lunch & learn at imsc 2009 - agilent imsc... · agilent’s new 6540 ultra high definition...
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Welcome to Agilent’s
Lunch & Learn at IMSC 2009
Agilent Lunch & Learn
Monday 13:00-14:00
Introducing the new Agilent 6540 and
6538 Ultra High Definition (UHD)
Accurate-Mass Q-TOF LC/MS Systems
• 1290 Infinity UHPLC
• Fast, Accurate, High Resolution Q-
TOF
• Using Peak Capacity to Improve
Screening and Complex Sample
Profiling
Ken Imatani & Pat Perkins, Agilent, CA
Agilent Hospitality Suite Monday
Time: 18:15-21:00
Room: Danzig
Agilent Elemental BingoStarts at 19:00
Great prizes to be won!
Drinks and Snacks will be
provided.
Enter & WinWe will be running daily competitions during IMSC
2009, ask a member of staff for more details!
Happy HourDaily from 16:00-18:00
at the Agilent booth (B)
Ultra High Definition
Optimizing all Analytical Dimensions
Signal Response
Mass Spectrum
• Sensitivity
• Dynamic Range
• Linearity
• Mass Accuracy
• Resolving Power
• Acquisition Rate
• Separation Speed
• Peak Capacity
August 2009
IMSC 2009 Infinity LC 6540 QTOF lunch and learn
Page 3
Agilent’s New 6540 Ultra High Definition
Accurate Mass Q-TOF
• 500 ppb mass accuracy
• femtogram sensitivity
• 5 decades dynamic range
• 40,000 resolving power
• 20 Spectra/sec
• Excellent Linearity and Isotopic
Fidelity
• Supports Agilent Jet Stream and
HPLC-Chip
Exceptional accurate mass, sensitivity,
dynamic range and resolution … perfect
match for 1290 Infinity UHPLC
August 2009
IMSC 2009 Infinity LC 6540 QTOF lunch and learn
Page 4
Ion Beam Compression (IBC)* Technology
Drives Higher Resolution
Up to 10-fold ion beam compression
Exit from collision cell
Into slicer and pulser region
Narrowed beam slit enables mass resolution
of 40K
Compressed and cooled ion beam ensures the best sensitivity
performance in high resolution mode
* Patent pending
August 2009
IMSC 2009 Infinity LC 6540 QTOF lunch and learn
Page 5
6540
UHD Q-
TOF
6530
Q-TOF
• 1ppm/C Expansion Coefficient for Inner
Flight Tube virtually eliminates calibration
drift due to flight tube elongation.
• 2nd Order Temporal Focusing Ion Mirror
uses high transmission Harp Grid for
maximum sensitivity
Enhanced Ion Flight Tube and Mirror TechnologyStable, Sensitive, High Resolution
DC Quad
Octopole 2
Ion Pulser
Ion Mirror
Detector
Turbo
August 2009Page 6
IMSC 2009 Infinity LC 6540 QTOF lunch and learn
Next Generation – Ultra High Speed Detector
Page 7
IMSC 2009 Infinity LC 6540 QTOF lunch and learn
New Bipolar TOF Detector
• New ultra fast and high efficiency scintillator
• New ultra fast response PMT design continues the tradition of high dynamic range and detector lifetime
• Developed by Photonis with Agilent TOF Technology
• Specifically enhances Resolution in 2Ghz Ext. Dynamic Range Mode
Single Ion Response
~800 psec FWHM
2 nsec/div
August 2009
6540 Ultra High Definition QTOF
Maintaining Resolving Power – Across the Mass Range
