ACQUITY System Solutions

ACQUITY UPLCTM
System Solutions
ACQUITY UPLCTM
For Complete Innovation
Ultra Performance LCTM is a new category of separation science

that brings novel and powerful capabilities to the laboratory. UPLCTM
leverages the theories and principles of HPLC. It incorporates small
particles, very low system volumes, and fast detection for increased
throughput, sensitivity and peak capacity.
ACQUITY Ultra Performance LCTM Systems have been holistically
designed to control and optimize all the parameters required to take
full advantage of the benefits of UPLC in todays laboratory.
Waters is leading the way into a new era of Separation Science

2004 Waters Corporation
For Complete Confidence

2004 Sales Launch Workshops
Waters Corporate Office

y ACQUITY UPLCTM Laboratory Workshop MassLynx\Empower
y ACQUITY UPLCTM System Overview Hardware
y ACQUITY UPLCTM System Overview - Software & Connections InsightTM
y ACQUITY UPLCTM System Solutions
Providence Biltmore Hotel

y WATERS Laboratory Informatics
y ACQUITY UPLCTM Column Chemistry
y ACQUITY UPLCTM Sales Collateral
y ACQUITY UPLCTM Sales Positioning
ACQUITY UPLCTM Can Fit Anywhere

Screening
CombiChem Lib.
Lib. Composition
Composition &
&
CombiChem
Purity
Purity
Physicochemical Properties
Properties
Physicochemical
in vitro ADME
Characterization
Toxicology & Disease

Markers
Natural Products
Products
Natural
in vivo Characterization
Stability
Purification
Natural Products
CombiChem Products
Synthesis Products
Metabs, Impurs,
Contams, Degrads
Quantification
ADME(T)
Delivery, Formulation &

Release
Bioanalysis
Routine Quan, QA/QC
Identification
Targets
Ntrl Prods,
Prods, Impurs,
Impurs,
Ntrl
Contams, Degrads
Degrads
Contams,
Metabolites
Metabolites
Patent Protection/
Product Extension
Method
Development
Monitoring
MD for Wide Range of

Compounds
Synthesis
Products
Formulation &
Release
Optimization
Purity in Bulk Drug
Analysis
Optimization & Validation
Storage Effects
Trace Analysis
Immediate Impact Applications

y These Application Areas will be Significantly Impacted by
ACQUITY UPLCTM
Peptide Mapping
Tandem Quadrupole Quantification
Metabolite Identification
Metabonomics
Agenda
y Customer Challenges
y ACQUITY UPLCTM System Solutions
Proof Statements
y Competition
There is none for UPLCTM
y Meeting Customer Need
Transition Velocity
Particle Size Evolution
Properly designed small particles allow us to achieve

higher speeds, AND increased resolution
Summary of Theoretical Trade-offs

y At constant column length (improving Resolution), 1.7 m
particles offer:
1.7x (+70%) improvement in Resolution vs. 5 m particles (1.3x vs. 3 m)
3x (+300%) improvement in Speed vs. 5 m particles (2x vs. 3 m)
1.7x (+70%) improvement in Sensitivity vs. 5 m particles (1.3x vs. 3 m)
y At constant L/dp (improving Speed), 1.7 m particles offer:

Same Resolution
9x (+900%) improvement in Speed vs. 5 m particles (3x vs. 3 m)
3x (+300%) improvement in Sensitivity vs. 5 m particles (2x vs. 3 m)
ACQUITY UPLC
Systems for Peptide
Mapping
Peptide Mapping
DISCOVERY
Basic & Target
Applied
ID
DEVELOPMENT
Lead
Lead
Lead
Lead
ID Isolation Characterization Testing
Lots of
searching &
identifying
Lead
Pilot
Optimization Scale
Lots of
characterizing
and optimizing
Peptide Mapping MS/MS
Peptide Mapping MS
Peptide Mapping UV
PRODUCTION
Clinical
Trials
QA &
QC
Full Scale
Manufacturing
Lots of
analyzing
Peptide Mapping Challenges in QC

y Samples are complex
Protein digests
y Need resolution of 10s 100s peptides

For stability, purity and ID
y Need sensitivity
For stability and purity
y Require reproducibility
QC means that you get the same answer each time
y Speed
Currently 1-2 hr runs
Has not been a feasible option.
High Resolution Peptide Mapping 100 pmol:

