INSTITUTE OF CHEMICAL TECHNOLOGYDepartment of Food Chemistry and AnalysisPrague, Czech Republic, jana.hajslova@vscht.cz

2005 - DART and DESI reviews introducing challenges of Ambientdesorption mass spectrometry published

2006 - 17th International mass spectrometry congress, PragueJeol exhibits DART - AccuTOFMS

2007 - ICT negotiates with Jeol, first tests carried out in Paris Centre

2008 - DART research initiated at ICT, first papers published

2008 - DART TOF MS incorporated into Biocop projecttogether with DESI, Analysis of strobilurins, commonICT, RIKILT Fera paper published in Anal. Chem.

2008 - DART TOF MS involved in Conffidence project for pesticide residue analysis

2009 - AOAC, Philadelphia, US - meeting Brian Musselman common steps planned

2009 - DART research introduced on occasion of RAFA„baby“ DART coupled with Orbitrap MS

Novel analytical strategy:

AMBIENT MASS SPECTROMETRY

- Reduced / minimal sample prep

- No chromatographic separation

…opens the doors to many challenging applicationsin various areas of food / environmental analysis

DESI Purdue Univ. USA

DART JEOL, USA

The ambient ionization methods retain the signature advantages of MS:speed

chemical specificitylow detection limits

moreover: NO SAMPLE SEPARATION IS EMPLOYED!!!or SAMPLE PPREPARATION IS MINIMAL

a Techniques where ESI mechanisms are mainly responsible for ionization.

b Methods where chemical ionization is responsible for ionization (photoionization - PI, ion evaporation - IE and electrical discharge)

AMBIENT DESORPTION IONIZATION METHODS

desorption production of ionizing chemical reagents

: ThermalDesorption with

Secondary Ionization

Liquid Extraction SurfaceSampling Probe /

Ionization

Laser Desorption(Ablation) with

Secondary Ionization

Atmospheric PressureMatrix Assisted Laser Desorption/ionization

Liquid and Gas JetDesorption /Ionization

Van Berkel et al J. Mass Spectrometry, 43, 1181, 2008

Spray and fluid velocity illustration. Droplets are black spheres eight times their actual size. Background color indicates velocity magnitude of surrounding fluid. The blue line shows the optimum collection angle from experiments (~ 10°)

DESI ▼

The DART Ion Source(Jeol)

The space where the sample is placed

Excited-state atoms or molecules M*interact with sample and atmosphere

Steps occurring together, or separately, under

the influence of a particular agent:

DESORPTION – a change in phase (e.g., solid to vapor)

IONIZATION - an acquisition of charge by neutral analyte molecules

Penning Ionization

• Metastable atoms or molecules react with analytes that posses ionization potentials less than the metastableenergy:

M* + S S+. + M + electron

Proton Transfer

• Metastable atoms react with atmospheric water to produce ionized water clusters

• Dominant reaction mechanism when helium carrier used: He(23S) energy = 19.8 eV

• Huge reaction cross section: 100 A2

He(23S) + H2O → H2O+• + He(11S) + electron

H2O+• + H2O H3O+ + OH•

H3O+ + nH2O [(H2O)nH]+

[(H2O)nH]+ + M MH+ + nH2O

The ionization process in DART is a variation of APCI, reagent-ion population originates from the gas phase reactions of the

metastable He atoms produced in the discharge

WP6 - Pesticides

Overall aim of the Work package

Development of rapid screening methods for a specific class of

fungicides (strobilurins) in cereals

Extraction time:

Maximum of 3 hours for 20 samples

Working characteristics:

LODs < 0.05 mg/kg (LOQs < 0.1 mg/kg)

Repeatability - %CV < 40

WP6 Pesticides: target values for Strobilurin SPR bioassay

Is it possible to achieve these objectives also by DART?

No. Description By StatusT6.18. To develop/optimise ambient mass spectrometry methods for

detection of strobilurins in grain.M 53

T6.19 To conduct an intra-laboratory validation of methods from T6.18.

M 55

T6.20 To produce draft SOPs for the ambient mass spectrometry methods.

M 57 /In progress

T6.21 Conduct an inter-laboratory trial of the optimised ambient mass spectrometry methods.

M 60 In progress

T6.22 To design, develop and characterise oligonucleotide-binding reagents for the target strobilurins.

M 52

T6.23 To produce sufficient quantities of the optimum binder for assay development.

M 54

T6.24 To develop a prototype assay incorporating the oligonucleotide binder.....

