Velos Pro - Thermo Fisherapps.thermoscientific.com/.../San_Mateo/Velos_Pro_SM.pdfVelos Pro: High...

Velos Pro - Multiple Fragmentation Techniques for Comprehensive QualTechniques for Comprehensive Qual and Confident QuantTim StrattonBay Area LCMS Users Meeting20 Sep 2011

The Velos Pro

C h i S ll M l l St t Id tifi ti• Comprehensive Small Molecule Structure Identification• High scan speed with data dependant acquisition means never having to

know what is present before hand.N bilit t i HCD f t ti h i ith i• New ability to mix HCD fragmentation mechanism with previous mechanisms at ALL MSn levels opens a whole new avenue for structure identification.

• High sensitivity for low level metabolites and impurities – obtainHigh sensitivity for low level metabolites and impurities obtain fragmentation data on previously hard to detect components.

• Confident Small Molecule Quantitation• Easy quantification setup with multiple fragment mechanism options toEasy quantification setup with multiple fragment mechanism options to

achieve a sensitive, selective, and robust method• Employ multiple fragmentation modes in the same run• Post acquisition selection of the best transition to minimize interference q

without reinjection• High dynamic range (5 orders of magnitude) and sensitivity (<50 fg on

column) comparable to triple quad

2 Proprietary & Confidential

The Velos Pro









New Fragmentation Possibilities

Multiple Fragmentation Velos Pro:

Ability to use CID and HCD Techniques

MSn and Fragment Heritage

b y o use C a d C

combinations at multiple

different MSn levels.

LC-MS:Li 10 i di f

Sample:Linear 10 minute gradient of Water:ACN w/ 0.1% Formic.

Hypersil Gold aQ 50X2 1.9µ column

p

Trifluopirazine metabolites from

human liver S9 incubationN Hypersil Gold aQ 50X2 1.9µ column

1.Full scan + CID only MSn2.Full Scan + HCD MS2 + CID MS3

3 F ll S + HCD MS2 + HCD MS3N

NN

F

F F


3.Full Scan + HCD MS2 + HCD MS3S

F

New Fragmentation Possibilities – HCD vs CIDTrifluopirazine_HCD-HCD #2817 RT: 6.43 AV: 1 NL: 1.95E5 Trifluopirazine_CID-CID #2411 RT: 6.50 AV: 1 NL: 2.07E4T: ITMS + p ESI d w Full ms2 [email protected] [50.00-420.00]

140000

160000

180000

113.1

280.3

T: ITMS + p ESI d w Full ms2 [email protected] [100.00-420.00]

14000

16000

18000

20000141.1Trifluopirazine

HCD MS2

TrifluopirazineCID MS2

40000

60000

80000

100000

120000

Inte

nsity

141.2

70.1

248.398 2 308 3 4000

6000

8000

10000

12000

Inte

nsity

368.3

280 1

50 100 150 200 250 300 350 400m/z

0

20000

40000 98.2 308.3408.4

239.384.1 368.4211.1 348.6184.9

50 100 150 200 250 300 350 400m/z

0

2000

000 280.1 308.2348.3

262.0139.1 408.4340.2230.1210.2147.0

Trifluopirazine_CID-CID #2412 RT: 6.50 AV: 1 NL: 1.47E4T: ITMS + c ESI d w Full ms3 [email protected] [email protected] [50.00-155.00]

113.1

HCD f t ti id

8000

10000

12000

14000

sity

CID MS3

408 141HCD fragmentation provides a broader mass range of fragmentation. CID fragmentation can provide similar fragment detail

2000

4000

6000

8000

Inte

ns

70.298 2

can provide similar fragment detail but may need additional MSn

steps. However, this isn’t always a bad thing…


50 100 150 200 250 300 350 400m/z

098.2 113.9 146.2

bad thing…

New Fragmentation Possibilities – HCD MSn

Trifluopirazine_HCD-CID #2217 RT: 5.83 AV: 1 NL: 1.86E4T: ITMS + p ESI d w Full ms2 [email protected] [50.00-435.00]“HCDn” allows for p @ [ ]

12000

14000

16000

18000113.0837

296.1679

HCDn allows for even more structure data.