Influence of Particle Size
0.08
5 m
Peaks = 70
Pc = 143
AU
0.06
0.04
0.02
HPLC
0.00
0.08
AU
0.06
0.04
UPLC
0.02
0.00
0.00
5.00
10.00
15.00
20.00
25.00
30.00
Minutes
35.00
40.00
45.00
50.00
55.00
60.00
1.7 m
Peaks = 168
Pc = 360
TM
High Resolution Peptide Mapping:

Influence of Particle Size (Enhanced Baseline)
This appears in the flip chart

0.020
5 m
Peaks = 70
Pc = 143
AU
0.015
0.010
0.005
0.000
0.020
1.7 m
Peaks = 168
Pc = 360
AU
0.015
0.010
0.005
0.000
0.00
5.00
10.00
15.00
20.00
25.00
30.00
Minutes
35.00
40.00
45.00
50.00
55.00
60.00
High Resolution Peptide Mapping:

Influence of Particle Size (Enhanced Baseline)
0.020
5 m
Peaks = 70
Pc = 143
AU
0.015
0.010
0.005
0.000
0.020
AU
0.015
1.7 m
Peaks = 168
Pc = 360
0.010
0.005
0.000
20.00
21.00
22.00
23.00
24.00
25.00
26.00
27.00
28.00
29.00
30.00
Minutes
A decrease in particle size yields higher resolution

Injection-to-Injection Reproducibility
50 pmol Enolase Tryptic Digest
0.070
0.060
0.050
Injection 5
0.040
AU
Injection 4
0.030
Injection 3
Injection 2
0.020
0.010
Injection 1
0.000
0.00
Std Dev of <0.03 minutes over 1 hr

5.00
10.00
15.00
20.00
25.00
30.00
Minutes
35.00
40.00
45.00
50.00
55.00
60.00
Injection-to-Injection Reproducibility
50 pmol Phosphorylase b Tryptic Digest
0.014
0.012
0.010
AU
0.008
0.006
0.004
0.002
Std Dev of <0.07 minutes over 2 hrs

0.000
0.00
10.00
20.00
30.00
40.00
50.00
60.00
Minutes
70.00
80.00
90.00
100.00
110.00
120.00
Deamidation T15 HPLC separation, 20 pmol

:120 min.
Isoaspartic Acid DL Iso VK
Aspartic Acid DLDVK
Asparagine DLNVK
Pc: 320
Deamidation T15 UPLC separation :60 min.
DLIsoDVK
DLNVK
DLDVK
Pc: 500
Deamidation T15 UPLC separation :30 min.
Pc: 375
DLIsoDVK
DLNVK
DLDVK
Meeting Customer Needs

y By utilizing ACQUITY UPLCTM column technology, the
ACQUITY UPLCTM System delivers the ultimate in
y Resolution
Will greatly increase peak capacity over HPLC
For stability, purity and ID
y Sensitivity
Resolution and peak shape
Pull out possible modifications earlier that will impact activity
y Reproducibility
<0.07 minute Std deviation over 2 hrs
y Speed
Can run in 30 minutes what previously took 2 hrs
ACQUITY UPLCTM
Systems for Tandem
Quadrupole Quantification
Tandem Quad Quantification Challenges

y ADME
Many samples to analyse
Many different compounds to characterize
May have complex matrices
y Pharmacokinetics
Less candidate diversity, more detailed experiments
Complex matrices
Carryover
y Regulated Bioanalysis
Many samples, tight deadlines