M 58

T6.25 To prepare a draft SOP for the procedure developed in T6.24. M 60

Stop decision taken by TMG

WP6 – Tasks in 2009

Kow logP

2,5

3,4

3,99

4,5

3,1

3,6

Work to be completed at ICT Prague…

(i) Inter-laboratory Comparison (samples delivered November 10, 2009)

(ii) Comparison of performance characteristics (e.g., repeatability of the measurement, limits of detection, exploiting of high mass resolving power) of DART–TOFMS and DART–Orbitrap MS

(iii) Comparison of extraction techniques: EtOAc extraction vs. QuEChERS

Work to be completed at ICT Prague…

(iv) Testing of a new generation of DART ion source “Baby DART” → improvement of performance characteristics expected…

LC-MS/MS

SLE(50 mL of EtOAC,

15g Na2SO4)

AccuTOF™ DART™

PLE(~50 mL AcN, 15g Na2SO4

at 90°C)

QuEChERS10 mL AcCN /

disp. SPE

5g of sample

Alternative sample handling Confirmation

Final volume adjustment to 25 mL

12.5g of sample

EXPERIMENTAL SET-UP

Direct analysis of solid sample

MS[1];0.195..0.225;-1.0*MS[1];0.290..0.327; / ESI+ / 20071116_SMPL13

320 340 360 380 400m/z

0

50

100

150

Area (158063)x103

409.13999327.17349

404.12625

388.10808368.11366

397.38513

355.28702

328.17700

370.30724 383.36849337.27645314.14176371.31562324.29151 357.30173

353.27294

Kresoxim methyl

Dimoxystrobin

Azoxystrobin

Picoxystrobin Pyraclostrobin

Trifloxystrobin

Identification of analytes in wheat extract

[M+H]+ mass spectra

Crude ethyl acetate extract spiked with strobilurins at 120 µg/kg

Polarity: positive (DART+)Helium flow rate: 2.6 L/minDischarge voltage: 2400 VPeaks volatage: 1100 VBeam temperature: 300°C

m/z 409.13993

Estimation of element composition

Mass difference from accurate trifloxystrobin

mass – 5.91ppm(m/z 409.13993 vs.

409.13752)

Identification of analyte at “baby food” level

MS[1];1.526..1.549;-1.0*MS[1];0.869..0.892; / ESI+ / 20071122_SMPL3

200 400 600 800 1000m/z

0

100

Area (140177)x103

177.11595

397.38164575.50274

617.51438 855.72741281.24888

881.73322856.72953263.23683 295.22619 398.38533 618.51816882.73773178.11645 551.50383 619.52623 853.71310

[M+H]+ mass spectrum, crude ethyl acetate extract spiked with strobilurins at

12 µg/kg

elemental composition

analyte ion

Identification of analyte at “baby food” level

trifloxystrobin

0

100

Intensity (130750)x10

409.1400

410.1438

411.1479410.3592409.6898

-10

0

Intensity (8310)x103

206.0849

More IP for trifloxystrobin (solvent standard, 50 µg/kg)

Oriffice 1 voltage20 V (standard set-up)

Oriffice 1 voltage50 V

m/z 409.1375

m/z 206.0830 m/z 186.0540

Cone induced fragmentation Enhanced confirmation

Oriffice 1 voltage50 V

Column: Discovery C18 150 × 3 mm, 5µm (Supelco)Mobile phase: 10mM amoniumacetate - methanolFlow rate: 0.3 ml/minInjection volume:5 µlColumn temperatuire: 40 °C

Ionization: ESIPolarity: positiveCapillary voltage: 3200V, one: 50VSource temperature: 120°CDesolvatation temp.: 350°C

Quattro Premier XE (Waters)

Confirmation method: LC-MS/MS

Trifloxystrobin

Pyraclostrobin

Kresoxim methyl

Azoxystrobin

Dimoxystrobin

Picoxystrobin

CALIBRATION – matrix matched standard

Internal standard (prochloraz) added into crude extracts, 200 µg/mL

Azoxystrobin

y = 0,0044x + 0,1433R2 = 0,9954

0

1

2

3

4

5

6

0 200 400 600 800 1000 1200 1400

404.

