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The power of fast separation and The power of fast separation and dynamic MRM for pesticide analysis based on 1290/6460QQQ based on 1290/6460QQQ Dr. Volker Gnau Produkt Spezialist LCMS Page 1

The power of fast separation andThe power of fast ... · The power of fast separation andThe power of fast separation and ... • Final method includes 11 pesticides with 2 ... Avermectin

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Page 1: The power of fast separation andThe power of fast ... · The power of fast separation andThe power of fast separation and ... • Final method includes 11 pesticides with 2 ... Avermectin

The power of fast separation andThe power of fast separation anddynamic MRM for pesticide analysis

based on 1290/6460QQQbased on 1290/6460QQQ

Dr. Volker GnauProdukt Spezialist LCMS

Page 1

Page 2: The power of fast separation andThe power of fast ... · The power of fast separation andThe power of fast separation and ... • Final method includes 11 pesticides with 2 ... Avermectin

Ch t hi diti ( d )3.) Quantitative analysis of pesticides (sMRM)

Chromatographic conditions (pos. mode)1200 Series HPLC system consisting of:

- vacuum degasserS- binary pump SL

- wellplate sampler SL- diode array detector SL (not used)

HPLC methodHPLC method

Separation column: ZORBAX Eclipse Plus C-18, 100 x 2.1 mm, 1.8 µm

Guard column: ZORBAX Eclipse Plus C-18, 12.5 x 2.1 mm, 5 µm

Mobile phase: A: 5 mM ammonium formate + 0.01% formic acidB: methanol + 5 mM ammonium formate + 0.01% formic acid

Flow: 0.3 ml/min

Gradient**: 0.00 min 10 % B1.00 min 10 % B

18.00 min 100 % B20.00 min 100 % B20.10 min 10 % B25.00 min 10 % B

Inj.Vol.: 5 µl

Page 2

Page 3: The power of fast separation andThe power of fast ... · The power of fast separation andThe power of fast separation and ... • Final method includes 11 pesticides with 2 ... Avermectin

A il t J t t diti ( d )3.) Quantitative analysis of pesticides (sMRM)

Agilent Jetstream conditions (pos. mode)

Spray chamber conditions:G 250°CGas temp.: 250°CDry gas: 5 l/minNebulizer: 35 psiSheath gas temp: 250°CSheath gas temp: 250 CSheath gas flow: 10 l/min

Positive NegativeC V lt 4500 V 3000 VCapVoltage: 4500 V 3000 VNozzle voltage 300 V 0 V

Th l i f h h b di i f l i id h d l h• The selection of the spray chamber conditions for a multi-residue method always have to be a trade-off between the optimum settings of the individual compounds.

• For this analysis parameters have been chosen based on the optimization of the least abundant ion signals (Cypermethrin, Avermectin B1a, Fenhexamid, and Diflubenzuron).

• For Spinosyn A and Bentazone (most abundant signals in positive and negative mode) the use of the optimal values would result in a signal increase of 27.2 % and 34.0%, respectively.

Page 3

Page 4: The power of fast separation andThe power of fast ... · The power of fast separation andThe power of fast separation and ... • Final method includes 11 pesticides with 2 ... Avermectin

MRM (Multiple Reaction Monitoring)

Quad Mass Filter (MS2)Quad Mass Filter (MS1)

Collision Cell

Spectrum with backgroundions (from ESI)

Q1 lets onlytarget ion 210pass through

Collision cell breaks ion 210apart

Q3 monitors onlycharacteristic fragments 158

210

222

210

and 191 from ion 210 for quant and qual.

170 210 250 290

268 280165

210 150 170 190 210

210158

191

160

158

190

191

170 210 250 290 210 150 170 190 210 160 90no chemical background

Page 4

Page 5: The power of fast separation andThe power of fast ... · The power of fast separation andThe power of fast separation and ... • Final method includes 11 pesticides with 2 ... Avermectin

MRM t t ( d t ti MRM)3.) Quantitative analysis of pesticides (sMRM)

