3 ± 3.7 3% 91%

PGE2 5 5.5 ± 0.1 4% 90% 4.9 ± 0.2 6% 97% 20 18.0 ± 0.5 4% 90% 22.9 ± 0.9 7% 86% 100 116.8 ± 4.0 7% 83% 113.8 ± 2.3 4% 86% 200 206.5 ± 1.2 1% 97% 209.5 ± 4.6 4% 95%

PGF2α 5 5.5 ± 0.1 2% 90% 5.2 ± 0.1 3% 97% 20 19.2 ± 0.5 4% 96% 25.6 ± 0.4 3% 72% 100 120.3 ± 1.7 3% 80% 123.8 ± 1.1 2% 76% 200 233.3 ± 3.1 3% 83% 220.8 ± 0.9 1% 90%

TXB2 5 4.7 ± 0.2 7% 94% 5.0 ± 0.01 0.3% 100% 20 16.8 ± 0.8 8% 84% 22.2 ± 1.3 10% 89% 100 111.2 ± 4.1 7% 89% 118.0 ± 4.5 8% 82% 200 219.0 ± 3.6 3% 91% 189.5 ± 6.9 7% 95%


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Compound name


(pg)


Measured (pg) %RSD Accuracy Measured (pg) %RSD Accuracy 15-‐HETE 5 4.1 ± 0.1 2% 82% 4.2 ± 0.05 2% 84%

20 16.6 ± 0.2 2% 83% 21.8 ± 0.4 3% 91% 100 109.3 ± 2.6 5% 91% 110.8 ± 1.3 2% 89% 200 212.8 ± 2.3 2% 94% 219.0 ± 0.8 1% 91%

12-‐HETE 5 4.5 ± 0.04 2% 89% 4.8 ± 0.04 2% 95% 20 17.7 ± 0.3 2% 89% 23.1 ± 0.6 4% 85% 100 113.8 ± 1.8 3% 86% 117.0 ± 1.1 2% 83% 200 216.5 ± 1.8 2% 92% 220.0 ± 1.7 2% 90%

5-‐HETE 5 4.9 ± 0.05 2% 97% 5.0 ± 0.1 4% 100% 20 20.3 ± 0.3 3% 98% 25.7 ± 0.3 2% 72% 100 125.0 ± 3.0 5% 75% 127.8 ± 1.4 2% 72% 200 232.5 ± 1.4 1% 84% 239.5 ± 4.7 4% 80%


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Day-‐0 Day-‐1 Day-‐2 Day-‐3 Day-‐4 Day-‐5 2-‐weeks 4-‐weeks DHA bioacEve metabolome Δ change (%) RvD1 49.5±1.3

with -‐1.0 -‐0.2 +3.2 -‐3.2 -‐1.1 without +0.8 -‐2.0 +2.4 -‐13.2 -‐15.5 -‐33.4 -‐56.8

RvD2 13.4±0.8 with -‐1.3 +1.1 +6.4 +2.3 +1.1

without -‐9.2 +2.5 +2.2 -‐1.2 -‐19.2 -‐39.5 -‐59.0 RvD3 68.5±7.5

with -‐3.1 +8.0 +6.6 -‐4.4 +6.0 without -‐6.8 -‐8.5 -‐3.3 -‐11.3 -‐12.6 -‐18.9 -‐25.4

RvD5 24.9±2.2 with +6.5 -‐10.5 -‐18.0 -‐8.4 -‐16.8

without -‐16.4 -‐14.8 -‐15.1 -‐20.2 -‐17.0 -‐38.7 -‐44.0 RvD6 21.6±2.0

with +1.7 +2.2 -‐1.1 +0.4 -‐11.8 without +4.6 -‐9.8 -‐12.5 -‐17.2 -‐14.3 -‐19.5 -‐85.3

PD1 6.1±0.1 with -‐3.0 +5.5 -‐8.3 -‐11.9 -‐10.4

without +6.7 +1.7 -‐18.3 -‐26.8 -‐23.7 -‐33.0 -‐36.7 MaR1 9.0±0.8

with +2.3 +0.9 -‐8.0 -‐6.5 -‐17.8 without -‐6.5 -‐2.5 -‐2.6 -‐15.7 -‐24.7 -‐67.9 -‐73.1

EPA bioacEve metabolome RvE1 13.1±0.9

with -‐3.3 -‐9.0 -‐13.9 -‐9.4 -‐13.1 without +7.6 -‐4.2 -‐22.0 -‐23.7 -‐24.6 -‐50.7 -‐57.1

RvE3 686.3±51.0 with -‐6.4 -‐7.6 -‐5.1 -‐7.2 -‐4.4

without -‐1.3 -‐5.2 -‐4.5 -‐3.5 -‐18.7 -‐40.9 -‐42.2 AA bioacEve metabolome LXA4 11.2±1.0

with 1.8 +0.5 -‐8.6 -‐8.3 -‐5.9 without 4.4 -‐3.8 -‐5.2 -‐5.7 -‐3.8 -‐14.4 -‐39.9

LXB4 117.6±2.6 with -‐1.7 -‐3.6 -‐1.9 +5.2 -‐5.5

without -‐1.3 -‐5.8 -‐3.6 -‐5.8 -‐11.3 -‐17.7 -‐57.1 PGD2 55.1±1.9

with +9.8 -‐6.8 +3.3 +3.7 -‐15.5 without +7.6 -‐2.8 +5.8 +2.1 -‐16.9 -‐10.9 -‐26.7

