Simultaneous Quantification and Qualification With QED-MSMS Nitrofurains

7/30/2019 Simultaneous Quantification and Qualification With QED-MSMS Nitrofurains

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The world leader in serving science

Kefei Wang, Ph.D.Environmental and Food Safety Marketing

San J ose, CA

April 23, 2007

42nd WCTOW, Calgary

Simultaneous Quantification andSimultaneous Quantification and

Qualification with QED MS/MSQualification with QED MS/MS

and Its Application in Pesticideand Its Application in Pesticideand Food Residue Analysisand Food Residue Analysis


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2

Outline

What is QED MS/MS for Triple Quad?

QED MS/MS with RER

Application in Pesticide Analysis

Introducing EQuan

Pesticide in drinking water with EQuan

Application in Food Residue Analysis

Nitrofurans in Turbot Fish


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3

What is QED MS/MS?

Quantitation-Enhanced Data-Dependant MS/MS Scan

SRM triggered data dependent MS/MS scan

Both Quantitative and Qualitative scans are collected in the same analytical run

0

10

20

30

40

50

60

70

80

90

100

Relative

Abundan

ce

9.8 10.0 10.2 10.4 10.6

min

RT:10.0

SRM

m/z

Inten

sity

QED MS/MS


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QED MS/MS with Reverse Energy Ramp (RER)

InverseCollision

EnergyRamp

CE

M/Z

The RER scan function linearly reduces the amount of collision energy (CE) while theproduct ions are scanned from low to high mass in Q3. The resulting spectrumcontains more fragment ions with high abundance in low mass region.


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QED MS/MS Advantages on TSQ Quantum

9 Faster than a Q-trap

Does not require Dynamic fill timethat consumes cycle time

Excellent performance at low levels

9 RER yields rich product ion spectra

Library searchable MS/MS spectra

Fragment-rich product ion spectra for identification

No space charge

9 With unique H-SRM for precise parent ion selection Cleaner MS/MS spectra which are easier to interpret


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Example of HExample of H--SRM: Chloramphenicol at various Q1SRM: Chloramphenicol at various Q1

318 319 320 321 322 323 324 325 326 327

m/z

0

50

1000

50

100

0

50

100

Relative

Abundance

0

50

100

0

50

100321.01

322.92

321.93 325.03324.11318.83 325.94320.10

321.05

322.90

322.00 324.90324.00319.00 325.85320.00

320.99

322.89

324.93322.08 323.91319.01 325.98319.97

320.93

322.95

321.95 324.98323.93 325.94318.96 319.95

321.00

323.00

322.02 324.99324.01 325.98319.00 320.11

NL: 4.18E4

NL: 3.88E4

NL: 3.61E4

NL: 3.42E4

NL: 3.11E4

Q1= 0.7

Q1= 0.5

Q1= 0.2

Q1= 0.4

Q1= 0.1

100%

93%

86%

82%

74%


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SRM vs. H-SRM: Chloramphenicol in Pig Kidney (0.03 ug/kg)

R T : 0 . 0 0 - 5 . 0 0

0 . 0 0 . 5 1 . 0 1 . 5 2 . 0 2 . 5 3 . 0 3 . 5 4 . 0 4 . 5

T im e ( m in )

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

Re

lativeAbundance

0

1 0

2 0

3 0

4 0

5 0

6 0

7 0

8 0

9 0

1 0 0

Relative

Abundance

2 . 0 8

1 . 5 4

2 . 8 1

2 . 2 6

3 . 4 53 . 7 5

4 . 0 91 . 0 5 4 . 7 40 . 3 0 4 . 4 50 . 6 5

R T : 2 . 8 8M A : 1 0 4 2S N : 4 8 1

1 . 5 3

2 . 0 4

1 . 8 1

1 . 3 14 . 1 12 . 4 1 3 . 5 3

3 . 0 90 . 4 3 4 . 5 80 . 7 20 . 1 4 4 . 8 7

N L : 2 . 6 8 E 3

B a s e P e a k m / z = 1 5 1 . 5 0 - 1 5 2 . 5 0F : - c E S I s i d = 8 . 0 0 S R M m s 23 2 1 . 0 0 @ 1 2 . 0 0 [ 1 5 1 . 9 9 - 1 5 2 . 0 0 ,1 9 3 . 9 9 - 1 9 4 . 0 0 , 2 5 6 . 9 9 - 2 5 7 . 0 1 ]M S 1 2 2 1 D 0 7