MRM parameter setup (pos. mode static MRM)Compound Name Prec Ion MS1 Res Prod Ion MS2 Res Dwell Frag (V) CE (V)Avermectin B1a 890.6 Unit 567.2 Unit 25 130 4Avermectin B1a 890 6 Unit 305 Unit 25 130 20Avermectin B1a 890.6 Unit 305 Unit 25 130 20Spinosyn D 746.5 Unit 142.1 Unit 25 195 24Spinosyn D 746.5 Unit 98.1 Unit 25 195 48Spinosyn A 732.4 Unit 142 Unit 25 140 35Spinosyn A 732 4 Unit 98 Unit 25 140 55Spinosyn A 732.4 Unit 98 Unit 25 140 55Cypermethrin 435.1 Unit 193 Unit 25 90 10Cypermethrin 433.1 Unit 191 Unit 25 90 10Diflubenzuron 311 Unit 158 Unit 25 80 10Difl b 311 U it 141 U it 25 80 15Diflubenzuron 311 Unit 141 Unit 25 80 15Fensulfothion 309 Unit 281 Unit 25 120 10Fensulfothion 309 Unit 157 Unit 25 120 25Fenhexamid 302 Unit 96.9 Unit 25 100 16F h id 302 U it 55 U it 25 100 40Fenhexamid 302 Unit 55 Unit 25 100 40Aldicarb 208 Unit 116 Unit 25 70 0Aldicarb 208 Unit 89.1 Unit 25 70 15Carbendazim 192.1 Unit 160 Unit 25 120 15C b d i 192 1 U it 132 U it 25 120 35Carbendazim 192.1 Unit 132 Unit 25 120 35Acephate 184 Unit 143 Unit 25 90 5Acephate 184 Unit 95 Unit 25 90 20Methamidophos 142 Unit 125 Unit 25 80 10M th id h 142 U it 94 U it 25 80 10

Page 5

Methamidophos 142 Unit 94 Unit 25 80 10

Page 6: The power of fast separation andThe power of fast ... · The power of fast separation andThe power of fast separation and ... • Final method includes 11 pesticides with 2 ... Avermectin

Pesticide MRM Data Base G1733AA

Page 6

Page 7: The power of fast separation andThe power of fast ... · The power of fast separation andThe power of fast separation and ... • Final method includes 11 pesticides with 2 ... Avermectin

MRM t f ti id f lib ti t d d QC 7 (25 / l)3.) Quantitative analysis of pesticides (sMRM)

MRM traces of pesticides of calibration standard QC_7 (25 ng/ml)

• Final method includes 11 pesticides with 2 MRM transitions each

• D ll ti 25 f h t iti I t d l 2• Dwell time 25 ms for each transition, Inter-scan delay 2 ms

Page 7

Page 8: The power of fast separation andThe power of fast ... · The power of fast separation andThe power of fast separation and ... • Final method includes 11 pesticides with 2 ... Avermectin

3.) Quantitative analysis of pesticides (sMRM)C lib ti f t ti MRM (9 i di id l lib ti l l )Calibration curves for static MRM (9 individual calibration levels)

Res

pons

es

Res

pons

es

MethamidophosR2 = 0.9998

AcephateR2 = 0.9998

Res

pons

es

Res

pons

esAldicarb

R2 = 0.9998 Carbendazim

R2 = 0.9999

Page 8

• Blue triangles show overlay of QC samples (20 replicates for QC 10 ng/ml, 4 replicates for QC 25 ng/ml)

Page 9: The power of fast separation andThe power of fast ... · The power of fast separation andThe power of fast separation and ... • Final method includes 11 pesticides with 2 ... Avermectin

C lib ti f t ti MRM (9 i di id l lib ti l l )3.) Quantitative analysis of pesticides (sMRM)

Calibration curves for static MRM (9 individual calibration levels)R

espo

nses

Res

pons

es

FensulfothionR2 = 0.9998

FenhexamidR2 = 0.9992

Res

pons

es

Res

pons

esSpinosyn AR2 = 1.0000

DiflubenzuronR2 = 0.9997

Page 9

• Blue triangles show overlay of QC samples (20 replicates for QC 10 ng/ml, 4 replicates for QC 25 ng/ml)

Page 10: The power of fast separation andThe power of fast ... · The power of fast separation andThe power of fast separation and ... • Final method includes 11 pesticides with 2 ... Avermectin

C lib ti f t ti MRM (9 i di id l lib ti l l )3.) Quantitative analysis of pesticides (sMRM)

