lc/qqq, tof and q-tof for the analysis of pesticide

1

LC/QqQ, TOF and Q-TOF forthe Analysis of Pesticide

Residues in Food and WaterE. Michael Thurman1 and Imma Ferrer1

1Center for Environmental Mass Spectrometry (CEMS), University of

Colorado, Boulder, CO, USA

The 43rd Western Canadian Trace Organic Workshop, Saskatoon, April

27-30, 2008

OutlineOOutlineutline

1. Introduction: our research deals with environmental mass spectrometry and the identification of pesticides in food and water samples.

2. Examples of identification: pesticides in food(vegetable samples and olive oil...) and waterusing a variety of instruments:

- LC/TOF-MS- LC/QqQ-MS - LC/Q-TOF-MS

3. Highlight advantages and disadvantages forevery instrument.

2

Southern Spain (Almería):Vegetable Garden of Europe: Our Food Story

Southern Spain (AlmerSouthern Spain (Almeríía)a):Vegetable Garden :Vegetable Garden of Europe: Our Food Storyof Europe: Our Food Story

Greenhouses and Drip Irrigation

30,000 Hectares

Environmental BackgroundEnvironmental Background

• Occurrence of pesticides in food is an important environmental issue. Use of agrochemicals at various stages of cultivation has an important impact in food protection and quality preservation.

• European Food Regulations (91/414/EEC) require MRL’s of <0.01 mg/kg (or 10 ppb) for non regulated pesticides in food.

Available Instruments, which one do we use? Advantages:

- LC/TOF-MS: provides elemental formula composition, specificity and selectivity (high resolving power).

- LC/QqQ-MS: routine analysis for target compounds, high sensitivity.

- LC/Q-TOF-MS: fragmentation information, screening and non-target analysis.

3

TargetsTargets

Types of analytes identified by LC-MS

PesticidesPesticides wewe are are lookinglooking forfor......

NonNon--targetstargets

PesticidesPesticides oror metabolitesmetabolites thatthat may show may show up in up in thethe samplesample......

UnknownsUnknowns

SomethingSomething wewe are are notnot expectingexpecting......

Identification by LC-MSR

outin

ean

alys

esN

ovel

Ana

lyse

s

LC/TOF-MS or LC/QqQ-MS

LC/TOF-MS or LC/Q-TOF-MS

LC/Q-TOF-MS or LC/TOF-MS

LC Conditions:• Column

Zorbax Eclipse XDB C8, 4.6 x 150 mm, 5 µm particles • Mobile phase

Acetonitrile and water with 0.1 % formic acid• Gradient

10 % organic isocratic for 5 min, then to 100 % in 25 min.Flow = 0.6 mL/min

Analytical MethodologyAnalytical MethodologyAnalytical Methodology

MS Conditions:

• Dual sprayer providing constant low flow ofreference solution for continuous auto-calibration.

• Positive ESI+, Capillary 4000V, Fragmentor 190V.

Extraction procedure:• Ethyl acetate method• QuEChERS method (S.J. Lehotay, Journal of AOAC International, 2005)

4

LC/TOF-MS (TOFFY I)Almeria, Spain: Food

2004

LC/TOF-MS (TOFFY II)Boulder, CO, USA: Water

2008

1 GHz to 4 GHz DetectorAnalyst to Mass Hunter Software1100 to 1200 HPLCImprovements in accurate mass

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Time, min

0.0

1.0e6

2.0e6

3.0e6

4.0e6

5.0e6

6.0e6

7.0e6

8.0e6

9.0e6

1.0e7

1.1e7

1.2e7

1.3e7

1.4e7

1.5e7

1.6e7

1.7e7

1.8e7

1.9e73.5

20.9

2.1

16.4

2.3

12.2 20.619.315.7 17.6

15.414.4 17.9 26.913.4

5.7 24.022.511.53.2 9.4

4.4 24.227.725.6

24.921.9 23.2

LC/TOF-MS Multi-residue Analysis LC/TOFLC/TOF--MS MultiMS Multi--residue Analysis residue Analysis