N L : 4 . 8 1 E 2

B a s e P e a k m / z = 1 5 1 . 5 0 - 1 5 2 . 5 0F : - c E S I s i d = 8 . 0 0 S R M m s 2

3 2 1 . 0 1 @ 1 2 . 0 0 [ 1 5 1 . 9 9 - 1 5 2 . 0 0 ,1 9 3 . 9 9 - 1 9 4 . 0 0 , 2 5 6 . 9 9 - 2 5 7 . 0 1 ]M S 1 2 2 1 g 0 6

HH--SRMSRM

SRM

321152

321152

ASMS Poster 2005


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Pesticide Analysis in

Drinking Water


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EQuanThermos Solution Kit to LC-MS/MS Water Analysis


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EQuan

HTC

or Accela

Launched IUPAC, Japan Aug 06

Significant reduction in sample preparation timesOn-line sample enrichment

Lowers cost of analysis by ca. 1-2 days


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EQuan: On-line load and inject


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12

Advantage of EQuan: Save Time and Labour for Sample Prep

40 samples~ 5h

1 Sample preparation: 1L samplefiltration

internal Std.Filtration

~ 2d

6 Elution:

5 Drying:

4 Washing:

3 Extraction:

2 Condit ioning:

Enrichm

ent

MeOH / H2O

60mg Oasis

H2O/MeOH (95/5)

air

MeOH

~ 1d7 Detection:LC-MS

LC-MSMS

Total ~ 3 days


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13

Advantage of EQuan: Lower Detection Limit

T ria z in eP es tM ix _ 10 pp t_ 10 00 ul _ 04 3 /2 4 /2 005 1: 10 :0 1 A M

R T : 5 . 9 8 - 8 . 0 0

6 .0 6 .5 7 .0 7 .5 8 .0

T im e (m in )

0

5

1 0

1 5

2 0

2 5

3 0

3 5

4 0

4 5

5 0

5 5

6 0

6 5

7 0

7 5

8 0

8 5

9 0

9 5

1 0 0

Relative

Abundance

N L : 8 . 1 0 E 4

T IC F : + c E S I

si d =- 1 0 .0 0 S R M m s2

2 1 6 . 1 3 @ - 1 8 .0 0 [

1 7 4 . 0 0 ] M S

t ri a z i n e p e s tm i x _ 1 0 p p t _

1 0 u l _ 0 5

R T : 5 . 9 8 - 8 . 0 2

6 .0 6 .5 7 .0 7 .5 8 .0

T im e (m in )

0

5

1 0

1 5

2 0

2 5

3 0

3 5

4 0

4 5

5 0

5 5

6 0

6 5

7 0

7 5

8 0

8 5

9 0

9 5

1 0 0

Relative

Abundance

R T : 6 .9 1

A A : 3 1 9 9 9 9

S N : 1 1 6 R MS

N L : 8 . 1 0 E 4

T IC F : + c E S I si d =-1 0 .0 0

S R M m s 2 2 1 6 .1 3 @ - 1 8 .0 0

[ 1 7 4 . 0 0 ] M S I C I S

T ri a z in e P e stM ix _ 1 0 p p t_ 1

0 0 0 u l _ 0 4

10 ul injection 1000 ul injection

10 ppt Atrazine in ground water


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tria z in e p e s tm ix _ 1 0 p p t_ 1 0 u l_ 0 5 4 / 5 / 2 0 0 5 1 1 : 2 4 : 4 8 A M

R T : 6 . 5 0 - 8 . 5 0

6 . 5 7 . 0 7 . 5 8 . 0 8 . 5

T i m e (m i n )

0

5

1 0

1 5

2 0

2 5

3 0

3 5

4 0

4 5

5 0

5 5

6 0

6 5

7 0

7 5

8 0

8 5

9 0

9 5

1 0 0

Relative

Abundance

7 . 6 5

8 . 3 9

7 . 0 5

8 . 0 5

7 . 3 9

8 . 2 3

7 . 3 56 . 6 47 . 5 3

6 .6 8 6 .8 4

8 . 0 3

N L : 8 . 9 5 E 2

T I C F : + c E S I

s id = -1 0 . 0 0 S R M m s 2

2 3 0 . 1 5 @ - 1 7 . 0 0 [

1 8 8 . 0 5 ] M S

t ri a z i n e p e s t m i x _ 1 0 p p t _

1 0 u l _ 0 5

R T : 6 . 4 8 - 8 . 4 8

6 . 5 7 . 0 7 . 5 8 . 0

T i m e (m i n )