PGE2 199.2±10.2 with +3.7 -‐1.2 +0.1 -‐11.8 -‐15.6

without -‐6.2 -‐12.2 -‐14.3 -‐20.8 -‐15.3 -‐11.9 -‐29.8 PGF2a 143.3±2.5

with -‐0.4 -‐0.2 +1.2 +8.9 -‐13.6 without +8.0 +1.1 +1.6 +4.8 -‐9.0 -‐15.6 -‐34.1

TxB2 6067.0±128.4 with -‐5.2 -‐7.8 +7.4 -‐5.5 -‐12.5

without +9.2 -‐3.2 +7.3 -‐16.6 -‐19.2 -‐17.8 -‐34.2

Table 9: Human serum (250µl) was stored under -‐30°C with or without 1ml methanol. On the designated day (Day-‐0 to 4-‐weeks), LC-‐MS-‐MS based LM metabololipidomics were carried out. Results are expressed as pg/ml serum; mean ± SEM of d = 3 determinaFons and Δ change (%) from Day-‐0.

MulEple ReacEon Monitoring TransiEons

37/104




Table 1: MRM transiFon for LC-‐MS-‐MS based metabololipidomics of AA, EPA and DHA derived lipid mediators.

Table 2: MRM transiFon for LC-‐MS-‐MS based metabololipidomics of AA and DHA derived aspirin-‐triggered lipid mediators.

38/104

Compound Q1 Q3 Compound Q1 Q3

AA 303.3 259.1 15-‐HEPE 317.2 219.1 PGD2 351.3 233.2 18-‐HEPE 317.2 259.1 PGE2 351.3 175.1 PGF2α 353.3 193.1 DHA 327.3 283.1 TXB2 369.3 169.1 RvD1 375.2 215.2 LTB4 335.3 195.2 RvD2 375.2 141.2 LXA4 351.2 235.1 RvD3 375.2 147.1 LXB4 351.2 221.1 RvD5 359.2 199.1 5,15-‐diHETE 335.2 115.2 RvD6 359.2 159.1 5-‐HETE 319.2 115.1 MaR1 359.2 250.1 12-‐HETE 319.2 179.1 4,14-‐diHDHA 359.2 221.1 15-‐HETE 319.2 219.1 PD1 359.2 153.1

4-‐HDHA 343.2 101.1 EPA 301.3 257.1 7-‐HDHA 343.2 141.1 LXA5 349.2 215.1 14-‐HDHA 343.2 205.1 LXB5 349.2 221.2 17-‐HDHA 343.2 245.1 5,15-‐diHEPE 333.2 235.2 RvE1 349.2 195.1 d4-‐PGE2 355.3 193.2 RvE2 333.3 253.2 d5-‐LXA4 356.3 115.2 RvE3 333.3 201.1 d4-‐LTB4 339.3 197.2 5-‐HEPE 317.2 115.1 d8-‐5S-‐HETE 327.3 116.1 12-‐HEPE 317.2 179.1 d5-‐RvD2 380.2 141.2


AT-‐LXA4 351.2 235.1 AT-‐RvD3 375.2 147.1 AT-‐LXB4 351.2 221.1 AT-‐RvD5 359.2 199.1

AT-‐RvD6 359.2 159.1 AT-‐RvD1 375.2 215.2 AT-‐RvD2 375.2 141.2 AT-‐PD1 359.2 153.1




AddiEonal references:

Specific lipid mediator signatures of human phagocytes: microparFcles sFmulate macrophage efferocytosis and pro-‐resolving mediators. Dalli J, Serhan CN. Blood. 2012 120(15)

Metabolomics-‐lipidomics of eicosanoids and docosanoids generated by phagocytes. Yang R, Chiang N, Oh SF, Serhan CN. Curr Protoc Immunol. 2011 Chapter 14:Unit 14.26.

Table 3: MRM transiFon for LC-‐MS-‐MS based metabololipidomics of further metabolites of AA, EPA and DHA derived lipid mediators.

39/104


20-‐OH-‐LTB4 351.2 195.1 Dihydro-‐RvD1 377.2 279.1 20-‐COOH-‐LTB4 365.2 195.1 7-‐oxo-‐RvD2 373.2 260.1 15-‐oxo-‐LXA4 349.2 233.1 16-‐oxo-‐RvD2 373.2 260.1 13,14-‐dihydro-‐LXA4 353.2 253.1 22-‐OH-‐RvD2 391.2 249.1 5-‐oxo-‐LXB4 349.2 219.1 Dihydro-‐RvD2 377.2 279.1

14-‐oxo-‐MaR1 357.2 221.1 18-‐oxo-‐RvE1 347.2 213.1 7-‐oxo-‐MaR1 357.2 219.1 19-‐OH-‐RvE1 365.2 205.1 22-‐OH-‐MaR1 375.2 177.1 20-‐OH-‐RvE1 365.2 291.1 22-‐COOH-‐MaR1 389.2 177.1 20-‐COOH-‐RvE1 379.2 237.1 Dihydro-‐MaR1 361.2 223.1 Dihydro-‐RvE1 351.2 225.1 22-‐OH-‐PD1 375.2 153.1

22-‐COOH-‐PD1 389.2 153.1 8-‐oxo-‐RvD1 373.2 231.1 Dihydro-‐PD1 361.2 263.1 17-‐oxo-‐RvD1 373.2 231.1




Lipid Mediator BiosyntheEc Pathways

40/104




Key eicosanoids that play pivotal roles in iniEaEng inflammaEon and its resoluEon.