Pepper matrix, spiking level: 0.05 mg/Kg

3.56.1 7.5

11.7

15.4

16.2

17.7 21.0

21.7

22.1

22.523.9

25.9

27.2

27.6

28.6

29.2

CY

RO

MA

ZIN

E

CA

RB

EN

DA

ZIM

TH

IAB

EN

DA

ZO

LE

ME

TH

OM

YL

IMID

AC

LO

PRID

AC

ET

AM

IPR

ID

TH

IAC

LO

PRID

SPIN

OSA

D A

SPIN

OSA

D D

DIM

ET

HO

MO

RPH

AZ

OX

YST

RO

BIN

TR

IFL

UM

IZO

L HEXAFLUMURON

LUFENURON

TEFLUBENZURON

FLU

FEN

OX

UR

ON

Inte

nsity

Time (min)

5

0.20.1167.1040167.1040[M + H]+C6H10N6Cyromazine

0.5

-0.3

0.1

-0.4

-0.3

0.2

-0.1

-0.6

-1.3

-0.1

-0.3

0.1

-0.1

-0.3

-0.1

mDaError

1.0489.0435489.0440[M + H]+C21H11N2O3F6ClFlufenoxuron

-0.6510.9857510.9854[M + H]+C17H8N2O3F8Cl2Lufenuron

0.2380.9815380.9816[M + H]+C14H6N2O2F4Cl2Teflubenzuron

-0.8460.9889460.9885[M + H]+C16H8N2O3F6Cl2Hexaflumuron

-1.0346.0928346.0925[M + H]+C15H15N3OF3ClTriflumizol

0.5404.1242404.1243[M + H]+C22H17N3O5Azoxystrobin

-0.1388.1310388.1310[M + H]+C21H22NO4ClDimethomorph

-0.8746.4838746.4832[M + H]+C42H67NO10Spinosyn D*

-1.8732.4681732.4668[M + H]+C41H65NO10Spinosyn A*

-0.5253.0309253.0308[M + H]+C10H9N4ClSThiacloprid

-1.3223.0745223.0742[M + H]+C10H11N4ClAcetamiprid

0.5256.0596256.0597[M + H]+C9H10N5O2ClImidacloprid

-0.1185.0355185.0355[M + Na]+C5H10N2O2SMethomyl

-1.7202.0433202.0430[M + H]+C10H7N3SThiabendazole

-0.3192.0767192.0767[M + H]+C9H9N3 O2Carbendazim

ppmm/z calculatedm/z experimentalSelected ionFormulaCompound

LC/TOF-MS Accurate Mass MeasurementsLC/TOFLC/TOF--MS Accurate Mass MeasurementsMS Accurate Mass Measurements

Max. 2.7e5 cps.

16.5 17.0 17.5 18.0 18.5 19.0 19.5 20.0 20.5 21.0 21.5 22.0 22.5 23.0 23.5 24.0 24.5 25.0 25.5 26.0 26.5Time, min

0.01.0e42.0e43.0e44.0e45.0e46.0e47.0e48.0e49.0e41.0e51.1e51.2e51.3e51.4e51.5e51.6e51.7e51.8e51.9e52.0e52.1e52.2e52.3e52.4e52.5e52.6e52.7e5

Intensity, cps

20.9

(a) mass window: 0.2 Dam/z 233.0-233.2

Olive Oil, 10 ppb diuronXIC m/z 233

diuron

Unknown m/z 233.1533

TIC of +TOF MS: from Sample 3 (Aceite MSPD 10 ppb) of Aceite MSPD calibration.wiff Max. 5.9e6 cps.

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Time, min

0.02.0e54.0e56.0e58.0e51.0e61.2e61.4e61.6e61.8e62.0e62.2e62.4e62.6e62.8e63.0e63.2e63.4e63.6e63.8e64.0e64.2e64.4e64.6e64.8e65.0e65.2e65.4e65.6e65.8e65.9e6

Intensity, cps

21.0

19.425.1 28.5

22.8 29.114.527.918.1 23.5 27.72.2 16.9 24.621.516.1 26.52.4 25.717.6

19.013.7 20.115.83.5 11.64.14.4 12.6

TIC

Max. 1.4e4 cps.