0

5

1 0

1 5

2 0

2 5

3 0

3 5

4 0

4 5

5 0

5 5

6 0

6 5

7 0

7 5

8 0

8 5

9 0

9 5

1 0 0

Relative

Abundance

R T : 7 .6 1

A A : 4 2 1 4 9 2

S N : 4 8 4 R M S

N L : 1 . 1 5 E 5

T I C F : + c E S I si d =- 1 0 .0 0

S R M m s 2 2 3 0 .1 5 @ -1 7 .0 0

[ 1 8 8 . 0 5 ] M S I C I S

T ri a z i n e P e st M i x _ 1 0 p p t_ 1

0 0 0 u l _ 0 4

10 ul injection 1000 ul injection

Advantage of EQuan: Lower Detection Limit

10 ppt Propazine in ground water


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Linearity

ProphamY =518.898*X R 2 =0.9998 W: Equal

0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500

pg/mL

0

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

2000000

2200000

2400000

2600000

2800000

Area

DiuronY =9883.64*X R 2 =0.9999 W: Equal

0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500

pg/mL

0

5000000

10000000

15000000

20000000

25000000

30000000

35000000

40000000

45000000

50000000

Area

Diuron, 1-5000fg/uL,1mL Injection

Propham, 5-5000 fg/uL,1mL Injection

R2=0.9998

R2=0.9999

i i C i l Q


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Triazine summary: Conventional vs. EQuan

LOQ < 10 PPT

Component Name

Parent Ion

m/z

Product Ion

m/z Conventional EQuan

Prometon 226.17 184.00 No Yes

Simazine 202.09 132.00 No Yes

Ametryn 228.12 186.00 No Yes

Prometryn 242.16 200.00 No Yes

Atrazine 216.10 174.00 No Yes

Propazine 230.12 188.00 No Yes

Methyl Parathion 262.03 138.00 No Yes

Thidiazuron 221.04 102.00 No Yes

Diuron 233.01 72.00 No Yes

Imidacloprid 256.04 209.00 No Yes

i id l i i i k i h d


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Pesticide Analysis in Drink Water with EQuan and QED-MS/MS

TricyclazoleCarbaryl

Carbofuran

AsulamDiuron

Siduron

Daimuron

CarpropamideThiodicarb

Azoxystrobin

FlazasulfuronBensulfuron-methyl

Halosulfuron-methyl

Ch t h C diti f EQ


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Chromatography Conditions for EQuan

Gradient Program for Loading Pump Gradient Program for Analysis PumpA: Water with 0.1% v/v Formic Acid B: Acetonitrile with 0.1% v/v Formic Acid

Loading Column:

Hypersil Gold 20x2.1 mm 12 m

Analytical Column:Hypersil Gold 50x2.1 mm 3 m


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Ion source: ESI +

Spray voltage: 4500 V

Sheath gas pressure: 45

Auxiliary gas pressure: 0

Ion transfer capillary

temperature:

330 oC

Source CID: 10 V

Scan Type: Event 1. SRM

Event 2. QED-MS/MS with RER

Q1 peak width 0.7 Da


Collision gas pressure: Ar at 1.0 mTorr

TSQ Quantum Discovery Max Conditions

Scan Event 1 SRM


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Scan Event 1: SRM

Scan Event 2: QED with RER


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Scan Event 2: QED with RER

Threshold of 5E4

San Time of 0.174 s CE = 10 V, Energy Ramp = 45 V, i.e. CE ramped from 55 V to 10 V

Chromatograms: 10 ppteach Pesticides in Drinking Water


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Chromatograms: 10 ppt each Pesticides in Drinking Water

Chromatograms: 10 ppteach Pesticide in Drinking Water


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Chromatograms: 10 ppt each Pesticide in Drinking Water

Calibration from 1 ppt


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Calibration from 1 ppt

QED-MS/MS of 5 ppt Carbofuran from EQuan


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QED-MS/MS of 5 ppt Carbofuran from EQuan

QED MS/MS Spectra for Library Search for Confirmation


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QED MS/MS Spectra for Library Search for Confirmation

Food SafetyEnvironmental


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LC-MS/MS of Nitrofuran

Metabolites in TurbotFish

Incident of Nitrofuran Contamination of Turbot


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Fish in Oct 2006 in China

Turbot fish, Scophthalmusmaximus (Linnaeus),

Southern Flounder

Sample Ppreparation


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Sample Ppreparation

2.0 g of sample with IS in 25 ml 0.125 N HClHomogenization + NBA50 mM addit ion (250 l)