AnF-‐Inflammatory and Pro-‐Resolving Lipid Mediators. Charles N. Serhan, Stephanie Yacoubian and Rong Yang Annu Rev Pathol. 2008 3: 279–312. PMCID: PMC2739396

BiosyntheEc scheme for the Prostaglandins, Leukotrienes and Lipoxins

41/104




Resolvins and ProtecFns in InflammaFon-‐ResoluFon Charles N. Serhan, Nicos A. Petasis Chem Rev. 2011 12 111(10): 5922–5943. PMCID: PMC3192290

E-‐series resolvin biosynthesis and metabolic inacEvaEon.

BiosyntheEc scheme for the E-‐series Resolvins

42/104





BiosyntheEc scheme for the D-‐series Resolvins

Pathways and enzymes in the biosynthesis of D-‐series resolvins.

43/104





BiosyntheEc scheme for the aspirin triggered D-‐series Resolvins

Pathways and enzymes involved in the biosynthesis of aspirin-‐triggered D-‐series resolvins.

44/104





Biosynthetic scheme for the Protectins

Pathways and enzymes involved in the biosynthesis of protecEn (PD1/NPD1) and 10S,17S-‐diHDHA.

45/104




Maresins: novel macrophage mediators with potent anFinflammatory and proresolving acFons. Serhan CN, Yang R, Mar6nod K, Kasuga K, Pillai PS, Porter TF, Oh SF, Spite M. J Exp Med. 2009 206(1):15-‐23. PMCID: PMC2626672

BiosyntheEc scheme proposed for MaR1 and related products.

Biosynthetic scheme for the Maresins

46/104




LC-‐MS-‐MS Spectra

47/104





Prostaglandins & Thromboxane

48/104




PGD2

10.6

351=M-‐H 333=M-‐H-‐H2O 315=M-‐H-‐2H2O 289=M-‐H-‐H2O-‐CO2 271=M-‐H-‐2H2O-‐CO2 235=279-‐CO2 233=251-‐H2O 217=279-‐H2O-‐CO2 189=251-‐H2O-‐CO2

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

271

189

315

233

333

351 289 217 251 235

100 140 180 220 260 300 340 380 m/z, Da

+H 251 -‐H

279

HO

O

COO-

OH

49/104




PGE2

10.5

351=M-‐H 333=M-‐H-‐H2O 315=M-‐H-‐2H2O 289=M-‐H-‐H2O-‐CO2 271=M-‐H-‐2H2O-‐CO2 243=279-‐2H2O 235=279-‐CO2 233=251-‐H2O 217=279-‐H2O-‐CO2 207=251-‐CO2 189=251-‐H2O-‐CO2

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

271

315 333

189

233 289 217 351 243

235

207 279

100 140 180 220 260 300 340 380 m/z, Da

+H 251 -‐H

279

O

HO

COO-

OH

50/104




PGF2α

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

10.9

353=M-‐H 335=M-‐H-‐H2O 317=M-‐H-‐2H2O 299=M-‐H-‐3H2O 291=309-‐H2O 273=309-‐2H2O 263=281-‐H2O 235=253-‐H2O 219=281-‐H2O-‐CO2 217=253-‐2H2O 209=253-‐CO2 193=253-‐H-‐CH3-‐CO2 191=253-‐H2O-‐CO2 173=253-‐2H2O-‐CO2

253 209 281

353

309

335 291

317 273 299 191 219 235 173

217

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

100 140 180 220 260 300 340 380 m/z, Da

HO

HO

COO-

OH

253 281

+H

-‐H

193

309

51/104

263




TxB2

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

10.1

369=M-‐H 333=M-‐H-‐2H2O 325=M-‐H-‐CO2 315=M-‐H-‐3H2O 307=M-‐H-‐H2O-‐CO2 289=M-‐H-‐2H2O-‐CO2 279=297-‐H2O 233=269-‐2H2O 195=213-‐H2O 189=269-‐2H2O-‐CO2 177=213-‐2H2O 151=169-‐H2O 133=169-‐2H2O 125=169-‐CO2

169

195

177

125 289

151 369 307

315 333 279 325 189 133 233

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

100 140 180 220 260 300 340 380 m/z, Da

269 297

+H

-‐H

213

169

O

OH

HO

COO-

OH

52/104

297





Leukotrienes

53/104




COO-

OHOH

LTB4

13.3

335=M-‐H 317=M-‐H-‐H2O 299=M-‐H-‐2H2O 291=M-‐H-‐CO2 273=M-‐H-‐H2O-‐CO2 255=M-‐H-‐2H2O-‐CO2 205=223-‐H2O 179=223-‐CO2 177=195-‐H2O 161=223-‐H2O-‐CO2 151=195-‐CO2 133=195-‐H2O-‐CO2

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

335

195

317

151 273 129

111 177 299 255 205

133 291 115

179

219 161

100 140 180 220 260 300 340 380 m/z, Da

115

219 -‐H

223

111 -‐H

+H 195

270

261 281 λmax

MeOH = 270 nm

ε = 40,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

270 230 250 290 210 330 310

54/104 Wavelength, nm




129

20-‐OH-‐LTB4

8.8

351=M-‐H 333=M-‐H-‐H2O 315=M-‐H-‐2H2O 289=M-‐H-‐H2O-‐CO2 271=M-‐H-‐2H2O-‐CO2 179=223-‐CO2 177=195-‐H2O 151=195-‐CO2 133=195-‐H2O-‐CO2