Complex matrices
Sensitivity
Carryover
Require reproducible methods
Compliance
Experimental Conditions
y ACQUITY for UPLC
y Quattro Micro for MS detection
y Bridged Hybrid C18 column,
y ESI+ with MRM data monitoring
2.1 x 30 mm, 1.7 m, 40oC
y 0.5 ml/minute flow rate

y 5 L Sample injection
y Mobile Phase
A: 10 mM NH4OAc in 20% MeOH

B: 10 mM NH4OAc in 80/20 MeOH/AcN
y Gradient
35%B to 100%B in 1 minute
y Alliance HT 2795 for HPLC

y Xterra C18 Column
2.1 x 30 mm, 3.5 m, 40oC
y 0.3 ml/minute flow rate

y 5 l Sample injection
y Mobile Phase
A: 10 mM NH4OAc in 20% MeOH

B: 10 mM NH4OAc in 80/20 MeOH/AcN
y Gradient
25%B to 100%B in 2.5 minute
y Dwell Time: 100 ms

y Inter-scan delay: 20 ms
y Inter-channel delay: 20 ms
y Source temperature: 130oC
y Desolvation temperature: 350oC
y Desolvation gas flow: 750 L/Hr
y Cone gas flow: 50 L/Hr
UPLCTM vs. HPLC*

Alprazolam-D5, 100 ppb, 314.11 > 210.11
100
0.66
Alprazolam, 10 ppb, 309.09 > 205.13

100
0.67
UPLCTM
%
100
100
HPLC
%
2.59
Time
0.50 1.00 1.50 2.00 2.50 3.00 3.50
2.62
Time
0.50 1.00 1.50 2.00 2.50 3.00 3.50
HPLC and UPLCTM 309.09>205.13*

1 ppb, 10 ppb, and 100 ppb
1 ppb, 10 ppb, and 100 ppb
6 Injection each
6 Injection each
Response
Response
r2=0.998
r2=0.998
Slope = 88.7
Slope = 148.2
ppb
ppb
0
20
40
60
HPLC
80
100
20
40
60
UPLC
80
100
HPLC and UPLCTM*

309.09>205.13
Average S/N 1 ppb = 32.9
Average S/N 1 ppb = 131.5 (4 times over HPLC)

0.67
2.61
100
100
1 ppb, and 10 ppb

6 Injection each
HPLC
Time
1.60
2.00
2.40
2.80
3.20
2
0.20
UPLC
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
1.10
Time
1.20
Average RT = 2.62, SD = 0.00428, RSD% = 0.163 Average RT = 0.670, SD = 1.62e-8, RSD% = 1.52e-6
Experimental Conditions
y Acquity UPLC
y Micromass Quattro Premier
y Bridged Hybrid C18 Column: 2.1 x

50 mms, 1.7 mm, 40oC
y Electrospray
y Injection Volume: 5 mL
y Flow Rate: 0.6 ml/min
Back Pressure at 10900 psi
No splitting
y Mobile Phase
10 mM NH4OAc at pH 5.0 in 20%
MeOH
10 mM NH4OAc at pH 5.0 in
MeOH/Acetonitrile at 80/20
y Gradient:
40% B to 50% A in 0.4 minute
2 minute total run time
y MRM Data Monitoring

y Capillary Voltage: 3 kv
y Source Temperature: 130oC
y Desolvation Temperature: 400oC
y Desolvation Gas Flow: 800 L/Hr
y Cone Gas Flow: 50 L/Hr
y Collision Gas Flow: 0.3 L/Hr
y Sample Prep.:
Standards were spiked into crashed
plasma
Rat plasma was crashed by
acetonitrile 2/1 ratio
100 ms vs. 5 ms Dwell Time

Alprazolam in Rat Plasma, 10 ng/mL, 5 ul Injection
100 ms Dwell Time, 10 ms Delay
Peas Area = 16791
0.63
0.63
Peak Width = 1.8 s
Convert the x axis

to scan number
Points Across Peak = 7

%
Time
0.25
0.75
1.25
100
1.75
105
110
115
120
125
130
Scan
5 ms Dwell Time, 5 ms Delay

Peak Area = 16262
0.63
Peak Width 1.8 s
0.63
Points Across Peak = 60

Convert the x axis
to scan number
%
0.25
0.75
1.25
1.75
Time
1100
1200
1300
1400
Scan
Calibration Curves: 100 ms vs 5 ms