Calibration curves for static MRM (9 individual calibration levels)R

espo

nses

Res

pons

es

Spinosyn DR2 = 0.9998

CypermethrinR2 = 0.9989

Res

pons

es

Avermectin B1aR2 = 0.9996

Page 10

• Blue triangles show overlay of QC samples (20 replicates for QC 10 ng/ml, 4 replicates for QC 25 ng/ml)

Page 11: The power of fast separation andThe power of fast ... · The power of fast separation andThe power of fast separation and ... • Final method includes 11 pesticides with 2 ... Avermectin

Comparison of MRM and D namic MRM4.) Quantitative analysis of pesticides (dMRM)

Comparison of MRM and Dynamic MRM

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 25Time Segment 1 Time Segment 2 Time Segment 3 Time Segment 4

MRM

Compounds (10/block)

Cycle Time (sec)50 800 5 0 8 1 0 7

100 70Cycle Time (sec)

Dynamic MRM

0.5 0.8 1 0.7

Max Coincident

Cycle Time (sec) 0.4 0.4 0.4 0.420 40 40

y

30

• 2 x shorter cycle times supports narrow chromatographic peaks, more analytes or longer dwell per analyte.

Page 11

Page 12: The power of fast separation andThe power of fast ... · The power of fast separation andThe power of fast separation and ... • Final method includes 11 pesticides with 2 ... Avermectin

Dynamic MRM

# of MRMs will vary:Dwell times are adjusted dynamically toadjusted dynamically to keep scan time constant for best quant data quality.

MRMs chosen based

on analyte RT window

y

o a a yte do

On average, the number of MRMs at any time are manyany time are many fewer than with time segment methods, allowing much faster

MS cycle times.

Page 12

Agilent LC/MS Seminar2009

Page 13: The power of fast separation andThe power of fast ... · The power of fast separation andThe power of fast separation and ... • Final method includes 11 pesticides with 2 ... Avermectin

Set p of D namic MRM methods4.) Quantitative analysis of pesticides (dMRM)

Setup of Dynamic MRM methods

• Create table with compound names, MRM transitions and parameters, retentionti d t ti ti i d i EXCEL ( b t i d f M H ttimes and retention time windows in EXCEL (can be automized from MassHunterQuant report)

Page 13

Page 14: The power of fast separation andThe power of fast ... · The power of fast separation andThe power of fast separation and ... • Final method includes 11 pesticides with 2 ... Avermectin

Set p of D namic MRM methods4.) Quantitative analysis of pesticides (dMRM)

Setup of Dynamic MRM methods

• Select “Dynamic MRM” as Scan type from MassHunter Acquisition (allows up to200 t MRM d t 4000 MRM i t t l f t t l ti )200 concurrent MRMs and up to 4000 MRMs in total for constant cycle times)

1. 2.

• Copy table from EXCEL and paste it into MassHunter Acquisition from clipboardCopy table from EXCEL and paste it into MassHunter Acquisition from clipboard

Page 14

Page 15: The power of fast separation andThe power of fast ... · The power of fast separation andThe power of fast separation and ... • Final method includes 11 pesticides with 2 ... Avermectin

Set p of D namic MRM methods4.) Quantitative analysis of pesticides (dMRM)

Setup of Dynamic MRM methods

• Pressing the “Apply” button creates MRM methodS t f ti t MRM t bl d d ll ti i d t ti ll b d• Setup of time segments MRM tables, and dwell times is done automatically basedon the specified cycle time, retention times and retention time windows.

3.

• Calculation of maximum number of concurrent MRMs, minimum and maximumdwell times

Page 15

Page 16: The power of fast separation andThe power of fast ... · The power of fast separation andThe power of fast separation and ... • Final method includes 11 pesticides with 2 ... Avermectin

Ch t hi diti ( d )4.) Quantitative analysis of pesticides (dMRM)

Chromatographic conditions (pos. mode)1200 Series HPLC system consisting of:

- vacuum degasserS- binary pump SL

- wellplate sampler SL- diode array detector SL (not used)