CID can do the same interpretation when going to MS4 (not shown)

4000

6000

8000

10000

Inte

nsity 141.1671

70.0834

264.1678

324 2514

MS . (not shown)MS2 of 424

50 100 150 200 250 300 350 400m/z

0

2000324.2514

424.3353235.0843 364.2516185.0007

264.2 264.1521

10000

12000

14000

16000

18000

nsity 30000

35000

40000

45000

50000

nsity

MS3 HCD-HCD424 296

MS3 HCD-CID424 296NH

•

FF

NN

F F

OHN

N

FOH

2000

4000

6000

8000

10000

Inte

n

235.3296.3

263.5

256.3228 2194 1 297 1121 3 183 0152 95000

10000

15000

20000

25000

Inte

n

296.1775227.2070204 0744139 2329

F

S

NF

F F

S

NF

F F


50 100 150 200 250 300m/z

0256.3228.2194.1 297.1121.3 183.0152.9

100 150 200 250 300m/z

0227.2070204.0744139.2329

Fragmentation and Metabolite Finding

• Finding the related components remains a challenging step in Metabolite Identification.

• Fragment based techniques are common and useful techniquesM f t id b tt d t ti• More fragments can provide better detection

• HCD fragmentation provides a richer MS2 spectraN

N

N

N

N

NF F

S

NF

F FS

F

N


N

FISh with HCD Fragmentation

• Parent + Observed MS2 (HCD) Fragments used – 9 total• Able to detect related materials immediately.Able to detect related materials immediately.



N

N

S

NF

F F

MSn Spectral Tree HCD MS2 Fragmentsp HCD MS2 Fragments



-14.097 –CH2N

NH

14.097 CH2

NF

F F

S


Inclusion of Simple Phase I Modifications

9 HCD Fragments

9 HCD Fragments9 HCD Fragments+ Phase I


100113.2

HCD CID vs HCD HCD – Mixing Fragment Techniques

60

80

100

Abu

ndan

ce 280.2

141.2

70.1 N

N

N

F F

50 100 150 200 250 300 350 4000

20

40

Rel

ativ

e

70.1

248.298.1 308.2 408.3239.196.2 368.3

S

F

m/z

70

80

90

100

e

248.3

80

90

100248.2

HCD 50% CID 35%

40

50

60

70

elat

ive

Abu

ndan

c

40

50

60

70

ativ

e A

bund

ance

50 100 150 200 2500

10

20

30Re

280.3

247.3 260.3

233.1210.2

0

10

20

30Rel

a

280.2211.2 246.1


50 100 150 200 250m/z

100 150 200 250m/z

Complementary Fragmentation – CID and HCD

Trifluopirazine_CID-CID #2503 RT: 6.72 AV: 1 NL: 2.44E5T: ITMS + p ESI d w Full ms2 [email protected] [100.00-420.00]

100141.1

Trifluopirazine_HCD-CID #2489 RT: 6.49 AV: 1 NL: 1.19E6T: ITMS + p ESI d w Full ms2 [email protected] [50.00-420.00]

100113.2

N

N

F F

70

80

90

ce

70

80

90

e

280.2

S

NF

F

CID 35%

40

50

60

ativ

e A

bund

anc

40

50

60

ativ

e A

bund

anc

141.2

70.1

HCD 50%CID 35%

20

30

40

Rel

a

368.3

308 2 348 2

20

30

40

Rel

a

248.298.1 308.2

408 3262 2

50 100 150 200 250 300 350 400m/z

0

10 308.2 348.2280.1 369.3262.1139.1 288.2

50 100 150 200 250 300 350 400m/z

0

10408.3262.2

239.196.2 368.3290.2139.156.1


MSn Fragmentation Heritage

Trifluopirazine_HCD-CID #2489 RT: 6.49 AV: 1 NL: 1.19E6T: ITMS + p ESI d w Full ms2 [email protected] [50.00-420.00]