Propranolol, External calibration, 0.05 500 ng/mL, 13 points
5 ms Dwell Time, 5 ms Inter-channel Delay
Slope = 118.6
Slope = 140.5
r2 = 0.995
r2 = 0.993
ng/mL
100
200
300
400
500
ng/mL
0
100
200
300
400
500
Calibration Curves: 100 ms vs 5 ms

Alprazolam, Internal calibration, 0.02 200 ppb, 13 points
Slope = 0.0109
Slope = 0.0115
R2 = 0.996
R2 = 0.998
ng/mL
0
40
80
120
160
200
ng/mL
0
40
80
120
160
200
No Detectable Carryover in Rat Plasma

First Blank After 1000 ng/mL Injection
1000 ng/mL, Plasma, 5 ms

CalP5ms0328058
0.63
100
Plasma Blank
MRM of 3 Channels ES+

314.1 > 210.2
6.91e6
CalNt5ms0328059

314.1 > 210.2
7.00e4
100
%
%
Peak Area = 97963

0
0
CalP5ms0328058
100
0.63
CalNt5ms0328059

309.0 > 205.2
7.00e4
100
309.1 > 205.2

4.97e7
Peak Area = 747309

0
CalP5ms0328058
0.48
100

260.2 > 116.0
6.27e6
CalNt5ms0328059

260.2 > 116.0
7.00e4
100
Peak Area = 96015

0
0.20
0.60
1.00
1.40
Time
Time
0.20
0.40
0.60
0.80
1.00
1.20
1.40
System Robustness
10 ng/mL Standard Spiked in Crashed Rat Plasma
Alprazolam, Internal Calibration, 5 ms Dwell Time, 5 ms Delay
Robustness03290007
Retention Time (Min.)
309.1 > 205.2
0.64
100
Time (Min)
0.8
n = 963
0.7
Peak Area = 9228

0
0.6
Robustness03290983
0.5
309.1 > 205.2
0.64
100
Average = 0.65 min, RSD% = 0.26%
0.4
1
150
300
450
600
750
900
Peak Area = 9204
Injection No.
0.50
Time
1.50
1.00
Calculated Conc. (ng/mL)
Calculated Concentration (ng/mL)

25
n=6
n = 963
20
15
Response
10
R2 = 0.999
5
Average = 10.83 ng/mL, RSD% = 4.43%
0
1
150
300
450
600
Injection No.
750
900
0.0
0
200
400
600
800
ng/mL
1000
AcQuity/QuattroPremier

y By utilizing ACQUITY UPLCTM column technology, the ACQUITY
UPLCTM System delivers the ultimate in
y Resolution
Greatly increases peak capacity over HPLC
Pull away possible interfering endogenous molecules
y Sensitivity
Able to quantify low level metabolites
Low dosing of drug
Important metabolites
y Speed
Can run in 1 minute and get very good data
Discovery and development labs can benefit
y Carryover
Minimal
ACQUITY UPLCTM
Systems for
Metabolite ID
Discovery/Development
Challenges
y Discovery
Many samples to analyse quickly

Low incubation levels which will mimic the real dosing scenario
Need to find expected and unexpected metabolites easily
The dirty matrices may degrade instrument performance
y Development
Difficult to find some metabolites in the complex matrix

May miss metabolites that are at very low levels
Cant afford to mis-assign metabolite structures
Processing of complex data takes up too much time
Highly polar metabolites are difficult to analyse
The dirty matrices may degrade instrument performance
Paracetamol Metabolism in Humans

O
O
HN
HN
CH3
O
CH3
HN
~35%
60%
OH
O-Glucuronide
Glucuronide
m/z = 232
Sulphate
O
N
OSO3H
Paracetamol
m/z = 152
m/z = 328
O
CH3
CH3
Cysteine
O
S
m/z = 150
CH3
OH
NH2
m/z = 271
S-Cysteine
Conjugation
Resolution
30 Min TIC
H040502a_JG_14
100
1.10
1: TOF MS ES+
TIC
2.15e4
HPLC
5.02
23.33
23.69
%
12.40
10.22
1.73
6.00
4.33
7.62 8.52
9.23
21.31 22.04
13.51
12.03
13.93
11.25
20.70
16.40 17.51 18.63