1246

(azo

xyst

robi

n) /

an

d 37

6.03

86 (p

roch

lora

z)

ng/g„On-line“ injections

Mass Chrom[1];409.10081..409.19919; / ESI+ / 20071116_SMPL12

1.0 1.5 2.0Time[min]

0

2

4

6

8

10

Intensity (10493)x103

0.960

2.021

1.179

1.841

0.677 0.800 1.5721.322

2.167

30 ng/mL

6 ng/mL

60 ng/mL

120 ng/mL

240 ng/mL

600 ng/mL

PEG

correction of mass drift

METHODRecoveries Repeatability

(RSD, n=5)LOQs

(ug/kg)

SLEAccuTOF™ DART™

82–91% 8–15% 12–30

PLEAccuTOF™ DART™

80–94% 14–19% 12–30

QuEChERS AccuTOF™ DART™

89–95% 8–11% 12–30

QuEChERS LC-MS/MS

93–97% 4–7% 1–4

PERFORMANCE CHARACTERISTICSwheat grains spiked by strobilurins at 60 µg/kg

no matrix effects related to ionization were observed in crude extracts possible interferences at ions of analyes (close m/z from matrix)

Incurred residue analysis- wheat grains (BIOCOP test material)

0

0,05

0,10,15

0,2

0,25

0,3

0,350,4

0,45

0,5

SLE (AccuTOF™DART™)

PLE (AccuTOF™DART™)

QuEChERS (LC-MS/MS)

Analytical method

Con

cent

ratio

n (m

g/kg

)

AzoxystrobinKresoxim methylPyraclostrobin

Further simplification DIRECT mesurement

?

Sample preparation for direct DART TOFMS measurement

fillinga paper

“envelope”

AccuTOF™DART™

cca 1g of sample

2´

a)d)

b)c)

Items used for analysis:

a) incurred wheat grain sample b) filtr paperc) spatulad) PEG standard solution

DIRECT SCREENING OF INCURRED RESIDUES - wheat grains (BIOCOP test material)

1st „hit“5th „hit“

Mass difference –0.35 ppm(429 µg/kg)

Azoxystrobin

Mass difference –1.56 ppm(170 µg/kg)

Pyraclostrobin

Incurred residues screening- wheat grains (BIOCOP test material)

Mass difference –0.37 ppm(concentration 52 µg/kg)

Kresoxym-Me

1st „hit“

AccuTOF™DART™ providessimple and fast confirmation of strobilurins in wheat at MRL

levels.

Schurek J. et al.,

Anal. Chem. 80,

9567–9575 (2008),

doi: 10.1021/ac8018137

DART Cone

AnalyserDART–TOFMS

DART–Orbitrap MSDART Heated

capillaryAnalyser

New problem emerged…

APPROACH #1

APPROACH #2

Transfer of the technique…

DART–Orbitrap MS:Influence of capillary temperature on the fragmentation

300 350 400m/z

0

10

20

30

40

50

60

70

80

90

100

404.1241

322.5298300 350 400

m/z

0

10

20

30

40

50

60

70

80

90

100

404.1243

372.0982

359.0900300 350 400

m/z

0

10

20

30

40

50

60

70

80

90

100

372.0984

344.1033404.1246

317.0753

50°C300 350 400

m/z

0

10

20

30

40

50

60

70

80

90

100

404.1249

372.0986

344.1029

100°C 150°C 250°C

3.1x106Intensity

6.8x104

372.0982

5.0x106

6.8x105

6.6x106

3.3x106

8.3x106

3.6x105

Example: Azoxystrobine, [M+H]+ 404.124

280 300 320 340 360 380 400 420 440 460m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

