The world leader in serving science

Simultaneous Quantification and

Qualification with QED MS/MS
and Its Application in Pesticide
and Food Residue Analysis

Kefei Wang, Ph.D.

Environmental and Food Safety Marketing
San Jose, CA
April 23, 2007
42nd WCTOW, Calgary

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

What is QED MS/MS?

Quantitation-Enhanced Data-Dependant MS/MS Scan
SRM triggered data dependent MS/MS scan
SRM

QED MS/MS
RT:10.0

100

Intensity

90

Relative Abundance

80
70
60
50
40
30
20

m/z

10
0
9.8

10.0

10.2
min

10.4

10.6

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

QED MS/MS with Reverse Energy Ramp (RER)

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

CE

Inv
ers
Ene e Co
rgy llisio
Ram n
p

M/Z

QED MS/MS Advantages on TSQ Quantum

9 Faster than a Q-trap

Does not require Dynamic fill time that 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

Example of H-SRM: Chloramphenicol at various Q1

100

Q1= 0.7

NL: 4.18E4

321.01

100%

322.92
50
318.83

0
100

321.93

320.10

324.11

325.03

325.94

321.05

NL: 3.88E4

Q1= 0.5

322.90

93%

50
319.00

Relative Abundance

0
100

322.00

320.00

324.00

324.90

325.85

320.99

NL: 3.61E4

Q1= 0.4

322.89

86%

50

0
100

319.01

322.08

319.97

323.91

324.93

325.98
NL: 3.42E4

320.93

Q1= 0.2

82%

322.95
50
0
100

318.96

321.95

319.95

323.93

324.98

325.94
NL: 3.11E4

321.00

Q1= 0.1

323.00

74%

50
319.00

0
318

319

322.02

320.11
320

321

322
m/z

323

324.01

324.99

325.98

324

325

326

327

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

R T:

0 .0 0

- 5 .0 0

1 0 0

2 .0 8

321152

9 0
8 0

.6 8 E 3
P e a k m /z = 1 5 1 .5 0 -1 5 2 .5 0
E S I s id = 8 . 0 0
S R M m s 2
0 @ 1 2 .0 0 [ 1 5 1 .9 9 -1 5 2 .0 0 ,
9 -1 9 4 .0 0 , 2 5 6 .9 9 -2 5 7 .0 1 ]
2 2 1 D 0 7

N L : 4
B a s e
F : - c
3 2 1 .0
1 9 3 .9
M S 1

.8 1 E 2
P e a k m /z = 1 5 1 .5 0 -1 5 2 .5 0
E S I s id = 8 . 0 0
S R M m s 2
1 @ 1 2 .0 0 [ 1 5 1 .9 9 -1 5 2 .0 0 ,
9 -1 9 4 .0 0 , 2 5 6 .9 9 -2 5 7 .0 1 ]
2 2 1 g 0 6

SRM

1 .5 4

7 0
Relative Abundance

N L : 2
B a s e
F : - c
3 2 1 .0
1 9 3 .9
M S 1

6 0
2 .8 1
5 0
2 .2 6
4 0
3 0
2 0
3 .4 5

1 0

3 .7 5
0 .3 0

0 .6 5

1 .0 5

4 .0 9

4 .4 5

4 .7 4

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

1 0 0

321152

9 0

H-SRM

8 0

Relative Abundance

7 0
6 0
5 0
1 .5 3
4 0
3 0
2 .0 4
2 0

1 .8 1

1 0
0

1 .3 1
0 .1 4
0 .0

0 .4 3
0 .5

2 .4 1

0 .7 2
1 .0

1 .5

2 .0

2 .5
T im e (m in )

3 .0 9
3 .0

ASMS Poster 2005

7

3 .5 3
3 .5

4 .1 1
4 .0

4 .5 8
4 .5

4 .8 7

The world leader in serving science

Pesticide Analysis in
Drinking Water

EQuan
Thermos Solution Kit to LC-MS/MS Water Analysis

EQuan
Launched IUPAC, Japan Aug 06
HTC

or Accela

Significant reduction in sample preparation times

On-line sample enrichment

Lowers cost of analysis by ca. 1-2 days

10

EQuan:

On-line load and inject

11

LC-MS

Enrichment

Filtration

Advantage of EQuan: Save Time and Labour for Sample Prep

1 Sample preparation:

1L sample
filtration
internal Std.