HPLC methodHPLC method

Separation column: ZORBAX Eclipse Plus C-18, 100 x 2.1 mm, 1.8 µm

Guard column: ZORBAX Eclipse Plus C-18, 12.5 x 2.1 mm, 5 µm

Mobile phase: A: 5 mM ammonium formate + 0.01% formic acidB: methanol + 5 mM ammonium formate + 0.01% formic acid

Flow: 0.3 ml/min

Gradient**: 0.00 min 10 % B1.00 min 10 % B

18.00 min 100 % B20.00 min 100 % B20.10 min 10 % B25.00 min 10 % B

Inj.Vol.: 5 µl

Page 16

Page 17: The power of fast separation andThe power of fast ... · The power of fast separation andThe power of fast separation and ... • Final method includes 11 pesticides with 2 ... Avermectin

A il t J t t diti ( d )4.) Quantitative analysis of pesticides (dMRM)

Agilent Jetstream conditions (pos. mode)Spray chamber conditions:Gas temp : 200°CGas temp.: 200 CDry gas: 5 l/minNebulizer: 35 psiSheath gas temp: 250°CSh h fl 10 l/ iSheath gas flow: 10 l/min

Positive NegativeCapVoltage: 4500 V 3000 VCapVoltage: 4500 V 3000 VNozzle voltage 300 V 0 V

• Automatic setup of MRM tables based on selected cycle time, retention times and retention time windows for the individual compoundstime windows for the individual compounds

• Cycle time 750 ms• Interscan delay 2 ms• Total No of MRMs 350• Total No. of MRMs 350• Maximum No. Of concurrent MRMs 66• Minimum Dwell time 7.86 ms• Maximum Dwell time 371 5 ms

Page 17

• Maximum Dwell time 371.5 ms

Page 18: The power of fast separation andThe power of fast ... · The power of fast separation andThe power of fast separation and ... • Final method includes 11 pesticides with 2 ... Avermectin

4.) Quantitative analysis of pesticides (dMRM)C d i l d d i th d i d i MRMCompounds included in method using dynamic MRMs

