Validation of screening qualitative methods - score …score-cost.eu/.../05/150429_2_Qualitative-validation_TPortoles.pdfValidation of screening qualitative methods ... Advantages

Validation of screening

qualitative methods

Dr. Tania Portolés NicolauResearch Institute for Pesticides and Water,

University Jaume I, Castellón, Spain

29th April 2015

Introduction

Analytical Challenges

Thousands of organic contaminants of very different physico-chemical characteristics

Reliable detection, identification and accurate quantificationin samples

Metabolites and Transformation/Degradation Products

Investigation of new (unknown) contaminants potentiallydangerous for the environment and human health

State-of-the-art

Analytical Techniques

State-of-the-art

Ideal for TARGET METHODSand quantification

purposes

QUAD TRIPLE QUAD ION TRAP

Ideal for SCREENING

METHODS

“KNOWN” and “UNKNOWN”

FULL SPECTRUM

ACQUISITION MODESIM MS/MS

“KNOWN” or suspected residue

TIME OF FLIGHT/ORBITRAP

Complementary use of LC & GC - MS

Analytical Approaches

State-of-the-art

Well-established analytical(quantitative) methods

with low LOQs and LODs based on tandem MS

Need of strategies for wide-scope screening able to demonstrate the presence/absence of residues for subsequent quantitative efforts

Processing data

Quality Controls

Recoveries

Matrix matched calibration curves

The results of monitoring programmesoften show that only a fraction of the scope are regularly found across all of

the different samples.

TIME-CONSUMING

WASTE OF TIME

Current trend to focus quantitative and identification

efforts on compounds previously detected during rapid screening methods with a high number of

compounds

CURRENT TREND

Universal detect all potential contaminants that might be present in the samples above a certain concentration

Rapid

Little sample handling

Reliable identification of compounds detected

If possible, accurate quantification

This ideal situation that can not be reached

Ideal screening?

Screening of organic contaminants

The closest approach to the “universal” screening

Magnetic sector(Q)Time-of-flight

Orbitrap(Q)Time-of-flight


Combined use of GC and LC both coupled to HR MS

GC - HR MS

Non polar-medium polarVolatile compoundsThermally stable

LC - HR MS

Polar-ionic compoundsNon-volatile

Thermally unstable

HR (Q)TOF MS

Advantages of HR (Q)TOF

MS/MS experiments (accurate-mass product ion

spectra)

Highly valuable for elucidation and confirmation purposes

Information on parent molecule (LE) and fragment ions (HE)

MSE acquisition (simultaneous LE&HE spectra;

collision cell)

Powerful technique for screening, identification/confirmation

(and elucidation of unknowns)

Full spectrum dataat high sensitivity

Elevated Resolution(>10000 FWHM)

Accurate massmeasurements


Qualitative Validation of

Screening Methods

EU guidelines for method validation

Widely accepted guidelines and criteria to be established for the validation of screening methods.

For initial validation, a set of at least 20 samples (10x2) needs to be taken, consisting of different commodities representative for the intended application.

There are no requirements with regard to recovery of the analytes

The screening detection limit (SDL) is the lowest concentration for which it has been demonstrated that a certain analyte can be in at least 95% of the samples

A false-negative rate of 5% is accepted

For false detects no criterion has been set because the code of practise is that any detect will not be reported as positive, but trigger a follow up for full identification and quantification.

Validation requirements in qualitative methods

Define the detection criteria to optimize the selectivity

Evaluate unfortified samples

Selectivity. Fit-for-purpose

CLEAN-UPd-SPE with MgSO4,PSA and C18

EXTRACTION(ACN,1% HAc+MgSO4 andNaAc)

SolventEXCHANGEto toluene

QuEChERS AOAC Official method(acetate buffered version)

Validate a qualitative screening method for 130 pesticides in different commodities according to SANCO/12495/2011.

Establish the screening detection limit (SDL) for each of the pesticides studied.