Overnight incubation at 37 oC (16 hrs)

pH =7.2 (w/ NaOH & K2HPO4)

Centrifuge

Collect supernatant LLE (with 7 ml ethyl acetate

Collect and evaporate ethyl acetate under N2at 50 oC

Reconsti tute in 1.0 ml Water:MeOH (95 : 5)

Centrifuge and f ilter (0.45 um)

LC/MS/MS Analysis on TSQ Quantum Access

LC/MS/MS Conditions: Chromatographic Parameters


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LC/MS/MS Conditions: Chromatographic Parameters

Column:Thermo Hypersil Gold (1002.1 mm, 5)

Mobile Phase: A: 0.5 mM NH4Ac in Water; B: MeOH

Gradient:

Injection: 20 L (with loop)

Time (min) % A % B Flow Rate(l/min)

0 80 20

80

80

2020

3 20

250

250

250

250

5 20

5.1 807 80 250

LC/MS/MS Conditions QuantumAccess


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Ion source: ESI +

Spray voltage: 4800 V

Sheath gas pressure: 30

Auxiliary gas pressure: 6

Ion transfer capillary

temperature:

350 oC

Source CID: 10 V

Scan Type: Event 1. SRM

Event 2. QED-MS/MS with RER



Collision gas pressure: Ar at 1.5 mTorr

LC/MS/MS Conditions Quantum Access

SRM Parameters for 4 Nitrofuran Metabolite Derivatives


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* Quantitation Ion; Scan Width = 0.002 m/Z and Scan Time (Dwell Time) = 0.05 s

Nitrofuran Parent ion (m/z)Product ion

(m/z)CE (V)

236.04 104 19

236.04 134* 22

NBAOZ-D4(IS) 240.04 134* 14

249.04 104 22

249.04 134* 14

NBAHD-13C3(IS) 252.04 134* 14

209.00 166* 11

209.00 192 13

NBSEM-13C-15N2(IS) 212.05 168* 11

335.09 262 19

335.09 291* 12

NBAMOZ-D5(IS) 340.13 296 12

NBAMOZ

NBSEM

NBAHD

NBAOZ

SRM traces for nitrofuran residues in turbot fish sample


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RT: 0.00 - 7.01

0 1 2 3 4 5 6 7

Time (min)

0

50

100

0

50

100

RelativeA

bundance 0

50

1003.48

3.562.97 4.331.80 2.06 5.574.48 6.450.900.07

3.48

2.952.54 4.222.101.82 5.07 5.990.38 6.31

3.48

2.93 3.72 4.292.211.930.14 5.25 5.64 6.691.17

RT: 0.00 - 7.01

0 1 2 3 4 5 6 7

Time (min)

0

50

100

0

50

100

RelativeA

bundance 0

50

1003.35

3.572.702.04 4.181.78 5.674.70 6.980.05 1.12 6.39

3.35

4.333.591.87 2.70 4.401.120.34 5.86 6.93

3.33

1.91

3.592.66 4.34 5.06 5.43 6.21 6.631.500.890.14

RT: 0.00 - 7.01

0 1 2 3 4 5 6 7

Time (min)

0

50

100

0

50

100

Relative

Abundance 0

50

1003.31

3.472.72 3.790.980.45 2.16 5.654.82 6.391.54

3.31

3.402.591.78 4.21 6.544.861.09 5.650.12

3.32

2.972.60 3.47 4.171.901.37 5.965.04 6.980.35

RT: 0.00 - 7.01

0 1 2 3 4 5 6 7

Time (min)

0

50

100

0

50

100

Relative

Abundance 0

50

1002.51

1.90 3.06 3.58 4.28 4.74 5.37 5.761.40 6.510.65

2.51

1.90 2.77 3.69 4.13 5.30 5.615.02 6.721.550.50

2.47

1.86 2.84 4.113.26 5.20 6.474.701.380.83 5.57

p

NBAHD

NBAOZ

NBAMOZ

NBSEM

209166

209192

212168

236104

236134

240134

249104

249134

252134

335262

335291

340296

Calibration curve(0.05 to 10 ng/mL std soln)


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( g )

Excellent linearity for All Four Nitrofuran Metabolites!