2 Conditioning:

MeOH / H2O

3 Extraction:

60mg Oasis

4 Washing:

H2O/MeOH (95/5)

5 Drying:

air

6 Elution:

MeOH

7 Detection:

40 samples
~ 5h

~ 2d
Total ~ 3 days

LC-MSMS
12

~ 1d

Advantage of EQuan: Lower Detection Limit

T ria z in e P e s t M ix _ 1 0 p p t _ 1 0 0 0 u l_ 0 4

3/24/20 05 1:10:01 A M
R T : 5 .9 8 - 8 .0 2

R T : 5 .9 8 - 8 .0 0
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 r i a z i n e p e st m i x _ 1 0 p p t _
1 0 u l_ 0 5

100

95

90

N L : 8 .1 0 E 4
T I C 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 IC IS
T r i a z i n e P e st M i x _ 1 0 p p t _ 1
0 0 0 u l_ 0 4

R T : 6 .9 1
AA: 319999
S N : 1 1 6 R MS

100

95

90
85
85
80
80
75
75
70
70
65

10 ul injection

60

1000 ul injection

65

55

Relative Abundance

Relative Abundance

60

50

45

55

50

45

40
40
35

35

30

30

25

25

20

20

15

15

10

10

0
6 .0

6 .5

7 .0

7 .5

8 .0

6 .0

T i m e (m i n )

6 .5

7 .0

7 .5

8 .0

T i m e (m i n )

10 ppt Atrazine in ground water

13

Advantage of EQuan: Lower Detection Limit

t ria z in e p e s t m 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 .4 8 - 8 .4 8

R T : 6 .5 0 - 8 .5 0
N L : 8 .9 5 E 2

7 .6 5

1 0 0

8 .3 9

9 5
7 .0 5

9 0

T IC F : + c E S I
si d = -1 0 . 0 0 S R M m s2
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 st m i x _ 1 0 p p t _
1 0 u l_ 0 5

N L : 1 .1 5 E 5

R T : 7 .6 1
A A : 4 2 1 4 9 2
S N : 4 8 4 R MS

1 0 0

T
S
[
T
0

9 5

8 .0 5

IC F : + c
R M m s2
1 8 8 .0 5 ]
ri a z i n e P
0 0 u l_ 0 4

9 0

8 5
8 5
8 0
8 0
7 5
7 5
7 0

7 .3 9
7 0

6 5
6 5
6 0

Relative Abundance

Relative Abundance

6 0
5 5
8 .2 3

5 0

4 5
7 .3 5

6 .6 4

5 5

5 0

4 5

7 .5 3

4 0

4 0
3 5
6 .6 8

3 5

6 .8 4

3 0

3 0

8 .0 3
2 5

2 5

2 0

2 0

1 5

1 5

1 0

1 0

0
6 .5

7 .0

7 .5

8 .0

8 .5

6 .5

T i m e (m i n )

7 .0

7 .5

8 .0

T i m e (m i n )

1000 ul injection

10 ul injection

10 ppt Propazine in ground water

14

E S I si d = -1 0 . 0 0
2 3 0 . 1 5 @ -1 7 . 0 0
M S IC IS
e st M i x _ 1 0 p p t _ 1

Linearity
Propham
Y = 518.898*X R^2 = 0.9998 W: Equal
2800000
2600000

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

R2=0.9998

2400000
2200000
2000000
1800000

Area

1600000
1400000
1200000

Diuron
Y = 9883.64*X R^2 = 0.9999 W: Equal

1000000
800000

50000000

R2=0.9999

600000

45000000

400000
200000

40000000

0
0

500

1000

1500

2000

2500
3000
pg/mL

3500

4000

4500

5000

5500

35000000

Area

30000000

25000000

Propham, 5-5000 fg/uL,

1mL Injection

20000000

15000000

10000000

5000000

0
0

15

500

1000

1500

2000

2500
3000
pg/mL

3500

4000

4500

5000

5500

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

16

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

Tricyclazole
Carbaryl
Carbofuran
Asulam
Diuron
Siduron
Daimuron

Carpropamide
Thiodicarb
Azoxystrobin
Flazasulfuron
Bensulfuron-methyl
Halosulfuron-methyl

17

Chromatography Conditions for EQuan

Gradient Program for Analysis Pump

Gradient Program for Loading Pump

A: Water with 0.1% v/v Formic Acid B: Acetonitrile with 0.1% v/v Formic Acid

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

Loading Column:
Hypersil Gold 20x2.1 mm 12 m
18

TSQ Quantum Discovery Max Conditions

Ion source:

ESI +

Spray voltage:

4500 V

Sheath gas pressure:

45

Auxiliary gas pressure:

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

Q3 peak width

0.7 Da

Collision gas pressure:

Ar at 1.0 mTorr

19

Scan Event 1: SRM

20

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

21

Chromatograms: 10 ppt each Pesticides in Drinking Water

22

Chromatograms: 10 ppt each Pesticide in Drinking Water

23

Calibration from 1 ppt

24

QED-MS/MS of 5 ppt Carbofuran from EQuan

25

QED MS/MS Spectra for Library Search for Confirmation

26

Environmental

The world leader in serving science

Food Safety

LC-MS/MS of Nitrofuran
Metabolites in Turbot
Fish

Incident of Nitrofuran Contamination of Turbot

Fish in Oct 2006 in China
Turbot fish, Scophthalmus
maximus (Linnaeus),
Southern Flounder

28

Sample Ppreparation
2.0 g of sample with IS in 25 ml 0.125 N HCl
Homogenization + NBA50 mM addition (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 N2
at 50 oC
Reconstitute in 1.0 ml Water:MeOH (95 : 5)
Centrifuge and filter (0.45 um)
LC/MS/MS Analysis on TSQ Quantum Access

29

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:
Time (min)

%A

%B

Flow Rate
(l/min)

80

20

250

20

80

250

20

80

250

5.1

80

20

250

80

20

250

Injection: 20 L (with loop)

30

LC/MS/MS Conditions Quantum Access

Ion source:

ESI +

Spray voltage:

4800 V

Sheath gas pressure:

30

Auxiliary gas pressure:

Ion transfer capillary

temperature:

350 oC

Source CID:

10 V

Scan Type:

Event 1. SRM
Event 2. QED-MS/MS with RER

Q1 peak width

0.7 Da

Q3 peak width

0.7 Da

Collision gas pressure:

Ar at 1.5 mTorr

31

SRM Parameters for 4 Nitrofuran Metabolite Derivatives

Nitrofuran
NBAOZ
NBAOZ-D4 (IS)
NBAHD
NBAHD-13C3 (IS)
NBSEM
NBSEM-13C-15N2 (IS)
NBAMOZ
NBAMOZ-D5 (IS)

Parent ion (m/z)

Product ion
(m/z)

CE (V)

236.04

104

19

236.04

134*

22

240.04

134*

14

249.04

104

22

249.04

134*

14

252.04

134*

14

209.00

166*

11

209.00

192

13

212.05

168*

11

335.09

262

19

335.09

291*

12

340.13

296

12

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

32

SRM traces for nitrofuran residues in turbot fish sample

NBSEM

NBAOZ

RT: 0.00 - 7.01

RT: 0.00 - 7.01

3.48

100

209166
0.07

1.80 2.06

0.90

100

3.56 4.33
4.48
3.48

5.57

6.45

209192

50

2.95
1.82 2.10 2.54

0.38

0
100

4.22

5.07

5.99 6.31

3.48

0.14

0
0

1.17 1.93 2.21

1

0.05

3.72 4.29

3
4
Time (min)

5.25 5.64
5

0.34

0
100

1.12

1.87

1.91
0.14 0.89
0

3.31

3.47 3.79

2.16 2.72

4.82

5.65

6.39

3.31

100

249134
1.09

1.78

3.40 4.21
3.32

2.59

6.21 6.63
6

335262

4.86

5.65

6.54

1.40 1.90

0.65

3.06 3.58

3.47 4.17

3
4
Time (min)

5.04

5.96

100

1.55 1.90

0.50

0
100

2.77

3.69 4.13 5.02 5.30 5.61

340296
0.83 1.38 1.86
0

33

6.72

2.47

5.37 5.76 6.51

335291

50

6.98

4.28 4.74

2.51

50

1.37 1.90 2.60 2.97

0.35

5.06 5.43

2.51

252134

50

3
4
Time (min)

50
Relative Abundance

Relative Abundance

0.45 0.98 1.54

0
100

3.59 4.34

2.66

1.50

100

249104

0.12

6.93

RT: 0.00 - 7.01

100

50

5.86

NBAMOZ

RT: 0.00 - 7.01

4.33 4.40

240134

NBAHD

50

3.59

2.70

3.33

0
7

5.67 6.39 6.98

236134

50

6.69

3.57 4.18 4.70

2.70
3.35

50
2.93

1.12 1.78 2.04

100

212168

50

236104

50
2.97

Relative Abundance

Relative Abundance

50

3.35

100

2.84 3.26

4.11

3
4
Time (min)

