application of hr-lc-ms in screening methods for pesticides in cereals anne kruse lykkeberg, mette...

Application of HR-LC-MS in screening methods for pesticides in cereals

Anne Kruse Lykkeberg, Mette Erecius PoulsenAlmeria, 12 June 2014

National Food Institute, Technical University of Denmark

Disposition

Analysis at the instrument

Standard mixtures

Validation plan

Validation

Virtual computers – for data treatment

Sample preparation of cereals – QuEChERS


LC-QTOF instruments


Disposition


Standard mixtures

Validation plan

Validation




PCD – CSV file


No selection

QTOF-analysis – MS scan

No collision

• Used to determine retention time


PCDL - library


PCDL – library - spectra


Selection of single mass

QTOF-analysis – Targeted MS/MS

Collision

10, 20 and 40 V

• Obtaining compound spectra for Library• Spectra of daughter ions from the single

ion isolated in the quadropole


No selection

QTOF-analysis – All ion MS

Collision

10, 20 and 40 V

• MS scan with collision energy• Screening samples• Spectra of daughter ions of all compounds

eluting on a certain time point


Chromatogram - TIC


Spectra – MSscan incl. collision

0V

10V

20V

40V

Ofurace, [M+H]+


Disposition


Standard mixtures

Validation plan

Validation




Standard mixtures

50-100 pesticidesMixtures

Avoiding isomers in the mixtures

Isomers

2-6 mixtures togetherValidation


Spectra of isomers in Agilents libraryLibrary spectrum

m/z

30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185 190 195 200 205 210 215 220 225 230 235 240 245 250

Ab

un

dan

ce

0

10

20

30

40

50

60

70

80

90

100

110

142.07234100.00 184.1192996.84

226.1662467.74

86.0348915.20100.0505412.10

68.024324.7957.044724.62 152.118231.62

Library spectrum

m/z

30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185 190 195 200 205 210 215 220 225 230 235 240 245 250

Ab

un

dan

ce

0

10

20

30

40

50

60

70

80

90

100

110

170.10364100.00

226.166246.72114.066185.83 142.072345.31100.050543.8275.055293.1185.076032.24 128.081832.1057.069881.58

Library spectrum

m/z

30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185 190 195 200 205 210 215 220 225 230 235 240 245 250

Ab

un

dan

ce

0

10

20

30

40

50

60

70

80

90

100

110

170.10364100.00

226.1662441.95

100.0505410.48114.066189.97 142.072349.5875.055294.9686.034894.38 128.081834.1257.069882.73

N

N

N

NHNH CH3

CH3

CH3

CH3

OCH3

N

N

N

NHNH CH3

CH3

CH3

OCH3

CH3

N

N

N

NHNH

CH3

CH3

OCH3

CH3

Prometon

Terbumeton

Secbumeton


Disposition


Standard mixtures

Validation plan

Validation




Validation• My alternative validation plan• 20-25 samples – spiked at 0.1 mg/kg• 10-20 blank samples

Spiked – 0.1 mg/kg

Blank

Rice 5 5Oat 5 5Barley 5 5Wheat 5 5Rye 5 5


Dilution of spiked samples

Spiked rye is diluted with Blank rye …

Matrix is the sameRecovery is assumed to be the same at the different concentration levels

Rye 0.1 mg/kg Blank

0.10 mg/kg 1000 µl -

0.02 mg/kg 200 µl 800 µl

0.01 mg/kg 100 µl 900 µl

Blank - 1000 µl


Disposition


Standard mixtures

Validation plan

Validation




Compound list – exported to Excel


All file gathered in one Excel-file


Area of all samples spiked at 10 ppb


Comparison of areas in spiked samples with blank


Disposition


Standard mixtures

Validation plan

Validation




Virtual computers for data-analysisChallenges• The Agilent software demands Windows Professional 64 bit

– english version• Back up of all laboratory computer on LAB-domain• Laboratory domain, with no access from personal

computers

Solution• Virtual computers from Citrix

– On the Laboratory domain– Windows Professional 64 bit– Access to all data – back up early in the morning– Few licenses are necessary– Access from all computers with internet


Disposition


Standard mixtures

Validation plan

Validation




FVII cocktail IV – project• Aim is to develop a cereal-method to analyze many

analytes in one method.

• Analytes:– Pesticides (The pesticides most present in cereal – in practice

the standard mixtures which we use for our quantitative methods).

– Mycotoxins: Most important mycotoxins in cereals.