16.5 17.0 17.5 18.0 18.5 19.0 19.5 20.0 20.5 21.0 21.5 22.0 22.5 23.0 23.5 24.0 24.5 25.0 25.5 26.0 26.5Time, min

0.0500.0

1000.01500.02000.02500.03000.03500.04000.04500.05000.05500.06000.06500.07000.07500.08000.08500.09000.09500.0

1.0e41.1e41.1e41.2e41.2e41.3e41.3e41.4e41.4e41.4e4

Intensity, cps

21.3

(b) mass window: 0.05 Dam/z 233.00-233.05

diuron

Exp. mass m/z 233.0245

SelectivitySelectivity by LC/TOFby LC/TOF--MSMS

Diuron

Cl

NH

N

ClO

Theoretical mass: 233.0243

6

Example: Unknowns in Cucumbers

2 14 18 22 26 30Time, min

1.00e6

3.00e6

5.00e6

7.00e6

9.00e6

1.10e7

Intensity, cps

2.1

2.4

0.73.5

22.5

27.818.6 30.528.627.526.021.1 24.9

4.0

6 10

100 140 180 220 260 300 340 380m/z, amu

2.00e4

4.00e4

6.00e4

8.00e4

1.00e5

Intensity, counts

331.0435

285.0016

99.0080

127.0389353.0256

257.0066

-21.9821(M+Na)+

MH+

Isotope distribution tool for matching peak intensities

Database search for the empirical formula of C10H19O6PS2

100 140 180 220 260 300 340 380

m/z, amu

2.00e4

4.00e4

6.00e4

8.00e4

1.00e5

Intensity, counts

331.0435

285.0016

99.0080

127.0389353.0256

257.0066

Fragment ions

-21.9821

(M+Na)+

MH+

OH

O

O

3.3 ppm

O

O

HO

0.2 ppm

OH

O

S

PS

H3CO

H3CO

0.1 ppm

O

O

HO

S

PS

H3CO

H3CO

0.8 ppmS

PS

H3COH3CO

O CH3

O

O

OH

CH3

7

294.0 295.0 296.0 297.0 298.0 299.0 300.0 301.0 302.0 303.0 304.0 305.0m/z, amu

5000.00

1.00e4

1.50e4

2.00e4

2.50e4

3.00e4

3.50e4

4.00e4

4.50e4

5.00e4

5.50e4

6.00e4

6.50e4

7.00e4

7.50e4

8.00e4

8.50e4

9.00e4

9.50e4

1.00e5

1.05e5

1.10e5

1.15e5

1.20e5

1.25e51.28e5 297.0558

299.0530

298.0587

301.0503300.0560

m/z = 297.0558

Chlorine isotopicpattern

2 Cl2 Cl

LCLC//TOFTOF--MS MS unknown unknown accurateaccurate mass mass sspectrpectrum from Strawberry sampleum from Strawberry sample

m/z = 299.0530

DatabaseDatabase ScreeningScreening SearchSearch

8

O

NN

H2C

Cl

Cl

C14H15Cl2N2O+

Exact Mass: 297.0556

Imazalil

Comparison for Target AnalysisLC/QqQ-MS vs LC/TOF-MS

9

5x10

0.20.40.60.8

11.21.41.61.8

Abundance vs. Acquisition Time (min)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

+ TIC Product Ion (** -> **) Linuron.d

0.255 0.806 1.9171.3581 1 2 2 3 3 4 4

Linuron, CE voltage optimization

QqQQqQ: : OptimizationOptimization forfor MRM MRM transitionstransitions

5 V 10 V 15 V 20 V

4x10

0

0.5

1

1.5

Abundance vs. Mass-to-Charge (m/z)50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300

+ Product Ion (0.214-0.304 min, 12 scans) (249.0000 -> **) Linuron.d

248.8970

181.8980159.798362.0998 87.8994 217.5974132.9987

3x10

0

2

4

6


248.8970159.7983 181.8980

61.9998 87.8994 217.8974132.9987 173.9981152.6984

3x10

02

46


159.8983 181.8980

62.0998 248.9970217.0975132.698787.9994 152.7984 174.8981

3x10

0246


159.7983 181.8980

132.798759.8998

216.997587.5994

5 V

10 V

15 V

20 V

...MRM ...MRM transitionstransitionsCompound Fragmentor Precursor Ion Product ions Collision V

3,4,5-Trimethacarb 80 194 137 5122 25

3-Hydroxycarbofuran 80 238 163 10220 5

Acephate 80 184 143 5125 10

Acetamiprid 80 223 126 1556 15

Acetochlor 120 270 148 10224 10

Acibenzolar-S-methyl 120 211 136 2091 25

Aclonifen 120 265 248 15193 15

Alachlor 80 270 162 15238 10

Aldicarb 80 116 89 570 5

Aldicarb Sulfone 80 223 148 576 5

Aldicarb Sulfoxide 80 207 89 5132 5

Aldoxycarb 80 223 149 15177 5

Ametryn 120 228 186 2096 25

Aminocarb 120 209 137 20152 10

Column: Agilent SB C18 4.6 x 150 mm (1.8 µm)Column temp: 25 CMobile phase: 10%ACN / 90%H2O (0.1% HCOOH)Flow-rate: 0.6 mL/minGradient: t0 = 10% ACN, t30 = 100% ACNInjection volume: 10 µL