100113.2

Trifluopirazine_CID-CID #2503 RT: 6.72 AV: 1 NL: 2.44E5T: ITMS + p ESI d w Full ms2 [email protected] [100.00-420.00]

100141.1

N

N

F F

70

80

90

e

280.2

70

80

90

e CID 35%S

NF

F

40

50

60

tive

Abu

ndan

ce

141.2

70.140

50

60

ive

Abu

ndan

ce

HCD 50%CID 35%

20

30

40

Rel

at

70.1

248.298.1 308.2

20

30

40

Rel

at

368.3

50 100 150 200 250 300 350 400m/z

0

10308.2

408.3262.2239.196.2 368.3290.2139.156.1

50 100 150 200 250 300 350 400m/z

0

10 308.2 348.2280.1 369.3262.1139.1 288.2


m/zm/z

Comprehensive Qual

• HCD and CID• Complementary Fragmentationp y g• More complete fragmentation coverage

• HCD and FISh• HCD and FISh• More fragment coverage = Better search results

• MSn Fragmentation• Fragment heritage aids in identification• Multiple fragment techniques increases linkage information• Multiple fragment techniques increases linkage information


The Velos Pro









Easy Quantitation Setup – No Prior MRM Tuning

Quantitation with No Tuning. Velos Pro:

High scan rate allows for Post Acquisition Transition

Selection

g sca a e a o s o

simultaneous CID and HCD

fragmentation acquisition.

LC-MS:Sample:Linear 5 minute gradient of Water:MeOH w/ 0.1% Formic.

Hypersil Gold aQ 50X2 1 9µ

p

Rat plasma spiked with

Dextromethorphan and Hypersil Gold aQ 50X2 1.9µ column

Full Scan + CID(258) + HCD(258)

Metabolites. N

O


Easy Quantitation Setup – No Prior MRM TuningRT: 0.49 - 4.67

4 25 NL:

40

60

80

100at

ive

Abu

ndan

ce4.25

4.23

2.10 3.143 78

NL:9.74E3Base Peak M S M ix3_Std5_01

Mix3_Std5_01 #933 RT: 3.14 AV: 1 NL: 3.34E3T: ITMS + c ESI t E Full ms2 [email protected] [70.00-260.00]

215 1182

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5Time (min)

0

20Rel

a 3.783.122.12 3.67 4.333.26 4.182.43 2.662.05 3.993.081.92 4.422.311.67

1000

1500

2000

2500

3000

Inte

nsity

215.1182

Look at the CID fragmentsSelect m/z 215 and extract

100 150 200 250m/z

0

500 240.1457147.0405104.2404 171.1284

RT: 2.45 - 4.10

100

e

3.14

20

40

60

80

Rel

ativ

e A

bund

ance

3.21 3.883.06 3 382 77


2.5 3.0 3.5 4.0Time (min)

03 3.883.06 3.382.77

Easy Quantitation Setup – No Prior MRM TuningRT: 0.49 - 4.67

4 25 NL:

40

60

80

100at

ive

Abu

ndan

ce4.25

4.23

2.10 3.143 78

NL:9.74E3Base Peak M S M ix3_Std5_01

Mix3_Std5_01 #934 RT: 3.14 AV: 1 NL: 2.03E2T: ITMS + c ESI t E Full ms2 [email protected] [50.00-260.00]

200147.1111

0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5Time (min)

0

20Rel

a 3.783.122.12 3.67 4.333.26 4.182.43 2.662.05 3.993.081.92 4.422.311.67

50

100

150

200

Inte

nsity

213.2606171.1915

121.1791

Look at the HCD fragmentsSelect m/z 147 and extract

50 100 150 200 250m/z

0

258.383586.2859

RT: 2.45 - 4.10 SM: 7B

100

e

3.14

20

40

60

80R

elat

ive

Abu

ndan

ce

4.013.05 3.673.582 63


2.5 3.0 3.5 4.0Time (min)

03 582.63

Easy Quantitation Setup – No Prior MRM Tuning

• Selection of CID vs HCD fragments can be performed after acquisition.• Often, one fragmentation technique provides a more intense transition

providing more sensitivity.• Having all the fragments available also allows reselecting fragment ions

to improve selectivity.

