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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
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
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
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
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
Pesticide MRM Data Base G1733AA
Page 6
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
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)
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)
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)
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
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
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
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
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
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
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
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
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
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
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
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)
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)
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)
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
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)
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
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
Page 28
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
Page 29
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
Page 30
~ 40 Pesticides Eluting in a 1 minute RT Window
Page 31
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
Page 32
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.
Page 33
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
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.
Page 35
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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