392.1941

404.1252

459.0879

372.3121

355.2855

388.1069

409.1381337.2747

312.1174285.1239 327.1717

296.0591

Metominostrobin E+ZFenamidone

Orysastrobin

Azoxystrobin

Trifloxystrobin

Fluoxastrobin

Fluacrypyrim

Dimoxystrobin

Kresoxim-methyl

Pyraclostrobin

Picoxystrobin

Purified (PSA, C18) QuEChERS extract (wheat) spiked with strobilurins at 1 mg/kg

Polarity: positive (DART+)Helium flow rate: 1.5 L/minBeam temperature: 300°C

DART–Orbitrap MS

APPROACH #2

DART–Orbitrap MS: matrix effects

1.00 1.05 1.100

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relat

ive A

bund

ance

1.06

1.011.08

2.30 2.35 2.40Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

Relat

ive A

bund

ance

2.37

2.34

1.7 1.8Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

Relat

ive A

bund

ance

1.77

1.73

0.8 0.9Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

Relat

ive A

bund

ance

0.87

0.84

Standard in pure solvent

Matrix-matched standard (wheat extract)QuEChERS

cleanup: 150 mg PSA + 50 mg C18 per mL

QuEChERS

cleanup: 50 mg PSA per mL

QuEChERS

crude extract

Azoxystrobine

[M+H]+ 404.124

1000 ng/mL 1000 ng/mL/0.5 g 1000 ng/mL/0.5 g 1000 ng/mL/0.5 g

DART–TOFMS vs. DART–Orbitrap MS:Influence of mass resolving power

403.0 403.5 404.0 404.5 405.0 405.5 406.0 406.5m/z05

50

100404.1265

405.3732

405.1269

406.3764403.3569

5,500 fwhm 10,000 fwhm

TOFMS Orbitrap MS

CONffIDENCE project

WP-1c

Simplified MS screening tool for the analysis of dithiocarbamates in intact vegetables/fruits

Overview of DTCs

G. Crnogorac, W. Schwack, TrAC-Trend Anal Chem, 28, 40–50 (2009)

Overview of DTCs

G. Crnogorac, W. Schwack, TrAC-Trend Anal Chem, 28, 40–50 (2009)

Activities and achievementsOptimisation of DART–TOFMS instrumental

parameters for thiram and ziram

Sample preparation for the determination of thiram and ziram in fruits (pears) using DART–TOFMS

Validation study

Thiram Ziram

Activities and achievementsOptimisation of DART–TOFMS instrumental parameters

Gas beam temperature → 300°C optimal

Helium flow → 3.5 litters per minute optimal

Activities and achievementsOptimisation of DART–TOFMS instrumental parameters

VAPUR interface (only 20% increasing of signal intensity, but higher background) → without VAPUR

Source of figure: www.ionsense.com

Activities and achievementsDART[+]–TOFMS profiles of standards

100 150 200 250m/z

0

100

200

Intensity (222826)x103

124.08

123.08

240.99

229.00172.13163.12125.09 195.93122.01 188.12151.96 197.93 228.19

100 150 200 250 300m/z

0

50

100

150

Intensity (183639)x103

122.01123.08

124.09

282.28

172.13163.12 304.92188.13 283.28138.00 280.26239.15113.07 189.12

305.0 310.0m/z

10

20

30Intensity (30539)x103

304.92

306.92

308.92

307.91305.92303.92 309.92

Ziram[C6H12N2S4Zn+H]+

Thiram[C6H12N2S4+H]+

1 µg/mL in MeCN

2 µg/mL in MeCN

2007/57/EC

mg/kg

First considerations for the determination of thiram and ziram in fruits (pears) using DART–TOFMS

Both compounds soluble in acetonitrile (this information not available in literature!)

Possibility to use acetonitrile for sample extraction (QuEChERS approach)

Internal standard needed for reliable quantification (triphenyl phosphate, TPP)

Sample preparation

Thiram Ziram

Sample preparation

10 g sample (pears) 10 mL acetonitrileShaking – 1 min

4 g MgSO4 + 1 g NaCl

Shaking – 1 min

Centrifugation – 5 min, 11,000 rpm

DART–TOFMSanalysis

QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe)

(1)

(2)

(3)

(4)

(5)

Activities and achievementsThiram5 mg/kg

Ziram1 mg/kg

TPP(internal

standard)1 mg/kg

DART–TOFMS spectrum of matrix-matched standard (pear extract)

Activities and achievements

m/z 240.99 Zoom

Repeated injections of

spike

1st 2nd 3rd 1st 2nd 3rd

1st 2nd 3rd 1st 2nd 3rd

Repeated injections of

blank

Thiram — 5 mg/kg (MRL)

Sample introduction variations compensated using an internal standard (TPP)

m/z 304.93

Activities and achievementsZiram — 1 mg/kg (MRL)

Repeated injections of

spike

1st 2nd 3rd 1st 2nd 3rd

Repeated injections of

blank

Sample introduction variations compensated using an internal standard (TPP)

Activities and achievements

Analyte MRL Spike level Recovery RSD LOQ(mg/kg) (mg/kg) (%) (%) (mg/kg)

Thiram 5 5 85.2 6.7 0.1

Ziram 1 1 82.7 8.9 0.5

*) Note: Quantification performed using matrix-matched standards with internal standard (TPP) correction

Validation studyMatrix: pears

DART offers a lot of challenges in food analysis

Qualitative screening

Quantitative analysis

Metabolomics (non-target) profiling

Rection dynamics monitoring

Method development