Method development - QuEChERS

• Water• AcetonitrileExtraction

• Magnesium sulphate• NaCl, Citrate buffer

Phase separation

• 1 h at -80 ºC• Centrifugation

Freeze out step

• PSA • Magnesium sulphatePSA clean up


0 2 4 6 8 10 12 14 16 18 200

200

400

600

800

1000

1200

1400

1600

Positive mode

Pos blank 1

Retention time

m/z

Extraction


0 2 4 6 8 10 12 14 16 18 200

200

400

600

800

1000

1200

Positive mode

Pos blank 1Pos blank 2

Retention time

m/z

Phase separation


0 2 4 6 8 10 12 14 16 18 200

200

400

600

800

1000

1200

Positive mode

Pos blank 1Pos blank 2Pos blank 3

Retention time

m/z

Freeze out step


0 2 4 6 8 10 12 14 16 18 200

200

400

600

800

1000

1200

1400

1600

Positive mode

Pos blank 1Pos blank 2Pos blank 3Pos blank 4

Retention time

m/z

PSA clean up


0 2 4 6 8 10 12 14 16 18 200

200

400

600

800

1000

1200

1400

1600

Positive mode

Pos blank 1Pos blank 2Pos blank 3Pos blank 4MycotoxinsPesticides

Retention time

m/z

Analytes


0 2 4 6 8 10 12 14 16 180

100

200

300

400

500

600

700

800

Negative mode

Neg blank 1

Retention time

m/z

Extraction


0 2 4 6 8 10 12 14 16 180

100

200

300

400

500

600

700

800

Negative mode

Neg blank 1Neg blank 2

Retention time

m/z

Phase separation


0 2 4 6 8 10 12 14 16 180

2

4

6

8

10

12

Negative mode

Neg blank 1Neg blank 2Neg blank 3

Retention time

m/z

Freeze out step


0 2 4 6 8 10 12 14 16 180

100

200

300

400

500

600

700

Negative mode

Neg blank 1Neg blank 2Neg blank 3Neg blank 4

Retention time

m/z

PSA clean up


0 2 4 6 8 10 12 14 16 180

100

200

300

400

500

600

700

800

Negative mode

Neg blank 1Neg blank 2Neg blank 3Neg blank 4MycotoxinsPesticides

Retention time

m/z

Analytes


TIC af blank i positiv mode

7x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

3

3.2

3.4

3.6

3.8

4

4.2

4.4

4.6

4.8

5

5.2

5.4+ESI TIC Scan pos_Blank4.d

2

Counts vs. Acquisition Time (min)1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5 17 17.5 18


TIC af blank i positiv mode

7x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8

3

3.2

3.4

3.6

3.8

4

4.2

4.4

4.6

4.8

5

5.2

5.4+ESI TIC Scan pos_Blank4.d

Counts vs. Acquisition Time (min)6.6 6.8 7 7.2 7.4 7.6 7.8 8 8.2 8.4 8.6 8.8 9 9.2 9.4 9.6 9.8 10 10.2 10.4 10.6 10.8 11 11.2 11.4 11.6 11.8 12 12.2 12.4 12.6 12.8


TIC af blank i negativ mode

7x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2

2.1

2.2

2.3

2.4

2.5

2.6-ESI TIC Scan neg_Blank4.d

1 1 2 2

Counts vs. Acquisition Time (min)0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5 17 17.5 18 18.5 19 19.5


TIC af blank i negativ mode

7x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2

2.1

2.2

2.3

2.4

2.5

2.6-ESI TIC Scan neg_Blank4.d

Counts vs. Acquisition Time (min)6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 7.8 8 8.2 8.4 8.6 8.8 9 9.2 9.4 9.6 9.8 10 10.2 10.4 10.6 10.8 11 11.2 11.4 11.6 11.8 12 12.2 12.4 12.6 12.8 13


Matrix effect & recovery – Mycophenol acid• Mycophenol acid• 6.63 min• Mass: 320.1260

Recovery Matrix effect

PSA 7 106

Freeze out 114 153

Phase separation 105 149

Extraction 123 159


Matrix effect & recovery – Fumonisin B2• Fumonisin B2• 5.07 min • Mass: 705.3936


PSA 6 111

Freeze out 36 151


Extraction 76 153


Matrix effect & recovery – Roquefortin C• Roquefortin C• 5.15 min • Mass: 389.1852


PSA 92 75

Freeze out 92 41


Extraction 153 31


Matrix effect & recovery – Clothianidin• Clothianidin• 4.46 min • Mass: 249.0087


PSA 89 83

Freeze out 95 61


Extraction 153 32

application of hr-lc-ms in screening methods for pesticides in cereals anne kruse lykkeberg, mette...

Documents

national food institute

validation slide

quadropole slide

h slide

retention time slide

certain time point slide

selection qtofanalysis

blank samples