5x10
Counts vs. Mass-to-Charge (m/z)
200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800
922
R=42424622
R=38702
1221
R=43674
1521
R=42750322
R=302182121
R=418251821
R=404052421
R=39332 2721
R=37207
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
5.5
Scan Rate Independent
Counts vs. Mass-to-Charge (m/z)
622.00822
623.01059
624.01219 625.01471
m/z 622 and isotopes
August 2009
IMSC 2009 Infinity LC 6540 QTOF lunch and learn
Page 8
6540 Ultra High Definition QTOF
Mass Accuracy – Repetitive Injections
+ESI EIC(609.28066)
Scan Frag=240.0V Reserpine_40pgms3.d
0.511
Counts vs. Acquisition Time (min)
0.0 0.25 0.5 0.75 1
5x10
0
0.2
0.4
0.6
0.8
1
1.2609.28081
610.28425
611.28650
612.28987
Counts vs. Mass-to-Charge (m/z)
609 610 611 612 613 614
613.29210
Isotope Obs % Calc % Obs m/z Calc m/z Diff (ppm)
1 100 100 609.28081 609.28066 0.25
2 35.87 37.24 610.28425 610.28393 0.52
3 8.58 8.59 611.28650 611.28671 -0.34
4 1.41 1.48 612.28987 612.28941 0.75
5 0.14 0.21 613.29210 613.29203 0.11
Run
Error (ppm)
1 0.96
2 -0.17
3 0.25
4 0.02
5 0.39
6 0.13
7 0.01
8 0.52
9 0.04
10 0.30
Mean 0.25
Std. Dev 0.32
40pg reserpine on-column, 10 injections
250 ppb mass accuracy calibration and very accurate isotopic ratios
August 2009
IMSC 2009 Infinity LC 6540 QTOF lunch and learn
Page 9
6540 Ultra High Definition QTOF
Sensitivity – Full Scan MS Mode- 1 picogram
3x10
0
1
2609.280347
(M+H)+
610.283168
611.287790
612.292649
Counts vs. Mass-to-Charge (m/z)
607 608 609 610 611 612 613 614 615 616
4x10
0
1
2
Counts vs. Acquisition Time (min)
0.0 0.5 1 1.5
S/N = 319 RMS
Ratio m/z Diff.Theor. Expt. Theor. Expt. (ppm)100.00 100.00 609.2807 609.2803 -0.5137.24 36.36 610.2839 610.2832 -1.258.59 8.39 611.2867 611.2878 1.771.48 1.45 612.2894 612.2926 5.29
Resolution ~ 33,000
Excellent sensitivity, accuracy and resolution at 1 pg on column
August 2009
IMSC 2009 Infinity LC 6540 QTOF lunch and learn
Page 10
Topics
• Example: Pesticides in Foods
• Example: Drug metabolite identification
• Example: Peptide Mapping
August 2009
IMSC 2009 Infinity LC 6540 QTOF lunch and learn
Page 11
Pesticides Screening in Foods
• Accurate-Mass Databases for Comprehensive Screening of Pesticide Residues in Food by Fast
Liquid Chromatography Time-of-Flight Mass Spectrometry, Milagros Mezcua, Octavio Malato, Juan F.
García-Reyes, Antonio Molina-Díaz, and Amadeo R. Fernández-Alba, Anal. Chem., 2009, 81 (3), 913-929
DOI: 10.1021/ac801411t
• Method for screening 297 pesticides in foods with Agilent 1100 HPLC, 4.6 x 50 mm XDB-C18, 1.8 µm
column, and TOF-MS detection
• Component identification using accurate mass/retention time (AMRT) matching against a database
• 0.6 mL/min flow rate, 12 minute gradient, approximately 23 minutes run time
• Comparable method performance when tested on 60 commercial produce samples (QuEChERS protocol
sample prep) and compared with results from QQQ [note: 45/60 samples tested positive!]
