Advanced Chemistry Development, Inc. (ACD/Labs)

www.acdlabs.comwww.acdlabs.com

Fragment Prediction and Assignment for Experimental Mass

Spectra

Dr Graham A. McGibbonASMS Seminar

3 June 2007Indianapolis, Indiana

2

Objectives

Automated Fragment Screening using IntelliXtractFragment Assignment of tandem MS dataCoupling of MS and NMR

3

Recognizing Structure and Spectrum Similarities

Most drug metabolites have some chemical structural similarities to the parent drug

Their mass spectra may thus have similarities

A modification to the parent compound will shift the mass of the metabolite by a set mass (e.g., a hydroxylated metabolite will differ from the mass of the parent by +16 Da).

Modified structural regions may yield fragment ions that are shifted by the same set mass. The metabolite and parent compound may have common substructures, and this may yield common fragment ions.

It is logical to use mass spectral similarities to identify potential metabolites and easy to understand how the similarities can be exploited to extract and elucidate metabolites…but in practice this is can be difficult.

Use the structure-spectrum relationship to identify the sites of modification on the parent drug and display this information, since it is difficult for the user to keep track of when it is difficult to visualize.

P+F+

P+Δ+F+

P+Δ+F+Δ+

Transformation(e.g. Δ = O)

N

N+Δ

N

4

ExperimentalMetabolite Incubation

A metabolism study was performed using the drug verapamil. The drug was incubated with human microsomes under the following conditions:

Incubation:Human microsomes/S937°C shaking water bath50 mM Tris buffer @ pH 7.4NADPH regenerating system (NRS) in 2%NaHCO3 (Sigma S-5761) Solution containing:0.5 mg/ml b-NADP (Sigma N-0505)2.0 mg/ml Glucose-6-phosphate (Sigma G7879)1.5 units/ml Glucose-6-phosphate dehydrogenase (Sigma G7877)Equal volume MeCN quenchDrug concentration 100 μl 0.1 mg/ml (final quench volume 2 ml)

5

ExperimentalInstrumental

Waters Acquity SDS UPLC™ & Waters Micromass LCT Premier oa-Tof mass spectrometer.

ESI +veCone voltage of 40 V to induce Source CIDAccurate Mass CentroidedInternal Calibrant Reference = Leucine enkephalin

A Waters Acquity UPLC BEH C18 column (100 x 2.1 mm, 1.7 μm particle size) was used for the separation, with the following gradient:

Time(min)

AH2O:0.1% HCOOH

BCH3CN:0.1%

HCOOH

0 95 5

15 5 95

18 5 95

18 95 5

21 95 5

6

IntelliXtract™ ActiveVerapamil Fragment Analysis

7

IntelliXtract Active Fragment Summary

8

Demethylation Variant #1

260.2165.2

9

Demethylation Variant #2

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Example Fragmentation Work FlowIntroduction

Extract fragmentation data – (not covered in this presentation)

Add potential StructureApply Auto-Assignment RulesVerify fragmentation assignmentUpdate fragmentation if requiredStore fragment assignmentsStore fragment schemasReport elucidations

11

Fragment-vendor integration

Ability to push spectra into MS Autoassignment in:

ABI & MDS Sciex LightSight™Waters MassLynx™Thermo Electron Corp. Xcalibur(Qual Browser)™Agilent Technologies ChemStation™(Ion Trap)

12

Adding a structure to a spectrum

Route 1 :- Access to a dictionary of public domain structures using ACD/Dictionary (V9 > 125,000 Entries)

Route 2 :- Cut & Paste from Corporate Structure Editor

Route 3 :- Manually Draw Structure

13

Example - SulfadimethoxineRoute 1

14

Add Structure

15

Attach structure to extracted spectrum

16

Fragment generation using Auto-Assignment rule set

17

Algorithmic based fragmentation analysisMS2 -Propanolol

18

Assignment Quality Index (AQI)

Useful for fragmentation spectra containing isotopic fragmentsAQI =

Calculates theoretical isotope contribution using fragment empirical formulaThe experimental contribution of ion intensities are analyzed as a function of the calculated isotope pattern and their m/z0.000 = no match1.000 = exact match

19

Impact of AQI in fragment assignment

Assignment of complete spectrum = 85.4 %

Isotopes are excluded in the fragmentation

analysis

20

Impact of AQI in fragment assignment

Assignment of complete spectrum = 98.8 %

21

Impact of AQI in spectral assignment

Isotopes are included in the fragmentation

analysis

Theoretical m/z & Intensity – supporting accurate mass data

22

Rule set improvements in N-Oxide fragmentation

23

Visualization of Golden PairsEase of analysis

Linked Golden Pair

24

Fragmentation Rules improvement history

Version 10Addition of Neutral Loss improvements for pre/post ionizationAddition of Neutral Loss during iterative fragment steps

Version 11 (End of year 2007)Addition of negative ionization fragmentationAddition of hydrogen radical loss for specific rules