Validation set: 10 commodities (X2)

Samples spiked in duplicate at 0.01, 0.05 and 0.20 mg/kg

MS E

Screening of pesticides in food commodities by

GC-(APCI)QTOF MS: Qualitative validation

Buffer during extraction, as is done in this work, the commodity group ‘high water content’ and ‘high acid content and high water content’ can be merged and considered as one group which then includes most of the fruit and vegetable commodities.

Overall detection

Percentage of pesticides detected based on one diagnostic ion

± 5 ppm

± 0.2 min

0

20

40

60

80

100

120

1 diagnostic ion

Nu

mb

er

of

pesti

cid

es

0,01 mg/kg

0,05 mg/kg

0,2 mg/kg

n.e

SDLs – 1 diagnostic ion

77%

14%

7%

Number of pesticides detected at 95% of confidence - 19 out of 20

SDLs – 2 diagnostic ions

62%

24%

6%

Number of pesticides detected at 95% of confidence - 19 out of 20

0

5

10

15

20

25

30

% o

f 1

32

pesti

cid

es

detection and identification of

the 64%.

51

35

28

18

2 diagnostic ions, mass accuracy and ion ratio

0,01 mg/kg 0,05 mg/kg 0,2 mg/kg n.e

Limit of Identification (LOI)

Number of pesticides identified at 95% of confidence - 19 out of 20

2 diagnostic ions±0,2 min±5 ppm

q/Q < 30%

Time18.00 19.00 20.00 21.00 22.00

%

0

100

18.00 19.00 20.00 21.00 22.00

%

0

100

SCREENVEG046 2: TOF MS AP+ 220.134 150PPM

5.51e4Area

20.024979


4.82e5Area

20.0346859

m/z100 120 140 160 180 200 220 240 260 280 300

%

0

100

m/z100 120 140 160 180 200 220 240 260 280 300

%

0

100

SCREENVEG046 1283 (20.020) 2: TOF MS AP+ 3.19e5220.1338

192.1387160.1124

148.1118134.0964

161.0964

162.1279

192.1325

193.1407

248.1290

221.1360

280.1551

249.1314 281.1569


161.0964

119.9936 143.1060

248.1290209.0812203.1072162.0992

221.1177249.1334

281.1588

282.1611

q/Q ratio (s): 0.106q/Q ratio (st): 0.134

21% LE

HE

Screening QTOF in fruits and vegetables

Carrot sample

[M+H]+

-1.4 ppm

METALAXYLC15H21NO4

MH+ = 280,1549

CH3

O

N+

CH3

CH3O

CH3

0 ppm

-0.5 ppm

-1.2 ppm

O

N

CH3

CH3CH2

+O

O

CH3

CH3

-1.2 ppm

N

O+

H

N+

0.17 mg/kg

GC(APCI)-QTOF MS approach easily allowed widening the number of compounds investigated (post-target screening)