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

351

195

333

151 315 289 179 177

271 133 115

100 140 180 220 260 300 340 380 m/z, Da

COO-

OHOH

OH

270

261 281

λmaxMeOH = 270 nm

ε = 40,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

270 230 250 290 210 330 310

115 195

223 -‐H

+H

-‐H





129

129

20-‐COOH-‐LTB4

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

7.75

365=M-‐H 347=M-‐H-‐H2O 329=M-‐H-‐2H2O 321=M-‐H-‐CO2 303=M-‐H-‐H2O-‐CO2 285=M-‐H-‐2H2O-‐CO2 205=223-‐H2O 177=195-‐H2O 161=223-‐H2O-‐CO2 151=195-‐CO2

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

365

347

151

329 303

285 141 161 205 249 177 321 115

195

100 140 180 220 260 300 340 380 m/z, Da

270

261 281

λmaxMeOH = 270 nm

ε = 40,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

270 230 250 290 210 330 310

115 195

223

141 -‐H

+H

-‐H 249





129

129

COO-

COOH

OH OH


Lipoxins

57/104




LXA4

11.1

351=M-‐H 333=M-‐H-‐H2O 315=M-‐H-‐2H2O 307=M-‐H-‐CO2 289=M-‐H-‐H2O-‐CO2 271=M-‐H-‐2H2O-‐CO2 261=279-‐H2O 233=251-‐H2O 217=235-‐H2O 215=251-‐2H2O 207=251-‐CO2 199=235-‐2H2O 189=251-‐H2O-‐CO2 127=145-‐H2O

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

115 351

217

235

289 189 271

307 333 315 251 207 127 145 233 261 215 199

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

100 140 180 220 260 300 340 380 m/z, Da

115

+H

235

-‐H -‐H 145

251

279 -‐H

COO-

HO OH

OH

301

288

315

λmaxMeOH = 301 nm

ε = 50,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

260 280 300 320 340 240 360 380





LXB4

10.5

351=M-‐H 333=M-‐H-‐H2O 315=M-‐H-‐2H2O 307=M-‐H-‐CO2 297=M-‐H-‐3H2O 289=M-‐H-‐H2O-‐CO2 271=M-‐H-‐2H2O-‐CO2 261=279-‐H2O 243=279-‐2H2O 233=251-‐H2O 217=235-‐H2O 215=251-‐2H2O 207=251-‐CO2 203=221-‐H2O 189=251-‐H2O-‐CO2 177=221-‐CO2 159=221-‐H2O-‐CO2

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

351

221

315

233

333

189 251 207 271 203 177

159 289 297 215

243 307 261 217 115

100 140 180 220 260 300 340 380 m/z, Da

279 251

-‐H 221

115

235

+H

-‐H

+H

COO-

OH

HO OH

301

288

314

λmaxMeOH = 301 nm

ε = 50,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

260 280 300 320 340 240 360 380





13.0

5S,15S-‐diHETE

335=M-‐H 317=M-‐H-‐H2O 299=M-‐H-‐2H2O 291=M-‐H-‐CO2 273=M-‐H-‐H2O-‐CO2 255=M-‐H-‐2H2O-‐CO2 227=263-‐2H2O 217=235-‐H2O 201=219-‐H2O 191=235-‐CO2 173=235-‐H2O-‐CO2

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

255

317

335

173

201

115

273 235

299 217

191 291

227 219

100 140 180 220 260 300 340 380 m/z, Da

COO-

OH

OH

219

235

115

-‐H +H

263

-‐H

244

λmaxMeOH = 244 nm

ε = 25,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

220 230 240 250 270 210 280 290 260






MonoHydroxyETE Products

61/104




5-‐HETE

16.4

319=M-‐H 301=M-‐H-‐H2O 275=M-‐H-‐CO2 257=M-‐H-‐H2O-‐CO2

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

257

319

301

115

203

275

100 140 180 220 260 300 340 380 m/z, Da

115 232

-‐H

203

-‐H

COO-

OH

236

λmaxMeOH = 236 nm

ε = 25,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

220 230 240 250 270 210 280 290 260

232





12-‐HETE

15.9

319=M-‐H 301=M-‐H-‐H2O 275=M-‐H-‐CO2 257=M-‐H-‐H2O-‐CO2 163=207-‐CO2 145=207-‐H2O-‐CO2 135=179-‐CO2

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

319

179 257

301

135

163 275

145

207

100 140 180 220 260 300 340 380 m/z, Da

-‐H 207

+H

179

COO-

OH

236

λmaxMeOH = 236 nm

ε = 25,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

220 230 240 250 270 210 280 290 260





15-‐HETE

15.6

319=M-‐H 301=M-‐H-‐H2O 275=M-‐H-‐CO2 257=M-‐H-‐H2O-‐CO2 203=247-‐CO2 175=219-‐CO2

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

319

301

257

175

219

203 275

247

100 140 180 220 260 300 340 380 m/z, Da

219 +H

247

-‐H

COO-

OH

236

λmaxMeOH = 236 nm

ε = 25,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

220 230 240 250 270 210 280 290 260






Lipoxins

65/104




LXA5

10.2

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

349=M-‐H 331=M-‐H-‐H2O 313=M-‐H-‐2H2O 305=M-‐H-‐CO2 287=M-‐H-‐H2O-‐CO2 269=M-‐H-‐2H2O-‐CO2 217=279-‐H2O-‐CO2 215=233-‐H2O 207=251-‐CO2 189=251-‐H2O-‐CO2 171=251-‐2H2O-‐CO2