3-OH CarbofuranAcephateAcetamiprid

ImidaclopridIndoxacarbIprovalicarb

EthofumesateEtoxazoleFamoxadone

ChlorfluazuronChlorotoluronChloroxuron

Spinosyn ASpinosyn DSpirodiclofen

OmethoateOxadixylOxamylp

AldicarbAldicarb sulfoneAldicarb sulfoxidAmetrynAminocarbA it

pIsoprocarbIsoproturonIsoxaflutoleKresoxim-methylLinuronL f

FamphurFenamidoneFenarimolFenazaquinFenbuconazolF h id

ClethodimClofentezineClothianidinCycluronCymoxanilC th i

pSpiromesifenSpiroxaminTebuconazoleTebufenozideTebufenpyradT b thi

yPenconazolePhenmediphamPhoratePhosmetPirimicarbPi i if th lAmitraz

Avermectin B1aAzinphos-ethylAzinphos-methylAzoxystrobinBenalaxyl

LufenuronMalathionMefenazetMepanipyrimMepronilMesotrione

FenhexamidFenitrothionFenoxycarbFenpropimorphFenpyroximatFensulfothion

CypermethrinCyproconazoleCyprodinilDeltamethrinDemethon-S-methyl

-sulfoxide

TebuthiuronTeflubenzuronTemephosTerbutrynTetraconazoleThiabendazole

Pirimifos-methylProchlorazPromecarbPrometonPrometrynPropamocarbBenalaxyl

BendiocarbBenfuracarbBenzoximateBifenazateBitertanol

MesotrioneMetalaxylMethabenzthiazuronMethamidophosMethidathionMethiocarb

FensulfothionFenuronFipronilFlufenoxuronFluometuronFluquinconazole

-sulfoxideDesmediphamDiazinonDichlobutrazolDicrotophosDiethofencarb

ThiabendazoleThiaclopridThiametoxamThidiazuronThiofanoxThiophanat methyl

PropamocarbPropargitePropiconazolePropoxurProthioconazolePymetrozine

BoscalidBromuconazoleBupirimateBuprofezinButocarboxymButoxycarboxim

MethomylMethoprotryneMethoxifenozidMetobromuronMetribuzinM i h

qFlusilazolFlutolanilFlutriafolForchlorfenuronFormetanateF b id l

DifenoconazolDiflubenzuronDimethoatDimethomorphDioxacarbDi

p yTriadimefonTriadimenolTricyclazoleTrifloxystrobinTriflumizoleT ifl

yPyracarbolidPyraclostrobinPyrazophosPyridabenPyrimethanilP i fButoxycarboxim

CarbarylCarbendazimCarbetamidCarbofuranCarboxine

MevinphosMonocrotophosMonolinuronMyclobutanilNeburonNitenpyram

FuberidazoleFurathiocarbHexaconazolHexythiazoxHydramethylnonImazalil

DiuronEdifenphosEmamectin B1aEpoxiconazoleEtaconazoleEthiofencarb

TriflumuronZoxamide

PyriproxyfenQuinoxyfenRotenonSecbumetonSiduronSimetrynCarboxine

Page 18

NitenpyramImazalilEthiofencarb Simetryn

Page 19: The power of fast separation andThe power of fast ... · The power of fast separation andThe power of fast separation and ... • Final method includes 11 pesticides with 2 ... Avermectin

MRM t t ( d d i MRM)4.) Quantitative analysis of pesticides (dMRM)

MRM parameter setup (pos. mode dynamic MRM)Compound Name Prec Ion MS1 Res Prod Ion MS2 Res Average Dwell Frag (V) CE (V)Avermectin B1a 890.6 Unit 567.2 Unit 66.2 130 4Avermectin B1a 890 6 Unit 305 Unit 66 2 130 20Avermectin B1a 890.6 Unit 305 Unit 66.2 130 20Spinosyn D 746.5 Unit 142.1 Unit 17.7 195 24Spinosyn D 746.5 Unit 98.1 Unit 17.7 195 48Spinosyn A 732.4 Unit 142 Unit 14.7 140 35Spinosyn A 732 4 Unit 98 Unit 14 7 140 55Spinosyn A 732.4 Unit 98 Unit 14.7 140 55Cypermethrin 435.1 Unit 193 Unit 35.5 90 10Cypermethrin 433.1 Unit 191 Unit 35.5 90 10Diflubenzuron 311 Unit 158 Unit 10.1 80 10Difl b 311 U it 141 U it 10 1 80 15Diflubenzuron 311 Unit 141 Unit 10.1 80 15Fensulfothion 309 Unit 281 Unit 25.8 120 10Fensulfothion 309 Unit 157 Unit 25.8 120 25Fenhexamid 302 Unit 96.9 Unit 11.6 100 16F h id 302 U it 55 U it 11 6 100 40Fenhexamid 302 Unit 55 Unit 11.6 100 40Aldicarb 208 Unit 116 Unit 73.0 70 0Aldicarb 208 Unit 89.1 Unit 73.0 70 15Carbendazim 192.1 Unit 160 Unit 66.2 120 15C b d i 192 1 U it 132 U it 66 2 120 35Carbendazim 192.1 Unit 132 Unit 66.2 120 35Acephate 184 Unit 143 Unit 148 90 5Acephate 184 Unit 95 Unit 148 90 20Methamidophos 142 Unit 125 Unit 185.5 80 10M th id h 142 U it 94 U it 185 5 80 10

Page 19

Methamidophos 142 Unit 94 Unit 185.5 80 10

Page 20: The power of fast separation andThe power of fast ... · The power of fast separation andThe power of fast separation and ... • Final method includes 11 pesticides with 2 ... Avermectin

Static MRM for selected pesticides (25 ng/ml)

7x104

• Final method includes 11 pesticides with 2 MRM transitions each

• Dwell time 25 ms for each transition, Inter-scan delay 2 ms

Page 20

Page 21: The power of fast separation andThe power of fast ... · The power of fast separation andThe power of fast separation and ... • Final method includes 11 pesticides with 2 ... Avermectin

Dynamic MRM for 175 pesticides (25 ng/ml)

8x104

• Final method includes 175 pesticides with 2 MRM transitions each (350 MRMs)

• Flexible dwell times with a fixed cycle time of 750 ms Inter scan delay 3 5 ms• Flexible dwell times with a fixed cycle time of 750 ms, Inter-scan delay 3.5 ms

Page 21

Page 22: The power of fast separation andThe power of fast ... · The power of fast separation andThe power of fast separation and ... • Final method includes 11 pesticides with 2 ... Avermectin