List of 300 additional GC-amenable pesticides

Widening the scope of the screening QTOF

TetraconazoleFlutriafol

FludioxonilTebuconazole

EtoxazoleBoscalid

Standard

13 pesticidesMH+ or M+·

< 5ppm mass error

At least one compatible fragment

ion <5ppm mass error

MiclobutanilBupirimateAclonifen

Acetamiprid

Fragmentation in agreement with literature

MethopreneAllethrin

Isomethiozin

m/z100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270

%

0

100

m/z100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270

%

0

100


182.0603

165.0449155.0512138.0338111.1242

194.0475218.0365

217.0273

195.0498219.0295 247.0269

265.0381

250.0324

251.0331

267.0350

268.0407


239.2374199.1704183.1433151.0989145.1220

129.0205117.0362

173.1497 208.0693264.0295

267.0357

268.0380

m/z100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270

%

0

100

m/z100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270

%

0

100


182.0603

165.0449155.0512138.0338111.1242

194.0475218.0365

217.0273

195.0498219.0295 247.0269

265.0381

250.0324

251.0331

267.0350

268.0407


239.2374199.1704183.1433151.0989145.1220

129.0205117.0362

173.1497 208.0693264.0295

267.0357

268.0380

Time25.00 26.00 27.00 28.00 29.00

%

0

100

25.00 26.00 27.00 28.00 29.00

%

0

100

25.00 26.00 27.00 28.00 29.00

%

0

100

25.00 26.00 27.00 28.00 29.00

%

0

100

25.00 26.00 27.00 28.00 29.00

%

0

100


2.99e4

26.55

27.10


8.59e3

26.55

24.9025.11 26.31

27.34


6.44e3

26.53

26.69


3.82e4

26.55


3.21e5

26.54

ACLONIFENC12H9N2O3Cl

MH+=265.0380

[M+H]+1.1 ppm

0.4 ppm

NO

O

O NH2

Cl

-3.7 ppm

O

C+

NH2

Cl

-2.6 ppm

O NH

NH

-1.6 ppm

O C+

NH

Confirmed by

standard

Time25.00 26.00 27.00 28.00 29.00

%

0

100

25.00 26.00 27.00 28.00 29.00

%

0

100

25.00 26.00 27.00 28.00 29.00

%

0

100

25.00 26.00 27.00 28.00 29.00

%

0

100

25.00 26.00 27.00 28.00 29.00

%

0

100


2.99e4

26.55

27.10


8.59e3

26.55

24.9025.11 26.31

27.34


6.44e3

26.53

26.69


3.82e4

26.55


3.21e5

26.54

Widening the scope of the screening QTOF

N

O

O NH2

Cl

Screening of Water Samples

250 mL centrifuged water

CONDITIONING: 5 mL MeOH + 5 mL H2O

LOADING SAMPLE: by gravity

Elution: 10 mL MeOH

SPE

(OASIS

H

LB)

200 m

g

Screening of water samples - Procedure

Evaporation at 35ºC under Nitrogen

Re-disolving250 µL ethyl acetate

5 mL MeOH

GC-(APCI)QTOF

Evaporation at 35ºC under Nitrogen

Re-disolving500 µL 10% MeOH

5 mL MeOH

UHPLC-QTOF

DRYING : Vacuum , 30 min

PAHs, pesticides, PCBs, PBDEs, octyl/nonylphenols, UV

filters, antimicrobials,

insect repellents, PCNs, musks

polar pesticides, pharmaceuticals

, antibiotics, illicit drugs, UV

filters, hormones,

metabolites/TPs

x 250x 500

500 1500

QTOF MS screening (MSE)

List of target compounds

Reference standardavailable?

Exact mass filtering

XICs for each compound (LE)M+·, [MH]+, [M-H]-

YES NO

Retention time filtering

Fragment ionsconfirmation (HE)

Confirmation of theidentity

Fragment ionsinterpretation (HE) +

comparison HE spectrawith MS/MS from

literature + predicted RT + isotope pattern

Tentative Identification

Detection and Identification workflow

SUSPECTSCREENING

TARGET ANALYSIS

Confirmation withstandards

Qualitative Validation

Screening Detection Limit (SDL)lowest concentration for which a compound is detected in all spiked

samples (commonly using the M+· or MH+ at the LE function)

9 water samples(3 groundwater, 3 surface water, 3 effluent

wastewater)

141 organic contaminants

Pesticides

Pharmaceuticals/antibiotics

Drugs of abuse and metabolites

UV filters

Hormones

UHPLC-QTOF

166 organic contaminants

PAHs

Octyl/nonyl phenols

PCBs

PBDEs

Pesticides and metabolites

GC-(APCI)QTOF

Reliable identification (Limit of Identification)At least two accurate-mass ions and accomplishment of ion ratio