NBSEM

Y = 0.0563971+0.114772*X

R 2 = 0.9996 W: 1/X

0 2 4 6 8 10ng/mL

0.0

0.2

0.4

0.6

0.8

1.0

1.2

A

reaRatio

NBAHD

Y = 0.00896535+0.214755*X

R 2 = 0.9996 W: 1/X

0 2 4 6 8 10

ng/mL

0.0

0.5

1.0

1.5

2.0

AreaRatio

NBAMOZ

Y = 0.0285094+0.302227*X

R 2 = 0.9997 W: 1/X

0 2 4 6 8 10

ng/mL

0.0

0.5

1.0

1.5

2.0

2.5

3.0

AreaRatio

NBAOZ

Y = 0.0886324+0.29248*X

R 2 = 0.9998 W: 1/X

0 2 4 6 8 10ng/mL

0.0

0.5

1.0

1.5

2.0

2.5

3.0

AreaRatio

Nitrofuran Residues Spiked in Turbot Fish: 0.25 g/kg


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p g g

0 2 4 6 8 10

Time (min)

05000

15000

25000

0

10000

20000

30000

0

10000

20000

300004.59

4.59

4.59

NBSEM

m/z 209166

NBSEMm/z 209192

NBSCA-13C-15N2m/z 212168

0 2 4 6 8 10

Time (min)

0

5000

10000

0

10000

20000

0

4000

80004.27

4.27

4.25

NBAOZ

m/z 236104

NBAOZm/z 236134

NBAOZ-D4m/z 240134

0 2 4 6 8 10Time (min)

0

2000

6000

0

5000

10000

0

2000

60004.09

4.09

4.09

NBAHDm/z 249166

NBAHDm/z 249192

NBAHD-13C3m/z 252134

0 2 4 6 8 10Time (min)

020000

60000

100000

050000

150000

250000

020000

60000

100000 5.33

5.33

5.29

NBAMOZm/z 335262

NBAMOZm/z 335291

NBAMOZ-D5m/z 340296

QED-MS/MS at 1 ppb Full scan confirmation


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pp

50 100 150 200 250 300

m/z

0

20

40

60

80

100

0

20

40

60

80

100

0

20

40

60

80

100

RelativeAbundance

0

20

40

60

80

100208.81

209.45

192.36

104.07 166.59149.1692.45

39.60 76.44 132.92 213.19

104.26

133.85

78.41 236.0876.30 123.87 146.8992.3751.59 158.10 203.89 239.58

78.66

249.37

104.13

134.23

171.19

130.87 150.08 230.4199.69 205.1952.32 252.63

100.43127.92

261.9286.14

114.08 128.64 290.4678.5070.42 335.19159.13 262.48178.19 291.59233.59

NL: 1.74E4

std-1ppb_03#3514 RT: 3.63AV: 1 F: +c ESI sid=-10.00 dFull ms2 [email protected][30.000-214.000]

NL: 8.53E4

std-1ppb_03#3430-3469 RT:3.48-3.52 AV: 3 F: +c ESIsid=-10.00 d Full [email protected][30.000-241.000]

NL: 1.50E4

std-1ppb_03#3442 RT: 3.46AV: 1 F: +c ESI sid=-10.00 dFull ms2 [email protected][30.000-254.000]

NL: 1.24E5

std-1ppb_03#3014-3036 RT:2.85-2.89 AV: 3 F: +c ESIsid=-10.00 d Full [email protected][30.000-340.000]

SRM MS/MS

NBSEM

NBAOZ

NBAHD

NBAMOZ

RT: 0.00 - 7.01 SM: 7G

0 1 2 3 4 5 6

Time (min)

0

50

100

0

50

100

0

50

100

0

50

100

RT: 3.48MA: 715889

2.95 3.78 4.332.562.04 5.07 5.990.38 6.451.10

RT: 3.35MA: 479007

3.57 4.332.702.04 5.121.120.34 5.86 6.93

RT: 3.31

AA: 317931

RT: 2.51AA: 12124353

Library search results for positivesample


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y p p

Library search results for positivesample


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Summery of QED-MS/MS Benefits


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Allows for simultaneous Quan/Qual data to be collected

Provides structural confirmation

Powerful tool for multi-residue screening experiments

Can be used for library searching

Helps to eliminate false positives

Acknowledgment


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Fei Liu, Thermo Scientific China

Seafood Research Institute of Yellow Sea, Shanghai, China

J onathan Beck, Thermo Scientific, San J ose, USA

Michiko Yamaguchi, Kaori Saito, Thermo Scientific J apan

Documents

Simultaneous Quantification and Qualification With QED-MSMS Nitrofurains