4.70 5.20 5.57

5

6.47
6

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

Y = 0.0563971+0.114772*X
R^2 = 0.9996 W: 1/X

1.2

0.8
0.6
0.4

2.0
1.5
1.0

0.2

0.5

0.0

0.0
0

6
8
ng/mL
Y = 0.00896535+0.214755*X
R^2 = 0.9996 W: 1/X

10

6
ng/mL

10

Y = 0.0285094+0.302227*X
R^2 = 0.9997 W: 1/X
3.0

2.0

NBAHD

NBAMOZ

2.5
Area Ratio

Area Ratio

NBAOZ

2.5
Area Ratio

Area Ratio

3.0

NBSEM

1.0

Y = 0.0886324+0.29248*X
R^2 = 0.9998 W: 1/X

1.5
1.0

2.0
1.5
1.0

0.5
0.5
0.0

0.0
0

6
ng/mL

10

6
ng/mL

10

Excellent linearity for All Four Nitrofuran Metabolites!

34

Nitrofuran Residues Spiked in Turbot Fish: 0.25 g/kg

4.27

4.59

NBSEM
m/z 209166

30000
20000

NBAOZ
m/z 236104

8000
4000

10000

4.59

NBSEM
m/z 209192

30000
20000
10000

4.27

20000

NBAOZ
m/z 236134

10000
0

4.59

25000

10000

NBSCA-13C-15N2
m/z 212168

15000

4.25

NBAOZ-D4
m/z 240134

5000

5000

10

Time (min)
NBAHD
m/z 249166

4.09

NBAHD
m/z 249192

5000

NBAHD-13C3
m/z 252134

2000
0

5.33

NBAMOZ
m/z 335291

150000

5.29

NBAMOZ-D5
m/z 340296

60000
20000
0
10

Time (min)

Time (min)
35

10

NBAMOZ
m/z 335262

60000

50000
0
100000

4.09

(min)

250000

10000

0
6000

5.33
Time

20000
0

2000
0

100000

4.09
6000

10

QED-MS/MS at 1 ppb Full scan confirmation

MS/MS

SRM

RT: 0.00 - 7.01 SM: 7G

RT: 3.48
MA: 715889

100

209.45

80

NBSEM

50

192.36

60
40
20

1.10

3.78 4.33 5.07

5.99 6.45

RT: 3.35
MA: 479007

100

NBAOZ

50
0.34

1.12

2.04

3.57 4.33

2.70

5.12

5.86

6.93

Relative Abundance

0.38

2.04 2.56 2.95

39.60 76.44

104.07
92.45

149.16 166.59
132.92

100

NL: 8.53E4
std-1ppb_03#3430-3469 RT:
3.48-3.52 AV: 3 F: + c ESI
sid=-10.00 d Full ms2
236.000@cid13.26
[30.000-241.000]

80
60
133.85
78.41
20
76.30
123.87 146.89
92.37
51.59
158.10 203.89
0
40

236.08
239.58
NL: 1.50E4
std-1ppb_03#3442 RT: 3.46
AV: 1 F: + c ESI sid=-10.00 d
Full ms2 249.000@cid13.72
[30.000-254.000]

78.66
249.37

80

NBAHD

50

104.13

60

134.23

40
171.19

20
0

0
RT: 2.51
AA: 12124353

100

213.19

104.26

100

RT: 3.31
AA: 317931

100

NL: 1.74E4
std-1ppb_03#3514 RT: 3.63
AV: 1 F: + c ESI sid=-10.00 d
Full ms2 209.000@cid12.32
[30.000-214.000]

208.81

100

52.32

130.87 150.08

99.69

205.19 230.41

252.63
NL: 1.24E5
std-1ppb_03#3014-3036 RT:
2.85-2.89 AV: 3 F: + c ESI
sid=-10.00 d Full ms2
335.000@cid16.73
[30.000-340.000]

100.43

100

127.92

80
86.14

261.92

60

NBAMOZ

50

40
20

70.42 78.50

114.08 128.64
159.13 178.19

233.59

290.46
262.48 291.59

3
4
Time (min)

50

100

150

200
m/z

36

250

300

335.19

Library search results for positive sample

37

Library search results for positive sample

38

Summery of QED-MS/MS Benefits

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

39

Acknowledgment
Fei Liu, Thermo Scientific China
Seafood Research Institute of Yellow Sea, Shanghai, China
Jonathan Beck, Thermo Scientific, San Jose, USA
Michiko Yamaguchi, Kaori Saito, Thermo Scientific Japan

40