• Identified several pairs of “isobaric coeluting species” (ICS) which have similar retention times and
accurate mass values for their adduct ions or fragments
August 2009
IMSC 2009 Infinity LC 6540 QTOF lunch and learn
Page 12
Focused AMRT Databases
(Personal Compound Databases—PCD)
• Pesticides, Part # (G6854AA):
• 1600 analyte content (formulae & accurate mass)
• Structures
• CAS Numbers & Links to external website (pubchem)
• Forensic & Toxicology, Part # (G6855AA):
• ~7000 analytes content (formulae & accurate mass)
• Structures
• CAS Numbers & Links to external website (pubchem)
August 2009
IMSC 2009 Infinity LC 6540 QTOF lunch and learn
Page 13
Starting Conditions for Pesticides Analysis
• Configuration
• 1290 Infinity UHPLC, 6540 QTOF with Agilent jet stream technology
• Eclipse Plus C18 column, 2.1 x 50 mm or 100 mm, 1.8 µm
• Analysis conditions
• A = 0.1% formic acid in water, B = 0.1% formic acid in ACN; various flow
rates (1.2, 1.0, 0.5 mL/min); 60 ºC column temperature
• Gradient = 5% B to 95% B in various times (1.5, 3, 7 , 8.5, 17 minutes)
• 3200 m/z maximum mass, high resolution mode
• MS mode: 100-1000 m/z, various acquisition rates (10, 5, 3, 1
scans/second)
August 2009
IMSC 2009 Infinity LC 6540 QTOF lunch and learn
Page 14
RRLCUHPLC
August 2009
0.5 mL/min 2.1 x 100 mm column,
60 ºC, max pressure 410 bar
1.0 mL/min 2.1 x 100 mm column,
60 ºC, max pressure 780 bar
1.2 mL/min 2.1 x 100 mm column,
60 ºC, max pressure 930 bar
1.2 mL/min 2.1 x 50 mm column,
60 ºC, max pressure 510 bar
Page 15
IMSC 2009 Infinity LC 6540 QTOF lunch and learn
Mass Accuracy and Mass Resolution and Analysis
Speed Simultaneously
IMSC 2009 Infinity LC 6540 QTOF lunch and learn
August 2009
-0.60 ppm
R = 30,600-0.37 ppm
R = 31,400
O
O
O
NN
O
N
OCH3 CH3
azoxystrobin
1 spectrum/second
10 spectra/second
Page 16
Mass Accuracy is Invariant with Acquisition Rate
Pesticides Example
August 2009
-2.00
-1.50
-1.00
-0.50
0.00
0.50
1.00
1.50
2.00
0 1 2 3 4 5 6 7 8 9 10 11 12
Mass E
rro
r (p
pm
)
Acquisition Rate (scans/second)
azoxystrobin 404 m/z
cyproconazole 292 m/z
cyprodinil 226 m/z
diazinon 305 m/z
linuron 249 m/z
paclobutrazol 294 m/z
propazine 229 m/z
tetraconazole 372 m/z
tricyclazole 190 m/z
IMSC 2009 Infinity LC 6540 QTOF lunch and learn
Page 17
MS Resolution is Invariant with Acquisition Rate
Pesticides Example
August 2009
0
5000
10000
15000
20000
25000
30000
35000
0 1 2 3 4 5 6 7 8 9 10 11 12
Reso
luti
on
Acquisition Rate (scans/second)
azoxystrobin 404 m/z
cyproconazole 292 m/z
cyprodinil 226 m/z
diazinon 305 m/z
linuron 249 m/z
paclobutrazol 294 m/z
propazine 229 m/z
tetraconazole 372 m/z
tricyclazole 190 m/z
IMSC 2009 Infinity LC 6540 QTOF lunch and learn
Page 18
Methanol Mobile Phase for Pesticides Analysis
• Configuration
• 1290 Infinity UHPLC, 6540 QTOF with Agilent jet stream technology
• Eclipse Plus C18 column, 2.1 x 50 mm, 1.8 µm
• Analysis conditions
• A = 2 mM ammonium formate, 0.01% formic acid in water, B = 2 mM
ammonium formate, 0.01% formic acid in methanol; flow rate 0.8 mL/min,
40 ºC column temperature
• Gradient = 5% B to 95% B in 3 min, stop time 4 min (standard) or 10 min
(sample with matrix)
• 1700 m/z maximum mass, high resolution mode
• MS mode: 100-1000 m/z, 3 scans/second)
August 2009
IMSC 2009 Infinity LC 6540 QTOF lunch and learn
Page 19
Chamberlain, P., Hall, T., Zavitsanos, P. Agilent Technologies application note 5989-1688EN, January 2005.