RT: 2.45 - 4.10 SM: 7B

90

100

• Reselect, don’t reinject.

m/z 215 m/z 147HCD

RT: 2.45 - 4.10 SM: 7B

1003.13

50

60

70

80

90

e A

bund

ance

CID HCD

50

60

70

80

90

Abu

ndan

ce

Same injectionDiff t l ti

0

10

20

30

40

Rel

ativ

e3.14

10

20

30

40

50

Rel

ativ

e A

3.22 3.80 3.903.462.75 3.532.72 3.00

Different selectionSame Scale


2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0Time (min)

0

2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0Time (min)

0

MSn Quantitation – Ultimate Selectivity in Real Matrix

MSn Quantitation Velos Pro:

Robust MSn fragmentation Ultimate Selectivity

obus S ag e a o

and multiple fragmentation

techniques.

LC-MS:Sample:Linear 5 minute gradient of Water:MeOH w/ 0.1% Formic.

Hypersil Gold aQ 50X2 1 9µ

Human plasma standards with

Dextromethorphan and

Metabolites Hypersil Gold aQ 50X2 1.9µ column

CID (258) CID (210) MS3

Metabolites.N

OH OH


m/z 201m/z 258


RT: 0.90 - 4.13 SM: 7G

80

1002.65RT: 0.87 - 4.13 SM: 7G

1004.11

MS3 458 201 133MS2 458 201

Std 2

40

60

80

Rel

ativ

e A

bund

ance

40

60

80R

elat

ive

Abu

ndan

ce

4.04

2.653.813.67

3 48

Std 2100 fg/µL5 µL inj.Pl

RT: 0 91 4 09 SM: 7GRT 0 92 4 12 SM 7G

1.0 1.5 2.0 2.5 3.0 3.5 4.0Time (min)

0

20

1.0 1.5 2.0 2.5 3.0 3.5 4.0Time (min)

0

20

R 3.483.15

3.002.432.401.961.710.89Plasma

RT: 0.91 - 4.09 SM: 7G

60

80

100

unda

nce

2.66RT: 0.92 - 4.12 SM: 7G

60

80

100

ndan

ce

4.12

Std 110 fg/µL

0

20

40

Rel

ativ

e A

bu

2.52

3.933.593.422.92

0

20

40

Rel

ativ

e A

bu

4.053.443.39 3.963.31

3.082.972.642.391.971.711.27

10 fg/µL5µL inj.Plasma


1.0 1.5 2.0 2.5 3.0 3.5 4.0Time (min)

01.0 1.5 2.0 2.5 3.0 3.5 4.0

Time (min)

0


• Without using MS3, detection and quantitation was not possible below 0.1 fg/µL.

• With MS3, quantitation was possible down to 0.01 fg/µL and detection possible below that.

• Triplicate Standard curves were prepared and analyzed, the limit of Quantitation and Detection as well as the relative standard deviation (RSD) are shown below.

Std pg/µL MS2 MS3

1000 8% 5%1000 8% 5%100 5% 4%10 4% 7%1 11% 8%1 11% 8%

0.1 LOQ/LOD, 12% 14%0.01 X LOQ, 10%


0.005 X LOD

Confident Quantitation

• Multiple Fragment Techniques• More options for detection and development• More options for detection and development

• Full Scan MSn data• Full Scan MS data• Reselect, don’t Reinject

• MSn

• Selectivity• Selectivity


Acknowledgements

• Robert Mistrik – HighChem• Thermo Fisher Scientific

• Yingying Huang• Rose Herbold• Kate Comstock• Caroline Ding

•All of You•All of You


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