4.) Quantitative analysis of pesticides (dMRM)C lib ti f d i MRM (9 i di id l lib ti l l )

Res

pons

es

Res

pons

es

Calibration curves for dynamic MRM (9 individual calibration levels)

MethamidophosR2 = 0.9999

AcephateR2 = 0.9999

Res

pons

es

Res

pons

esAldicarb

R2 = 0.9999 Carbendazim

R2 = 0.9999

Page 22

• Blue triangles show overlay of QC samples (20 replicates for QC 10 ng/ml, 4 replicates for QC 25 ng/ml)

Page 23: The power of fast separation andThe power of fast ... · The power of fast separation andThe power of fast separation and ... • Final method includes 11 pesticides with 2 ... Avermectin

4.) Quantitative analysis of pesticides (dMRM)C lib ti f d i MRM (9 i di id l lib ti l l )

Res

pons

es

Res

pons

es

Calibration curves for dynamic MRM (9 individual calibration levels)

FensulfothionR2 = 1.0000

FenhexamidR2 = 0.9995

Res

pons

es

Res

pons

esSpinosyn AR2 = 1.0000

DiflubenzuronR2 = 0.9991

Page 23

• Blue triangles show overlay of QC samples (20 replicates for QC 10 ng/ml, 4 replicates for QC 25 ng/ml)

Page 24: The power of fast separation andThe power of fast ... · The power of fast separation andThe power of fast separation and ... • Final method includes 11 pesticides with 2 ... Avermectin

4.) Quantitative analysis of pesticides (dMRM)C lib ti f d i MRM (9 i di id l lib ti l l )

Res

pons

es

Res

pons

es

Calibration curves for dynamic MRM (9 individual calibration levels)

Spinosyn DR2 = 0.9999

CypermethrinR2 = 0.9958

Res

pons

es

Avermectin B1aR2 = 0.9992

Page 24

• Blue triangles show overlay of QC samples (20 replicates for QC 10 ng/ml, 4 replicates for QC 25 ng/ml)

Page 25: The power of fast separation andThe power of fast ... · The power of fast separation andThe power of fast separation and ... • Final method includes 11 pesticides with 2 ... Avermectin

R d ibilit t t f ik d l (10 / l)5.) Comparison of results for sMRM and dMRM

Reproducibility test for spiked sample (10 ng/ml)Peak Areas Solvent

(n = 20)Citron(n = 5)

Carrot(n = 5)

Cucumber(n = 5)

Tobacco(n = 5)

dMRM sMRM dMRM sMRM dMRM sMRM dMRM sMRM dMRM sMRM

MethamidophosAverage

CV276770 95

265792 22

229460 82

216752 40

229522 23

228792 16

224752 68

215911 40

3144 98

27315 1CV 0.95 2.22 0.82 2.40 2.23 2.16 2.68 1.40 4.98 15.1

AcephateAverage

CV248131.62

237652.17

238542.30

233281.36

252351.65

244610.75

254281.84

245820.82

19026.06

147812.6

CarbendazimAverage

CV897200.94

860271.66

534130.80

518371.20

538141.42

525320.92

919441.00

898120.61

64491.77

59202.63

AldicarbAverage

CV310691.21

298591.77

166870.75

162601.23

309241.76

302451.12

282141.37

267071.15

31991.20

27883.05

FensulfothionAverage 29533 28304 22060 21476 33777 32610 31789 30235 3661 3296g

CV 1.55 1.38 0.73 1.59 1.29 1.78 2.47 0.76 3.85 3.10

FenhexamidAverage

CV60132 43

57912 54

50091 14

46253 57

93314 48

94332 85

47008 50

54246 98

21656 83

26406 50CV 2.43 2.54 1.14 3.57 4.48 2.85 8.50 6.98 6.83 6.50

Page 25

Page 26: The power of fast separation andThe power of fast ... · The power of fast separation andThe power of fast separation and ... • Final method includes 11 pesticides with 2 ... Avermectin

R d ibilit t t f ik d l (10 / l)5.) Comparison of results for sMRM and dMRM

Reproducibility test for spiked sample (10 ng/ml)Peak Areas Solvent

(n = 20)Citron(n = 5)