100 140 180 220 260 300 340 380 m/z, Da

301

288

315 λmax

MeOH = 301 nm

ε = 50,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

260 280 300 320 340 240 360 380

349

COO-

OH

HO OH

115

+H

233 -‐H -‐H

145

251 279

-‐H

215 233

115

269 287

305 313

331 145 251

189

207 217 171





-‐H

5S,15S-‐diHEPE

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

333

100 140 180 220 260 300 340 380 m/z, Da

COO-

OH

OH

246 115 217

-‐H

235

+H -‐H 263

246

115

217

235

315

297

289

271

253 199

333=M-‐H 315=M-‐H-‐H2O 297=M-‐H-‐2H2O 289=M-‐H-‐CO2 271=M-‐H-‐H2O-‐CO2 253=M-‐H-‐2H2O-‐CO2 245=263-‐H2O 219=263-‐CO2 201=263-‐H2O-‐CO2 199=217-‐H2O 191=235-‐CO2 173=235-‐H2O-‐CO2

191

173

219

201

245

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

244

λmaxMeOH = 244 nm

ε = 25,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

220 230 240 250 270 210 280 290 260

12.2






E-‐Series Resolvins

68/104




RvE1

349=M-‐H 331=M-‐H-‐H2O 313=M-‐H-‐2H2O 305=M-‐H-‐CO2 295=M-‐H-‐3H2O 287=M-‐H-‐H2O-‐CO2 273=291-‐H2O 269=M-‐H-‐2H2O-‐CO2 255=291-‐2H2O 229=291-‐H2O-‐CO2 205=223-‐H2O 187=223-‐2H2O 179=223-‐CO2 177=195-‐H2O 161=223-‐H2O-‐CO2 151=195-‐CO2 133=195-‐H2O-‐CO2

291

195

223

115

+H

+H

-‐H

-‐H

8.3

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

349

195

331

161

205

291 313 179 287 273

187 151 295 115 255 229 177 305 269

100 140 180 220 260 300 340 380 m/z, Da

271

262 282 λmax

MeOH = 271 nm

ε = 40,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

270 230 250 290 210 330 310

237





COO-

HO

OH

OH

11.9

RvE2

333=M-‐H 315=M-‐H-‐H2O 297=M-‐H-‐2H2O 289=M-‐H-‐CO2 271=M-‐H-‐H2O-‐CO2 257=275-‐H2O 253=M-‐H-‐2H2O-‐CO2 231=275-‐CO2 213=275-‐H2O-‐CO2 199=217-‐H2O 173=217-‐CO2

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

333

271

213 253

115 315

199

275 297

257 289 231 217 173

100 140 180 220 260 300 340 380 m/z, Da

115 217 -‐H

275

+H

COO-

HO

OH

243

λmaxMeOH = 243 nm

ε = 25,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

220 230 240 250 270 210 280 290 260





RvE3

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

100 140 180 220 260 300 340 380 m/z, Da

303 275

-‐H 245 +H

+H

333

315

297

289

271

253

245

201

275

257

231

213

259

333=M-‐H 315=M-‐H-‐H2O 297=M-‐H-‐2H2O 289=M-‐H-‐CO2 271=M-‐H-‐H2O-‐CO2 259=303-‐CO2 257=275-‐H2O 253=M-‐H-‐2H2O-‐CO2 231=275-‐CO2 213=275-‐H2O-‐CO2 201=245-‐CO2

270

260 281 λmax

MeOH = 270 nm

ε = 40,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

250 270 290 310 230 330

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

13.2

71/104

COO-

HO OH

Wavelength, nm





MonoHydroxyEPE Products

72/104




5-‐HEPE

15.4


Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

255

317 115

201 299

273

100 140 180 220 260 300 340 380 m/z, Da

115 201

-‐H

COO-

OH

236

λmaxMeOH = 236 nm

ε = 25,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

220 230 240 250 270 210 280 290 260





12-‐HEPE

15.0

317=M-‐H 299=M-‐H-‐H2O 273=M-‐H-‐CO2 255=M-‐H-‐H2O-‐CO2 189=207-‐H2O 163=207-‐CO2 145=207-‐H2O-‐CO2 135=179-‐CO2

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

317

273 145 109

179

255

135

299 163

207

189

100 140 180 220 260 300 340 380 m/z, Da

179 +H

207 -‐H

COO-

OH

236

λmaxMeOH = 236 nm

ε = 25,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

220 230 240 250 270 210 280 290 260





15-‐HEPE

14.8

COO-

OH -‐H

247 219

+H


Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

317

255

219 175

299

247

203 273

185 229

100 140 180 220 260 300 340 380 m/z, Da

237

λmaxMeOH = 237 nm

ε = 25,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

220 230 240 250 270 210 280 290 260





18-‐HEPE

14.7

317=M-‐H 299=M-‐H-‐H2O 273=M-‐H-‐CO2 255=M-‐H-‐H2O-‐CO2 243=287-‐CO2 215=259-‐CO2

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

317

255

215

299

259

273

243

100 140 180 220 260 300 340 380 m/z, Da

259

+H

287 -‐H

COO-

OH

236

λmaxMeOH = 236 nm

ε = 25,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

220 230 240 250 270 210 280 290 260






D-‐Series Resolvins

77/104




RvD1

11.1

375=M-‐H 357=M-‐H-‐H2O 339=M-‐H-‐2H2O 331=M-‐H-‐CO2 313=M-‐H-‐H2O-‐CO2 295=M-‐H-‐2H2O-‐CO2 261=305-‐CO2 259=277-‐H2O 243=305-‐H2O-‐CO2 241=277-‐2H2O 215=233-‐H2O 197=233-‐2H2O 185=203-‐H2O 153=171-‐H2O 135=171-‐2H2O 127=171-‐CO2 123=141-‐H2O