10

5x10

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0.55

0.6

0.65

0.7

0.75

0.8

0.85

0.9

0.95

1

1.05

1.1

1.15

1.2

1.25

1.3

1.35

1.4

1.45

1.5

1.55

1.6

1.65

Abundance vs. Acquisition Time (min)1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

+ MRM (282.0 -> 212.0) mix100_100pg_5May.d

1 1 2 25x10

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0.55

0.6

0.65

0.7

0.75

0.8

0.85

0.9

0.95

1

1.05

1.1

1.15

1.2

1.25

1.3

1.35

1.4

1.45

1.5

1.55

1.6

1.65

Abundance vs. Acquisition Time (min)1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33

+ MRM (282.0 -> 212.0) mix100_100pg_5May.d

1 1 2 2

ExtractedExtracted Ion Ion chromatogramschromatograms100 100 CompoundsCompounds by by QqQQqQ

100 pg on column or 0.01 ppm (Baby food levels)

Ab

un

da

nce

Acquisition Time (min)

6x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

24.4 24.6 24.8 25 25.2 25.4

305 -> 169.0 , 153.0

Ratio=52.2

+ MRM (25.047-25.408 min, 18 scans) 300 pesticide

Ab

un

da

nce

Mass-to-Charge (m/z)

5x10

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

150 175 200 225 250 275 300 325

169.0

153.0

De Brabander et al., TRAC, 20 (8) 2001

Ion Ratios by Ion Ratios by QqQQqQ

N

NCH3

CH3

CH3

OP

S

OH3CO

H3C

m/z=153

m/z=169

Diazinon

11

Diazinon - 6 Levels, 6 Levels Used, 6 Points, 6 Points Used, 0 QCs

Re

spo

nse

s

Concentration (ng/ml)

7x10

0

0.25

0.5

0.75

1

1.25

1.5

1.75

2

-10 -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115

y = 208335.0516 * x - 152717.0728R^2 = 0.99956076

3-Hydroxycarbofuran - 6 Levels, 6 Levels Used, 6 Points, 6 Points Used, 0 QCs

Re

spo

nse

s

Concentration (ng/ml)

5x10

0

1

2

3

4

5

6

7

-5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100

y = 8408.3900 * x - 1375.3510R^2 = 0.99945613

CalibrationCalibration Data by Data by QqQQqQDiazinon (concentration range: 0.1-100 ppb)

3-Hydroxycarbofuran (concentration range: 5-100 ppb)

Green pepper matrix

Tomato matrix

QuantitationQuantitation by by QqQQqQ

12

MS/MS Interesting Isobaric Interference and example of how LC/TOF-MS could help

Diuron 233->160233->72

Fluometuron 233->160233->72

Diuron and fluometuron have the same isobaric precursor ion and same qualiferwith nearly identical ion ratios and similar retention times.

HN

N

O Cl

Cl

Diuron

C9H11Cl2N2O+


O

N NH

F

FF

C10H12F3N2O+


Fluometuron

1

10

100

400

Number ofTransitionsper group

Dwell time per ion100 mSec200 mSec 10 mSec

Dwell Time versus Number of MRM Transitions:“Where MRM meets Scan is Y”

ScanY

13

5x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

Abundance vs. Acquisition Time (min)1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

+ MRM (353.0000 -> 228.0000) 300 pesticides_Tomato matrix_1 ppb.d

28.557

1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8

LODLOD’’ss by by QqQQqQ

1 ppb in tomato matrixCompound

Precursor Ion

Product ions

Pepper LOD

LOD Tomato r2

ppb ppb 3,4,5-Trimethacarb 194 137 0.9 0.5 0.999 122 3-Hydroxycarbofuran 238 163 5.0 5.0 0.999 220 Acetamiprid 223 126 0.2 0.5 1.000 56 Aclonifen 265 248 20.0 10.0 0.979 193 Atrazine 216 174 0.8 0.6 1.000 132 Bendiocarb 224 109 1.0 1.0 0.996 167 Buprofezin 306 201 0.7 0.8 0.999 116 Carbaryl 202 145 5.0 5.0 0.999 117 Diazinon 305 169 0.5 0.2 1.000 153 Dimethoate 230 199 0.7 0.7 0.994 171 Fuberidazole 185 157 1.1 1.0 1.000 156 Imazalil 297 159 10.0 10.0 0.850 255 Irgarol 254 198 1.0 1.0 0.999 156 Malathion 331 99 0.8 2.0 0.998 127 Propazine 230 146 1.0 0.5 1.000 188 Terbuthylazine 230 174 0.1 0.5 0.999 132