Carrot(n = 5)

Cucumber(n = 5)

Tobacco(n = 5)

dMRM sMRM dMRM sMRM dMRM sMRM dMRM sMRM dMRM sMRM

Diflubenzuron Average

CV71302.46

67202.40

16396.14

17443.76

68024.68

63981.40

66092.02

62791.57

15034.98

24319.87

Spinosyn AAverage

CV153897

1.12151002

1.23142868

0.86143027

0.47114625

0.97114595

0.28112952

0.76109072

0.84231941.57

236130.80

Spinosyn Dp yAverage

CV442890.75

432481.88

410650.48

408070.61

230300.51

230671.13

259790.35

251540.92

58401.94

58283.51

CypermethrinAverage 1921 1817 2657 2775 1605 1457 1725 1684 1313 963

CV 4.20 4.46 4.26 4.30 6.96 2.61 1.11 5.00 36.79 14.62

Avermectin B1aAverage

CV105153.34

42825.90

113941.43

55561.06

105402.50

49024.65

70823.49

35622.55

121510.26

6982.63

• Cycle time has been 750 ms for both methods (resulting in about 15 data points across the chromatographic peak)

• Peak areas have been very similar except for Avermectin B1a which showed bigger response for dMRM method (peak elutes very late in chromatogram and therefore dwell time was adjusted to significantly higher

Page 26

(p y g j g y gvalues from acquisition software)

Page 27: The power of fast separation andThe power of fast ... · The power of fast separation andThe power of fast separation and ... • Final method includes 11 pesticides with 2 ... Avermectin

Agilent 1290 Infinity LC Attributes for MS Infinitely Better for LC/MS- Infinitely Better for LC/MS

LowestDelay

Pump (w/o) mixer: 10 µLPump, Fixed Loop 20 µLDelay

VolumePump, Fixed Loop 20 µLPump, Fixed Loop, JetWeaver 55 µL

ALS precision for small volumes:Highest

Precision

ALS precision for small volumes: <1.5% from 0.5-1µLm, <0.7% from 1-2µL, <0.25% @ 2-20 µl (40 ul) **

Pump Active Damping:RT stability < 0 2 % (1 5 min runs)**

<0.002% carry-over with ChlorhexidineOptional needle seat backflushing with FlexCube

RT stability < 0.2 % (1.5 min runs)

BestAutosampler Optional needle seat backflushing with FlexCube

Fixed Loop or Variable Loop Injections

1200bar @ 2mL/min for highest resolution per time

pPerformance

1200bar @ 2mL/min for highest resolution per timeReduced Ion & Matrix Suppression

HT-Solution for up to 2000 samples/day (ACR)Complete Integration and control from MassHunterEnables method conversion from/to any (U)HPLC

GreatestProductivity

Enables method conversion from/to any (U)HPLC

** preliminary Minimum Specs

Page 27

Page 28: The power of fast separation andThe power of fast ... · The power of fast separation andThe power of fast separation and ... • Final method includes 11 pesticides with 2 ... Avermectin

TIC t ( / d ) f diff t UHPLC di tAgilent 1290 Infinity LC methods for pesticides

TIC traces (pos/neg mode) for different UHPLC gradients4x10

3

+ESI TIC MRM CID@** (** -> **) pos_Cal_06.d

1 1Gradient program

0 min 10% B

1

2

3 0 min 10% B1 min 10 % B

18 min 100 % B20 min 100 % B

5x10

0.8

1

1.2+/-ESI TIC MRM CID@** (** -> **) pest test-0016.d

1 1 Gradient program0 min 20% B

0.2

0.4

0.6

5x10 +/-ESI TIC MRM CID@** (** -> **) pest test-0015.d

10 min 100 % B12 min 100 % B

0.5

0.75

1

1.25 1 1 Gradient program0 min 20% B5 min 100 % B

• Fi l th d i l d 11 ti id ith 2 MRM t iti h

0.25

Counts 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

7 min 100 % B

• Final method includes 11 pesticides with 2 MRM transitions each

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MRM t f A ti B1 f C l 6 (10 / l)Agilent 1290 Infinity LC methods for pesticides

MRM traces of Avermectin B1a for Cal_6 (10 ng/ml)2x103 5x103 8x103

RRLC method(25 min gradient)RT 18 98 min

1290 method(12 min gradient)RT 9 70 min

1290 method(7 min Gradient)

RT 5 51 minRT 18.98 minWidth 0.346 min

RT 9.70 minWidth 0.127 min

RT 5.51 minWidth 0.091 min

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Agilent 1290 Infinity LC + 6460A Triple Quad with Agilent Jet Stream TechnologyAnalysis of 300 Pesticides in 15 min.