25

Manual addition of Fragment Ionscomplimentary with algorithmic assignment

Lasso potential fragment region on structure

Adjust for proton addition/loss

Adjust for charge addition/loss

If fragment mass is present in the spectrum <equals +/-mass accuracy> mass of selected fragment then fragment structure can be added to the fragment table

26

Neutral Loss Spectrum ModeUseful for spotting classical losses

-2[H2O] -[H2O]

-[C3H6]

Parent Mass Referenced

at 0 Da mass shift

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Advanced Spectral Annotation

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Partial Structure Representations in support of Elucidation

NH

O

CH3

O

OHOH

OH

OH

O

OH

NH

O

CH3

O

OHOH

OH

OH

O

+[17]

29

Linking Partial Structures and Fragmentation spectra

Manual Fragmentation Analysis Method

30

31

ACD/MS Fragmenter example outputStructure based fragmentation schema creation

43N

5 N2

S1

N12

O8

NH19 20

O15

109 18

13 17OH11

1614

CH321

CH3 22

CH323

O6 O

7m/z 348

C13H24N4O5SM

43N

5 N2

S1

N12

O8

NH2+

19 20

O15

109 18

13 17OH11

1614

CH321

CH3 22

CH323

O6 O

7m/z 349

C13H25N4O5SM + H

43N

5 N2

S1

N12

O8

NH3+

19

O15

109 18

13 17OH11

1614

O6 O

7m/z 293

C9H17N4O5SM + H - C4H8

-C4H8

43N

5 N2

S1

N12

O8

NH19 20

O15

109 18

13 17OH11

1614

CH321

CH3 22

CH323

O6 O

7m/z 348

C13H24N4O5SM

43N

5 NH+

2S1

N12

O8

NH19 20

O15

109 18

13 17OH11

1614

CH321

CH3 22

CH323

O6 O

7m/z 349

C13H25N4O5SM + H

43N

5 NH2+

2S1

N12

O8

O15

13 17

1614

O6 O

7m/z 220

C6H10N3O4SM + H - C7H15NO

-C7H15NO

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Databasing fragmentation schemasACD/ChemFolder

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Templated Report

350300250200150100m/z

20

40

60

80

100

Rel

ativ

e In

tens

ity (%

)

332.1

293.2

275.1235.0

220.1

200.0174.1

130.2

43

N5

N2S

1

N12

O8

NH19 20

O15

109 18

13 17OH11

1614

21

2223

O6 O

7

Fragmentation Analysis Report

Operator pdp Parent Ion 349 Plot Type StickSample ID tim p1 ms2 Scan 358Spectrum Assigned 98.8 % [99-355/0-100/0] Spectrum Title TIMP1MS2.D

43N

5 N2

S1

N12

O8

NH19 20

O15

109 18

13 17OH11

1614

CH321

CH3 22

CH323

O6 O

7m/z 348

C13H24N4O5SM

43N

5 N2

S1

N12

O8

NH2+

19 20

O15

109 18

13 17OH11

1614

CH321

CH3 22

CH323

O6 O

7m/z 349

C13H25N4O5SM + H

43N

5 N2

S1

N12

O8

NH3+

19

O15

109 18

13 17OH11

1614

O6 O

7m/z 293

C9H17N4O5SM + H - C4H8

-C4H8

43N

5 N2

S1

N12

O8

NH19 20

O15

109 18

13 17OH11

1614

CH321

CH3 22

CH323

O6 O

7m/z 348

C13H24N4O5SM

43N

5 NH+

2S1

N12

O8

NH19 20

O15

109 18

13 17OH11

1614

CH321

CH3 22

CH323

O6 O

7m/z 349

C13H25N4O5SM + H

43N

5 NH2+

2S1

N12

O8

O15

13 17

1614

O6 O

7m/z 220

C6H10N3O4SM + H - C7H15NO

-C7H15NO

Parent Structure

34

Possible Data Input

13C NMR (standard, APT, DEPT)1H NMR1H/13C HS/MQC, HETCOR1H/13C HMBC, long-range HETCOR or variants MW or MF 1H/1H COSY, DQF-COSY, TOCSYNOESY, ROESY, XCORFE, INADEQUATEMultinuclear NMR: 15N, 17O, 31P, 29Si, 19F, 33SIR, MS, elemental compositionPartially assigned NMR data

35

Initial Data

MF acquired from an HR/MSStarting material1H, 13C, HMQC, HMBC NMR

36

MCD (Molecular Connectivity Diagram)

37

Final Result

MF (13C)3.171 ppm1.579 ppm

#1 #2

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Summary

ACD/MS Manager & ACD/MS Fragmenter offer extensive capabilities for manual and assisted spectral interpretationFragmentation analysis for spectra including isotopic peaks can significantly improve fragment assignment quality

39

Summary

Combination reporting can enable complex reports to be created to meet a demanding range of applicationsFragmentation schemas can be databased for future reference

40

Acknowledgements

Patrick Perkins - Agilent Technologies Inc. for access to example data