Better Response with Methanol Mobile Phase
IMSC 2009 Infinity LC 6540 QTOF lunch and learn
August 2009Page 20
0
100000
200000
300000
400000
500000
600000
700000
800000
Res
po
nse
(are
a c
ou
nts
)
MeOH gradient, 2 mM ammonium formate, 0.01% formic acid ACN gradient, 0.1% formic acid
Chromatographic Observations using Standards
Same Instrument and Column
August 2009
IMSC 2009 Infinity LC 6540 QTOF lunch and learn
Page 21
3.0642.726
Acquisition Time (min)
1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2
2 mM ammonium
formate + 0.01%
formic acid
pH ~ 3.5
2 mM ammonium
acetate
pH ~ 5.0
2 mM ammonium
acetate + 0.02%
acetic acid
pH ~ 4.0
Thiabendazole, pKa 4.73
N
NH
N
S
4.564
4.4743.543
Acquisition Time (min)
3.4 3.6 3.8 4 4.2 4.4 4.6 4.8
OH
CH3
N
NN
Cl
HOH
H
N
NN
Cl
CH3
Cyproconazole
ACN gradient
0.1% formic acid
Methanol gradient
2 mM ammonium
formate + 0.01%
formic acid
37Cl isotopomer
Isobaric Co-eluting Species
Example: 63 ppm Mass Difference (18 mDa)
IMSC 2009 Infinity LC 6540 QTOF lunch and learn
August 2009Page 22
Isobaric Co-eluting Pair m/z ppm
Difference
Differentiate by
RT No fragment m/z
Tricyclazole
Propazine
190.0433
190.0667
123 X
(1.3 min)
X
Methiocarb
Diethofencarb fragment
226.0896
226.1074
79 X X
Diethofencarb fragment
Cyprodinil
226.1074
226.1339
117 X
(0.53 min)
X X
Cyproconazole -37Cl
Paclobutrazol
294.1182
294.1368
63 X
Clofentezine-37Cl
Diazinon
305.0618
305.1083
300 X
Chlorfenvinphos
Triflumuron
358.9768
359.0405
177 X
Tetraconazole
Azoxystrobin fragment
372.0288
372.0979
186 X X
294.13690
294.11840
Mass-to-Charge (m/z)
294.08294.10
294.12294.14
294.16294.18
Paclobutrazol
0.44 ppm
R=29,600
Cyproconazole-37
Cl
0.66 ppm
R=30,500
Identify These Pesticides By Isotopic Pattern And
Mass
cyproconazole
paclobutrazol
Page 23 August 2009
IMSC 2009 Infinity LC 6540 QTOF lunch and learn
Pesticides in Pepper Matrix, 10 ppb
3 Scans/sec Acquisition Rate
IMSC 2009 Infinity LC 6540 QTOF lunch and learn
August 2009Page 24
163.05348(M+H)+
164.05652(M+H)+ 165.04954
(M+H)+
163 164 165
Methomyl
-0.90 ppm
R =24,200
258.05655
(M+H)+
257.06237
(M+H)+259.05936
(M+H)+
(M+H)+
256 257 258 259 260
256.05959(M+H)+
260.06128
Imidacloprid
-0.06 ppm
R =28,900
(M+H)+
269.15754(M+H)+
(M+H)+
268 269 270
268.15425
270.13975
Diethofencarb
-0.33 ppm
R =29,100
(M+H)+
361.03783(M+H)+
360.04448(M+H)+
362.04095(M+H)+
(M+H)+
359 360 361 362 363
363.03121
359.04026
Triflumuron
-0.60 ppm
R =31,700
489.04371(M+H)+
491.04126(M+H)+
490.04738(M+H)+ 492.04627
(M+H)+
489 490 491 492
Flufenoxuron
-0.34 ppm
R =34,500
2.872 3.162
1.048
1.2131.232
1.318
1.331
1.5221.897
2.069
2.233
2.233 2.470
2.483
2.589
2.661
2.780
2.8392.8651.700
Acquisition Time (min)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 2.4 2.5 2.6 2.7 2.8 2.9 3 3.1 3.2 3.3
2.549
2.865
2.464
2.4642.457
Acquisition Time (min)1 2 3 4 5 6 7 8 9
TIC
Matrix Suppression Effect
Response of Standard = 1
IMSC 2009 Infinity LC 6540 QTOF lunch and learn
August 2009Page 25
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
Atr
azin
e
Azo
xys
tro
bin
Ca
rba
ryl
Ca
rbe
nd
azim
Ca
rbo
fura
n
Ch
lorf
en
vin
ph
os
Clo
fen
tezin
e
Cyp
roc
on
azo
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Cyp
rod