0%

20%

40%

60%

80%

100%

0

20

40

60

80

100

120

140

160

0.0

2 µ

g/L

0.1

µg

/L

0.5

µg

/L

0.0

2 µ

g/L

0.1

µg

/L

0.5

µg

/L

0.0

2 µ

g/L

0.1

µg

/L

0.5

µg

/L

0.0

2 µ

g/L

0.1

µg

/L

0.5

µg

/L

0.0

2 µ

g/L

0.1

µg

/L

0.5

µg

/L

0.0

2 µ

g/L

0.1

µg

/L

0.5

µg

/L

0.0

2 µ

g/L

0.1

µg

/L

0.5

µg

/L

>0

.5 µ

g/L

Fre

qu

en

cy

Screening Detection Limit (SDL)PESTICIDES

PHARMACEUTICALS DRUGS OF ABUSE PCBs PAHs OTHERS

TOTAL

Screening Detection Limit

m/z100 120 140 160 180 200 220 240 260 280 300 320 340 360 380

%

0

100

m/z100 120 140 160 180 200 220 240 260 280 300 320 340 360 380

%

0

100

DAVFLET317 1363 (21.261) 2: TOF MS AP+ 3.20e4197.9280

114.9619

124.9826

153.0139

199.9253

201.9221

349.9346

323.8995215.9022 295.8680257.8958

353.9278


277.1447

353.9284

m/z100 120 140 160 180 200 220 240 260 280 300 320 340 360 380

%

0

100

m/z100 120 140 160 180 200 220 240 260 280 300 320 340 360 380

%

0

100


114.9619

124.9826

153.0139

199.9253

201.9221

349.9346

323.8995215.9022 295.8680257.8958

353.9278


277.1447

353.9284

Chlorpyriphos ethylC9H12Cl3NO3P

[M+H]+ 349.9341

H4O3PS

0 mDaC5H3NOCl3

0 mDa

C2H6O2PS

0 mDa C4H10O2PS

0 mDa

Time20.50 21.00 21.50 22.00

%

0

100

20.50 21.00 21.50 22.00

%

0

100

20.50 21.00 21.50 22.00

%

0

100

20.50 21.00 21.50 22.00

%

0

100

20.50 21.00 21.50 22.00

%

0

100

DAVFLET317 Sm (Mn, 1x1) 2: TOF MS AP+ 114.962 150PPM

5.23e3

21.26


2.78e3

21.26


3.37e3

21.26

21.64


1.11e4

21.26


5.02e4

21.25

GC-(APGC)QTOF target analysis

Time20.50 21.00 21.50 22.00

%

0

100

20.50 21.00 21.50 22.00

%

0

100

20.50 21.00 21.50 22.00

%

0

100

20.50 21.00 21.50 22.00

%

0

100

20.50 21.00 21.50 22.00

%

0

100


5.23e3

21.26


2.78e3

21.26


3.37e3

21.26

21.64


1.11e4

21.26


5.02e4

21.25

1.4 mDa

LE

HE

HE

HE

HE

Standard available Target analysis

m/z100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400

%

0

100

m/z100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400

%

0

100

DAVFLET155 2239 (33.165) Cm (2238:2243-(2252:2281+2212:2234)) 2: TOF MS AP+ 1.19e5232.0756

204.0807

105.0340

177.0703

146.0820111.6446

178.0754205.0850

250.0858

251.0887

380.0728360.1818277.3729

327.7480 410.1026


232.0755223.0489140.8842153.4109

201.5325

362.2115

251.0896

313.0682252.0922 299.0319 360.1976363.2148

414.3727

Pou Borriana

Time32.00 32.50 33.00 33.50 34.00 34.50

%

0

100

32.00 32.50 33.00 33.50 34.00 34.50

%

0

100

32.00 32.50 33.00 33.50 34.00 34.50

%

0

100

32.00 32.50 33.00 33.50 34.00 34.50

%

0

100

32.00 32.50 33.00 33.50 34.00 34.50

%

0

100

32.00 32.50 33.00 33.50 34.00 34.50

%

0

100

DAVFLET155 Sm (Mn, 1x1) 2: TOF MS AP+ 105.034 0.02Da

3.66e3

33.19

31.92


2.14e3

33.19

33.49


6.19e3