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

100 140 180 220 260 300 340 380 m/z, Da

215

375

233

141

135

277 357 171 295 331

313 123 197 259 339 241 153 127 185 243

113

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

COO-

HO

OH

HO

141

233 -‐H

+H 277

171 -‐H

113 +H

-‐H

305

203

301

288

316

λmaxMeOH = 301 nm

ε = 50,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

260 280 300 320 340 240 360 380

78/104

261

Wavelength, nm




COO-

HO

OH

OH

RvD2

10.6

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

100 140 180 220 260 300 340 380 m/z, Da

375

128

277

295

233 357 339 197 203

313 331 185

215

110 321

375=M-‐H 357=M-‐H-‐H2O 339=M-‐H-‐2H2O 331=M-‐H-‐CO2 321=M-‐H-‐3H2O 313=M-‐H-‐H2O-‐CO2 295=M-‐H-‐2H2O-‐CO2 287=305-‐H2O 259=277-‐H2O 241=277-‐2H2O 215=233-‐H2O 203=247-‐CO2 197=233-‐2H2O 185=247-‐H2O-‐CO2 110=128-‐H2O

233

+H 247

277 +H

305

-‐H

-‐H 262

128 -‐H

301

288

315

λmaxMeOH = 301 nm

ε = 50,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

260 280 300 320 340 240 360 380

287

259

241

79/104

141 -‐H

141

247

Wavelength, nm




RvD3

277 +H

195

181 165

209 -‐H

+H

101 -‐H

375

147

137

357 165 215 181 277 313 295 259 159 177

339 101 191

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 m/z, Da

375=M-‐H 357=M-‐H-‐H2O 339=M-‐H-‐2H2O 313=M-‐H-‐H2O-‐CO2 295=M-‐H-‐2H2O-‐CO2 259=277-‐H2O 215=277-‐H2O-‐CO2 191=209-‐H2O 177=195-‐H2O 159=195-‐2H2O 147=165-‐H2O 137=181-‐CO2

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

10.8

80/104

220 240 260 280 300 320 200

Rel. Abs. (%)

100%

0%

271

λmaxMeOH = 271 nm

ε = 40,000 abs.L/mol.

281 260

234

Wavelength, nm




OH

HO

COO-HO

COO-

HO

HO

141

-‐H 289

+H

+H 246

113

261

-‐H

-‐H

217

RvD5

13.0

359=M-‐H 341=M-‐H-‐H2O 323=M-‐H-‐2H2O 315=M-‐H-‐CO2 297=M-‐H-‐H2O-‐CO2 279=M-‐H-‐2H2O-‐CO2 245=289-‐CO2 243=261-‐H2O 228=246-‐H2O 227=289-‐H2O-‐CO2 199=217-‐H2O

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

199

279 359

141 341 297

246 261 217

113 228

245 315 323

227

243

100 140 180 220 260 300 340 380 m/z, Da

242

λmaxMeOH = 242 nm

ε = 25,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

220 230 240 250 270 210 280 290 260





RvD6

359=M-‐H 341=M-‐H-‐H2O 315=M-‐H-‐CO2 297=M-‐H-‐H2O-‐CO2 279=M-‐H-‐2H2O-‐CO2 245=289-‐CO2 243=261-‐H2O 239=257-‐H2O 227=289-‐H2O-‐CO2 217=261-‐CO2 209=289-‐2H2O-‐CO2 199=261-‐H2O-‐CO2

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

100 140 180 220 260 300 340 380 m/z, Da

359

341

315

297

279

289

261

101

257

239

243

217

199

245

227

101

257

-‐H

+H 261

-‐H 289

209

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

240

λmaxMeOH = 240 nm

ε = 25,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

220 230 240 250 270 210 280 290 260

13.4





OH

OH

-OOC


Maresins

83/104




OHOH

COO-

MaR1

250 113

246 -‐H

221

+H

+H 141 -‐H

13.1

359=M-‐H 341=M-‐H-‐H2O 323=M-‐H-‐2H2O 315=M-‐H-‐CO2 297=M-‐H-‐H2O-‐CO2 279=M-‐H-‐2H2O-‐CO2 232=250-‐H2O 228=246-‐H2O 203=221-‐H2O 177=221-‐CO2 159=221-‐H2O-‐CO2 123=141-‐H2O

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

359

177

250 297

123 341

228 113 246 221 315 232 279

141 323

203 159

100 140 180 220 260 300 340 380 m/z, Da

270

261 281

λmaxMeOH = 270 nm

ε = 40,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

270 230 250 290 210 330 310





13.6

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

244

λmaxMeOH = 244 nm

ε = 25,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

220 230 240 250 270 210 280 290 260

4S,14S-‐diHDHA

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

359

341

100 140 180 220 260 300 340 380 m/z, Da

297

279

101

221

138 109 323 315

239

213 203

187

159

177

359=M-‐H 341=M-‐H-‐H2O 323=M-‐H-‐2H2O 315=M-‐H-‐CO2 297=M-‐H-‐H2O-‐CO2 279=M-‐H-‐2H2O-‐CO2 239=257-‐H2O 231=249-‐H2O 213=249-‐2H2O 203=221-‐H2O 187=249-‐H2O-‐CO2 177=221-‐CO2 159=221-‐H2O-‐CO2