Accurate mass at 100 picograms with extracted ions

NN

H2NNO2

NCl

Imidacloprid

C9H11ClN5O2+


N

N

N

Cl

NH

NH

C8H15ClN5+


Atrazine

In Buffer Atrazine LOD is 20 pg and Imidacloprid is 100 pg.

14

QQQ inScan

1

10

100

300

Instrument Detection Limit10 pg1 pg 100 pg

Screening Pesticides:TOF detection limit is X and the “Crossover Point is XY”

QQQ inMRM

TOF is Full Scan2 ppm Accuracy

QQQ inScan

Scan

Number ofTransitionsper group

Examples of Environmental Samples byLC/Q-TOF-MS

15

Q-TOF Targeted Analysis300 compounds in 25 minutes

N

N

N

Cl

NH

NH

C8H15ClN5+


Atrazine

Atrazine Frag ions

Answering our “Puzzle” by LC/TOF-MS and LC/TOF-MS-MS

N N

N

HN CH3

H2N

C5H8N5+


N N

NHNH3C

H2N CH3

Cl

CH3

N

NH2N

Cl

C2H3ClN3+


N

NNH3C

C4H6N3+

Exact Mass: 96.0556

NH2

H2N Cl

CH4ClN2+

Exact Mass: 79.0058

HN N

NH2N

HN CH3

Cl

C5H9ClN5+


EE

N N

NH2N

NH3

Cl

C3H5ClN5+


N

NNHH3C

Cl

C4H7ClN3+


C8H15ClN5+


N

NHN

C2H2N3+


N

HNH3C

C3H7N2+

Exact Mass: 71.0604

Atrazine Pathway

C3H4N5+


N

HNN

N

H2N

Atrazine pathwayof fragmentationfrom standard andfrom the analysisof a water sampleLawrence, KS

16

TOF compared with QQQ...

Screening and ID of pesticide by QqQ is favorite method but there are limits to dwell times and number of ions.

LC/TOF-MS offers another approach, which takes full scan data and searches database by accurate mass or by formula. Example of 100+ compounds including fragment ions.

QqQ more sensitive by 10-100 times, if transitions are few (1-20).

Cross-Over Point is 100-150 transitions and 10 msec dwell per window or about 20-40 pg on column.

TOF compliments QqQ for food analysis, especially non-targets and large screenings (isotope clusters useful) .

Q-TOF offers additional structural information, specially for fragment ions (good structural elucidation capability).

Rapid Resolution is easily possible with Q-TOF in food matrices from 25 to 5 minutes for a large number of compounds.

Accuracy remains excellent even with 2 to 3 second peaks, something not possible by QqQ with so many compounds.

Either TOF or Q-TOF can be used for screening and confirmation in food matrices.

Q-TOF offers additional information…

17

Antibiotic in Water

Example of 4 GHz High Resolution for Antibiotic Analysis in Water

H2N

SNH2

O O

N

N

OCH3

OCH3C12H15N4O4S+


H3N N

N

OCH3

OCH3

C6H10N3O2+


H2N

S

O O

C6H6NO2S+


4x10

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+ Scan (7.929-8.012 min, 8 scans) Sulfadimethoxine_4GHz_190V.d

156.0769

156.0115

Counts vs. Mass-to-Charge (m/z)155.7 155.8 155.9 156 156.1 156.2 156.3 156.4 156.5

H2N

SNH2

O O

N

N

OCH3

OCH3C12H15N4O4S+


H3N N

N

OCH3

OCH3

C6H10N3O2+


H2N

S

O O

C6H6NO2S+


18

AcknowledgmentsAcknowledgmentsAcknowledgments

Funding from the Ministerio de Educacion y Ciencia(Project AGL-2004-04838)

Dr. Jerry Zweigenbaum, Agilent Technologies, Inc

Department of Civil, Environmental, andArchitectural Engineering, University of Colorado, Boulder, CO USA

lc/qqq, tof and q-tof for the analysis of pesticide

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