inil
De
iso
pro
pyla
tra
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Dia
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on
Die
tho
fen
ca
rb
Flu
dio
xo
nil
Flu
fen
ox
uro
n
Ima
za
lil
Ima
za
pyr
Imid
ac
lop
rid
Lin
uro
n
Me
thio
ca
rb
Me
tho
myl
Mo
lin
ate
Mo
nu
ron
Pa
clo
bu
tra
zo
l
Pro
ch
lora
z
Pro
pa
zin
e
Te
tra
co
na
zo
le
Th
iab
en
da
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le
Tri
cycla
zo
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Tri
flu
mu
ronR
es
po
ns
e r
ela
tive
to
sta
nd
ard
spiked pepper matrix spiked tap water matrix
Metabolite ID
August 2009Page 26
IMSC 2009 Infinity LC 6540 QTOF lunch and learn
Experimental Conditions for Verapamil Metabolite
ID Analysis
• Configuration
• 1290 Infinity UHPLC, 6540 QTOF with Agilent jet stream technology
• Eclipse Plus C18 column, 2.1 x 50 mm, 1.8 µm
• Analysis conditions
• A = 0.1% formic acid in water, B = 0.1% formic acid in ACN; 1.2 mL/min;
60 ºC column temperature
• Gradient = 5% B to 95% B in 1.0 minute, stoprun 1.5 minutes, postrun
0.5 minutes
• 1700 m/z maximum mass, extended dynamic range mode
• MS mode: 100-1000 m/z, 3 scans/second
August 2009
IMSC 2009 Infinity LC 6540 QTOF lunch and learn
Page 27
Verapamil: Major and Minor Metabolites (Phase I)
August 2009
desmethyl
Isopropyl
dealkylation
monohydroxy
dihydroxy
TICverapamil
IMSC 2009 Infinity LC 6540 QTOF lunch and learn
Page 28
6x10
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
2.8 455.290321
(M+H)+
441.274704
471.284287487.279822
(M+H)+
Counts vs. Mass-to-Charge (m/z)435 440 445 450 455 460 465 470 475 480 485 490 495 500 505
Coeluting Metabolites with Parent Drug: Need
Wide Dynamic RangeFive decades of response in a
single scan
2.68 million
counts
25 counts
verapamil dihydroxy metabolite of verapamil
desmethyl
metabolite
monohydroxy
metabolite
400
counts
August 2009
IMSC 2009 Infinity LC 6540 QTOF lunch and learn
Page 29
Metabolite ID Results
August 2009
IMSC 2009 Infinity LC 6540 QTOF lunch and learn
Page 30
Rapid Peptide Mapping
August 2009Page 31
IMSC 2009 Infinity LC 6540 QTOF lunch and learn
6530 vs. 6540: Comparison of Peptide Mass
Spectra
CCTKPESERMPCTEDYLSLILNR
+4
+4
+3
+3
+2
+2
6530 Q-TOF
6540 Q-TOF
6530 Q-TOF
6540 Q-TOF
6530 Q-TOF
6540 Q-TOF
August 2009Page 32
IMSC 2009 Infinity LC 6540 QTOF lunch and learn
Peptide Mapping: Serotransferrin
Peak width 1.1 sec
27 data points
0.68 ppm
August 2009Page 33
IMSC 2009 Infinity LC 6540 QTOF lunch and learn
Summary
• Unmatched chromatographic peak capacity and resolving power—can
even resolve diastereomers when parameters are adjusted appropriately
• Superb MS mass accuracy and resolution—invariant with acquisition rate
• Sophisticated identification software and algorithms—handle complex
samples, overlapping isotopic patterns
• Sum (product?) is greater than the parts
• Run faster chromatography for higher throughput
– MS can keep up without sacrificing resolution
– MS can keep up without sacrificing mass accuracy
– MS has the dynamic range to handle coeluting peaks
– Software has the power to identify components of complex
chromatograms and spectra
– Result: more information and results per analysis and per unit time