231

COO-

OH

OH

249 -‐H

221 +H

101

-‐H

257

-‐H 138

109






ProtecEns

86/104




PD1

13.0

COO-

OH

OH

153

206 -‐H

+H

-‐H

+H

177 181

261

359=M-‐H 341=M-‐H-‐H2O 323=M-‐H-‐2H2O 315=M-‐H-‐CO2 297=M-‐H-‐H2O-‐CO2 279=M-‐H-‐2H2O-‐CO2 243=261-‐H2O 217=261-‐CO2 199=261-‐H2O-‐CO2 188=206-‐H2O 159=177-‐H2O 137=181-‐CO2 119=181-‐H2O-‐CO2

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

153

359

137

206

297 188

341 261 217

159 323 315

199 119 177

181 279 243

100 140 180 220 260 300 340 380 m/z, Da

270

261 281 λmax

MeOH = 270 nm

ε = 40,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

250 270 290 310 230 330






MonoHydroxyDHA Products

88/104




4-‐HDHA

16.7


Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

343

281

101

299

325

241

100 140 180 220 260 300 340 380 m/z, Da

101 241 -‐H

COO-

OH

235

λmaxMeOH = 235 nm

ε = 25,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

220 230 240 250 270 210 280 290 260





7-‐HDHA

16.1

343=M-‐H 325=M-‐H-‐H2O 299=M-‐H-‐CO2 281=M-‐H-‐H2O-‐CO2 123=141-‐H2O

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

281

343

201

141

325

299 113

123 230

100 140 180 220 260 300 340 380 m/z, Da

141 201

113 113

230 -‐H

+H

-‐H

COO-

OH

236

λmaxMeOH = 236 nm

ε = 25,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

220 230 240 250 270 210 280 290 260





14-‐HDHA

15.9

343=M-‐H 325=M-‐H-‐H2O 299=M-‐H-‐CO2 281=M-‐H-‐H2O-‐CO2 215=233-‐H2O 189=233-‐CO2 161=205-‐CO2

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

343

281

161

325 205

299

233

189

215

100 140 180 220 260 300 340 380 m/z, Da

233 -‐H

205 +H

COO-

OH

237

λmaxMeOH = 237 nm

ε = 25,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

220 230 240 250 270 210 280 290 260





17-‐HDHA

15.7


Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

343

255 211

281

201

325 245

299

229 273

100 140 180 220 260 300 340 380 m/z, Da

-‐H 273

+H 245

COO-

OH

236

λmaxMeOH = 236 nm

ε = 25,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

220 230 240 250 270 210 280 290 260





Aspirin-‐Triggered Special Pro-‐resolving Mediators

93/104




AT-‐LXA4

11.3

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

351

115

217

235

189 289 307

271 333 315

233

127

251 199 145

171 279 215

207 243 261

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

351=M-‐H 333=M-‐H-‐H2O 315=M-‐H-‐2H2O 307=M-‐H-‐CO2 289=M-‐H-‐H2O-‐CO2 271=M-‐H-‐2H2O-‐CO2 261=279-‐H2O 243=279-‐2H2O 233=251-‐H2O 217=235-‐H2O 215=251-‐2H2O 207=251-‐CO2 199=235-‐2H2O 189=251-‐H2O-‐CO2 171=251-‐2H2O-‐CO2 127=145-‐H2O

100 140 180 220 260 300 340 380 m/z, Da

COO-

HO OH

OH

115

+H

235

-‐H -‐H 145

251

279 -‐H

301

288

315

λmaxMeOH = 301 nm

ε = 50,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

260 280 300 320 340 240 360 380





AT-‐LXB4

11.1

351=M-‐H 333=M-‐H-‐H2O 315=M-‐H-‐2H2O 307=M-‐H-‐CO2 297=M-‐H-‐3H2O 289=M-‐H-‐H2O-‐CO2 271=M-‐H-‐2H2O-‐CO2 261=279-‐H2O 243=279-‐2H2O 233=251-‐H2O 217=235-‐H2O 215=251-‐2H2O 207=251-‐CO2 203=221-‐H2O 189=251-‐H2O-‐CO2 177=221-‐CO2 159=221-‐H2O-‐CO2

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

351

221

315

233

333

189 251 207 271 203 177

159 289 297 215

243 307 261 217 115

100 140 180 220 260 300 340 380 m/z, Da

301

288

314

λmaxMeOH = 301 nm

ε = 50,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

260 280 300 320 340 240 360 380


279 251

-‐H 221

115

235

+H

-‐H

+H

COO-

OH

HO OH




AT-‐RvD1

11.2

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

375=M-‐H 357=M-‐H-‐H2O 339=M-‐H-‐2H2O 331=M-‐H-‐CO2 313=M-‐H-‐H2O-‐CO2 295=M-‐H-‐2H2O-‐CO2 261=305-‐CO2 259=277-‐H2O 243=305-‐H2O-‐CO2 241=277-‐2H2O 215=233-‐H2O 197=233-‐2H2O 185=203-‐H2O 153=171-‐H2O 135=171-‐2H2O 127=171-‐CO2 123=141-‐H2O