Agilent 1290 Infinity LC + 6460A Triple Quad with Agilent Jet Stream Technology~600 Dynamic MRM Transitions

9 li t l d t 200 l l9 replicates analyzed at 200 pg levelMean area %RSD = 3.2, Mean height %RSD= 3.6

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~ 40 Pesticides Eluting in a 1 minute RT Window

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5 pg Atrazine on-column

••

Avg. Signal Area: 6,069Avg. Signal Height: 2,371 7 data points above FWHM

7 data points above FWHM2.2 sec FWHM

• •

4.8 sec @10% valley (14 data pts)10.2 sec peak width baseline-to-baseline (27 data points total)

• •

2.2 sec FWHM

baseline (27 data points total)

S/N: 119:1 (5*RMS noise) •

••

•Estimated LOQ: 500 fg or less

••

••

• • • • •• • •• •

10% valley

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Fast Polarity switching and fast MRMs with 6460 6430 performance should be nearly identical6430 performance should be nearly identical

The verapamil peak eluted in

Avg. W1/2 = 0.72 sec

p ptwo seconds from a sub-2-micron column at pressures exceeding a 1,000 bar with Agilent’s new 1290 InfinityAvg. W1/2 0.72 sec Agilent s new 1290 Infinity LC System. The verapamilion transition from 455 to 165 was monitored more than fifteen times across the peak with multiple injections demonstrated a peak area RSD of 5% The cycle time

W = 2.1 sec

RSD of 5%. The cycle time for six ion transitions and positive/negative polarity switching was less than 115

Cycle time < 115 ms

gmilliseconds.

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Assay Precision (1290/6460, dynamic MRM)

Diazepam, 500fg on-columnDiazepam, 100fg on-column

1x10

6 556.6

6.656.7

6.75

N= 5 (@500fg), %RSD (area) = 2.67

N= 35 (all Cal levels), %RSD (RT) = 0.03

unts

5 855.9

5.956

6.056.1

6.156.2

6.256.3

6.356.4

6.456.5

6.55

Diazepam - 7 Levels, 7 Levels Used, 35 Points, 35 Points Used, 0 QCs4x10

1.3

1.4

1.5

y = 143.2177 * x - 1.2275R^2 = 0.99970154

Cou

5.155.2

5.255.3

5.355.4

5.455.5

5.555.6

5.655.7

5.755.8

5.85

0.7

0.8

0.9

1

1.1

1.2

4 44.45

4.54.55

4.64.65

4.74.75

4.84.85

4.94.95

55.05

5.1

0.1

0.2

0.3

0.4

0.5

0.6

R2=0.99970154Quant & Qual transitions20% outlier ratio boundary

4.054.1

4.154.2

4.254.3

4.354.4

Counts vs. Acquisition Time (sec)271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306

Concentration (ppb)-5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105

-0.1

0

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SUMMARYAgilent LC/MS has achieved a new, higher level of performance

Combined operation in MassHunter software

Agilent 1290 Infinity LC allows fast, hi resolution separationsHuge peak capacity for complex separationsHuge peak capacity for complex separationsExcellent injection and RT precisionLow delay volume for very fast methodsy yVery low AS carry-over

Agilent MS systems can keep up!g y p pFast pos/neg switchingFast MRM and Dynamic MRM methods for triple quady p qExcellent sensitivity and linear dynamic range

Fast scanning TOF and Q-TOF without loss of spectral quality or resolution.

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SUMMARY

Agilent LC/MS can meetthe most demandingthe most demandingapplication needs.

Agilent 1290 Infinity LC and Agilent 6530 Accurate Mass Q-TOF MSThe ultimate Qual solution

Agilent 1290 Infinity LC and Agilent 6460 Triple Quad MSThe ultimate Quan solution

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