33.19

32.29


3.22e4

33.19


1.31e4

33.19


2.03e4

33.19

Pou Borriana

Time32.00 32.50 33.00 33.50 34.00 34.50

%

0

100

32.00 32.50 33.00 33.50 34.00 34.50

%

0

100

32.00 32.50 33.00 33.50 34.00 34.50

%

0

100

32.00 32.50 33.00 33.50 34.00 34.50

%

0

100

32.00 32.50 33.00 33.50 34.00 34.50

%

0

100

32.00 32.50 33.00 33.50 34.00 34.50

%

0

100


3.66e3

33.19

31.92


2.14e3

33.19

33.49


6.19e3

33.19

32.29


3.22e4

33.19


1.31e4

33.19


2.03e4

33.19

m/z100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400

%

0

100

m/z100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400

%

0

100


204.0807

105.0340

177.0703

146.0820111.6446

178.0754205.0850

250.0858

251.0887

380.0728360.1818277.3729

327.7480 410.1026


232.0755223.0489140.8842153.4109

201.5325

362.2115

251.0896

313.0682252.0922 299.0319 360.1976363.2148

414.3727

C24H27NO2

-0.5 mDa

C16H12NO2

-1.0 mDa

C16H10NO-0.6 mDa

C15H10N-0.6 mDa

C14H9

-0.6 mDa

C7H5O-0.6 mDa

HE

LE

LE

HE

HE

HE

HE

HE

Octocrylene?C24H27NO2

MH+=362.2120

Standard NOT available Suspect screening

GC-(APGC)QTOF suspect screening

Octocrylene 5 ppm

Time24.00 26.00 28.00 30.00 32.00

%

0

100

24.00 26.00 28.00 30.00 32.00

%

0

100

DAVFLET352_43 1: TOF MS AP+ 362.212 0.02Da

2.56e6

28.49


1.87e5

28.51

29.52

StandardOctocrylene

Sample

m/z70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270

%

0

100

m/z70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270

%

0

100

DAVFLET352_43 1827 (28.499) Cm (1823:1831-(1843:1866+1792:1813)) 2: TOF MS AP+ 3.27e7232.0757

204.0803

203.0729

105.0337

77.0389104.0498

78.0424

177.0691

176.0619

151.0542106.0373

129.0340 157.0284

178.0748

202.0649

205.0833

206.0943

233.0790 250.0858

234.0825 251.0891


204.0801

203.0729

177.0687105.0336

104.049877.0390

151.0541106.0366129.0340 157.0284

178.0749

202.0651

205.0834

206.0950

233.0789250.0860

234.0818 251.0900

HE

HE

GC-(APGC)QTOF standard confirmation

Octocrylene 5 ppm

Time24.00 26.00 28.00 30.00 32.00

%

0

100

24.00 26.00 28.00 30.00 32.00

%

0

100


2.56e6

28.49


1.87e5

28.51

29.52

StandardOctocrylene

Sample

m/z70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270

%

0

100

m/z70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270

%

0

100


204.0803

203.0729

105.0337

77.0389104.0498

78.0424

177.0691

176.0619

151.0542106.0373

129.0340 157.0284

178.0748

202.0649

205.0833

206.0943

233.0790 250.0858

234.0825 251.0891


204.0801

203.0729

177.0687105.0336

104.049877.0390

151.0541106.0366129.0340 157.0284

178.0749

202.0651

205.0834

206.0950

233.0789250.0860

234.0818 251.0900

HE

HE

GC-(APGC)QTOF standard confirmation

EDDP

C20H24N

-0.6 mDa

C18H19N

-0.5 mDa

C17H16N

-0.4 mDa

C13H16N

-0.5 mDa

C6H12N

-0.3 mDa

(a) (b)