215

375

233

141

135

277

185 127 357 171 295 331

113 313 123 197

153 259 339 241

243 0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

100 140 180 220 260 300 340 380 m/z, Da

COO-

HO

OH

HO

141

233 -‐H

+H 277

171 -‐H

113 +H

-‐H

305

203

301

288 316

λmaxMeOH = 301 nm

ε = 50,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

260 280 300 320 340 240 360 380





AT-‐RvD3

375=M-‐H 357=M-‐H-‐H2O 339=M-‐H-‐2H2O 313=M-‐H-‐H2O-‐CO2 295=M-‐H-‐2H2O-‐CO2 259=277-‐H2O 215=277-‐H2O-‐CO2 191=209-‐H2O 177=195-‐H2O 159=195-‐2H2O 147=165-‐H2O 137=181-‐CO2

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

10.5

97/104

220 240 260 280 300 320 200

Rel. Abs. (%)

100%

0%

271

λmaxMeOH = 271 nm

ε = 40,000 abs.L/mol.

281 260

234

375

147

137

357 165 215 181 277 313 295 259 159 177

339 101 191

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 m/z, Da

Wavelength, nm

277 +H

195

181 165

209 -‐H

+H

101 -‐H

HO

COO-

OH

HO




COO-

OH

OH

AT-‐PD1

12.4

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

270

261 281 λmax

MeOH = 270 nm

ε = 40,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

250 270 290 310 230 330

359=M-‐H 341=M-‐H-‐H2O 323=M-‐H-‐2H2O 315=M-‐H-‐CO2 297=M-‐H-‐H2O-‐CO2 279=M-‐H-‐2H2O-‐CO2 243=261-‐H2O 217=261-‐CO2 199=261-‐H2O-‐CO2 188=206-‐H2O 159=177-‐H2O 137=181-‐CO2 119=181-‐H2O-‐CO2 109=153-‐CO2

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

153

359

137

206

297 188

341 261 217

159 323 315

199 119 177

181 279 243

100 140 180 220 260 300 340 380 m/z, Da


153

206 -‐H

+H

-‐H

+H

177 181

261




Deuterium Labeled Internal Standards

99/104




d4-‐PGE2

+H 255 -‐H

283

O

HO

COO-

OH

D D

D D

275

319 337

193

239 211 355 293

237

283

355=M-‐H 337=M-‐H-‐H2O 319=M-‐H-‐2H2O 293=M-‐H-‐H2O-‐CO2 275=M-‐H-‐2H2O-‐CO2 239=283-‐CO2 237=255-‐H2O 211=255-‐CO2 193=255-‐H2O-‐CO2

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

100 140 180 220 260 300 340 380 m/z, Da

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

10.4

Rel. Int. (%

)

100/104




COO-

HO OH

OH

D D

D

D

D

d5-‐LXA4

10.1

356=M-‐H 320=M-‐H-‐2H2O 312=M-‐H-‐CO2 294=M-‐H-‐H2O-‐CO2 276=M-‐H-‐2H2O-‐CO2 233=251-‐H2O 222=240-‐H2O 189=251-‐H2O-‐CO2

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

356

115

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

100 140 180 220 260 300 340 380 m/z, Da

115

+H

240

-‐H

251

301

288

315

λmaxMeOH = 301 nm

ε = 50,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

260 280 300 320 340 240 360 380

320 312

294

276 251

240

233

222

189


-‐H 145

145




d4-‐LTB4

COO-

OHOH

D

D

D D

142

197 113

225 -‐H

+H 223

115 -‐H -‐H

339=M-‐H 321=M-‐H-‐H2O 303=M-‐H-‐2H2O 295=M-‐H-‐CO2 277=M-‐H-‐H2O-‐CO2 259=M-‐H-‐2H2O-‐CO2 207=225-‐H2O 205=223-‐H2O 181=225-‐CO2 179=197-‐H2O 163=225-‐H2O-‐CO2 153=197-‐CO2 135=197-‐H2O-‐CO2 124=142-‐H2O

339

197

321

153 277 207 181

115 303 259 163 124 135 295 142

113 205 179 0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

100 140 180 220 260 300 340 380 m/z, Da

270

261 281 λmax

MeOH = 270 nm

ε = 40,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

270 230 250 290 210 330 310 0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

13.1

Rel. Int. (%

)





d8-‐5S-‐HETE

COO-

OH

D D D D

D D D D

116 210

-‐H 327

309 116 265

210

283


0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

100 140 180 220 260 300 340 380 m/z, Da

236

λmaxMeOH = 236 nm

ε = 25,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

220 230 240 250 270 210 280 290 260

16.2

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

Rel. Int. (%

)





d5-‐RvD2

10.6

Rel. Int. (%

)

0%

50%

100%

2 4 6 8 10 12 14 16 18 20 22

Time, min

0

380

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Rel. Int. (%

)

100 140 180 220 260 300 340 380 m/z, Da

301

288

315

λmaxMeOH = 301 nm

ε = 50,000 abs.L/mol.

Rel. Abs. (%)

0%

50%

100%

260 280 300 320 340 240 360 380

362





COO-

OH

HO

OH

D

D D

DD

380=M-‐H 362=M-‐H-‐H2O 344=M-‐H-‐2H2O 336=M-‐H-‐CO2 318=M-‐H-‐H2O-‐CO2 300=M-‐H-‐2H2O-‐CO2 259=277-‐H2O 241=277-‐2H2O 215=277-‐CO2-‐H2O 185=247-‐H2O-‐CO2

238

+H 247

277 +H

133 -‐H

141 -‐H

344 336 318

300

141 277

133 113

113 +H

247 238

259 241

215

185

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