EDDP

C20H24N

-0.6 mDa

C18H19N

-0.5 mDa

C17H16N

-0.4 mDa

C13H16N

-0.5 mDa

C6H12N

-0.3 mDa

(a) (b)

LC-QTOF suspect screening

XX

EDDP

C20H24N

-0.6 mDa

C18H19N

-0.5 mDa

C17H16N

-0.4 mDa

C13H16N

-0.5 mDa

C6H12N

-0.3 mDa

(a) (b)

EDDP

C20H24N

-0.6 mDa

C18H19N

-0.5 mDa

C17H16N

-0.4 mDa

C13H16N

-0.5 mDa

C6H12N

-0.3 mDa

(a) (b)

X

X

LE

HE

HE

HE

HE

HE

HE

HE

LE

EDDPMetabolite methadone

C20H24NMH+ = 278.1906

m/z100 120 140 160 180 200 220 240 260

%

0

100

m/z100 120 140 160 180 200 220 240 260

%

0

100

NORMANGCC020 1732 (27.011) Cm (1730:1734-1709:1721)2.11e4194.0960

192.0806

191.9905

237.1023

195.0989238.1076

NORMANGCC020 1732 (27.004) Cm (1730:1738-1706:1722)8.52e4237.1027

220.1110

238.1058

249.0924

Time22.50 25.00 27.50 30.00 32.50

%

0

100

22.50 25.00 27.50 30.00 32.50

%

0

100

22.50 25.00 27.50 30.00 32.50

%

0

100

NORMANGCC020 2: TOF MS AP+ 192.081 150PPM

1.83e3Area

27.00197.35


4.91e3Area

27.00730.38


1.35e4Area

27.002008.48

m/z60 80 100 120 140 160 180 200 220 240

%

0

100

m/z60 80 100 120 140 160 180 200 220 240

%

0

100

NORMAN012 620 (8.694) Cm (619:620-612:614) 2: TOF MS ES+ 2.83e5194.0965

192.0811

125.1066103.0399

170.1649

195.0998 237.1022

207.1248

NORMAN012 620 (8.687) Cm (620-613:614) 1: TOF MS ES+ 2.36e5237.1024

227.1243

175.0766

238.1055

239.1073

Carbamazepine

C15H12N2OMH+ 237.1028

0.1 mDa(0.4 ppm)

- HNCO

UPLC-ESI-QTOF MSE

LE

HE

LC & GC

GC-APGC-QTOF MSE

Screening applied to 33 water samples

78 pesticides (metabolites/TPs); 24 pharmaceuticals (metabolites/TPs)4 drugs abuse; 4 preservatives; 5 UV-filters; 2 sweeteners 3 PAHs; 3 musks; 2 X-ray agents;1 antimicrobial; 2 insect repellents

The most frequently detectedPesticides

Triazine herbicides (particularly, terbuthylazine and terbutryn)Insecticides diazinon and chlorpyrifos-ethylFungicides thiabendazol, carbendazim and propiconazole

PharmaceuticalsAntibiotic ofloxacinAnti-inflammatory/analgesic diclofenacAngiotensin II receptor antagonists valsartan and irbesartanAntidepressant venlafaxineAnti-epileptic carbamazepine

OthersBenzoylecgonine (the main metabolite of cocaine)Tonalide (musk) and octocrylene (UV filter)

(12 GW, 12 SW, 9 EWW)

Positive findings in ground water and surface water

3%

Positive findings in effluent wastewater

Effluent wastewater

F HernándezJV Sancho

I Cervera

M Ibáñez

F López

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Validation of screening qualitative methods - score …score-cost.eu/.../05/150429_2_Qualitative-validation_TPortoles.pdfValidation of screening qualitative methods ... Advantages