19.21
15.67
24.59
27.49
25.8226.68
0
H042102a_RP_10
100
1: TOF MS ES+
TIC
3.51e4
0.67
UPLCTM
22.92
0.78
26.01
2.09
23.15
11.20
21.10
8.62
2.57
20.31
1.30
3.55
17.13
4.20
5.61 6.49 7.337.93
5.15
12.71
14.51
21.66
22.23
26.51
24.40
25.17
18.26 19.43
27.34
28.77 29.98
28.12
16.32
0
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
22.00
24.00
26.00
28.00
Time
30.00
Resolution
XIC 30 Minute Run
H040502a_JG_14
1: TOF MS ES+
232
1.11e3
HPLC
10.28
100
10.37
11.02
11.14
3.22
9.80
21.58
Time
2.50
5.00
7.50
10.00
12.50
15.00
17.50
20.00
22.50
25.00
27.50
30.00
1: TOF MS ES+
232
744
H042102a_RP_10
2.36
100
UPLCTM
6.22
2.05
1.51
6.38
6.59
2.68
0.56
23.42
6.80
0.73
22.50
10.39
24.35
7.83

0
Time
2.00
4.00
6.00
8.00
10.00
12.00
14.00
16.00
18.00
20.00
22.00
24.00
26.00
28.00
Sulphates
Speed
UPLCTM 10 Minute Run
H042102a_RP_13
100
1: TOF MS ES+
TIC
4.83e4
0.40
8.71
0.48
9.85
8.81
%
5.95
10.04
1.52
6.32
4.95
10.31
6.87
8.20
8.54
9.02 9.33
5.74
0.77
9.48
8.35
7.79 8.00
6.74
7.54
7.28
3.28
2.67
2.90
3.93 4.34
0
Time
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
Speed
30min HPLC Vs 10min UPLCTM
H040502a_JG_14
1: TOF MS ES+
232
1.11e3
10.28
100
10.37
HPLC
30 min
11.02
3.22
9.80
21.58
0
H042102a_RP_13
100
1: TOF MS ES+
232
974
1.65
1.75
3.65
UPLCTM
10 min
%
1.96
3.70
1.13
3.86
8.48 8.77
5.82
10.89
Time
2.50
5.00
7.50
10.00
12.50
15.00
17.50
20.00
22.50
25.00
27.50
30.00
Sensitivity
H040502a_JG_14
1: TOF MS ES+
31.92 441
44.6
HPLC
100
26.89
H042102a_RP_10
UPLCTM
100
1: TOF MS ES+
441
187
21.45
31.75
6.36
31.71
5.19
0.79
6.46
26.93
27.10
14.71
5.25
0.90
31.53
31.34
6.49
20.99
31.23
25.95
31.96
25.67
14.66
22.37
31.11
17.56
5.38
5.14
10.49
6.61
8.36
5.02
4.69
16.57
17.66
20.24 22.16
8.83
28.19
25.17
15.69
19.25
20.34
22.69
0.48
29.87 30.29
29.09
8.90
11.77
6.19
1.98 2.15
2.88
3.243.83
Time
5.00
7.50
10.00
12.50
17.87 18.66 19.47
6.47
7.66
15.00
17.50
20.00
22.50
25.00
27.50
30.00
5.75
4.68
5.35
6.51
6.63
7.98
7.47
8.96
9.10
11.56
13.19 14.58
11.06 12.12
13.78 15.86
17.57
16.34
21.98
20.31
24.15
23.06
26.46
23.64
26.67
Time
2.00
4.00
6.00
8.00
10.00
12.00
Glutathione Conjugate (sign of tox)

24.73
20.79
1.24
3.53
4.07
2.50
26.09
22.49
18.65
12.46
27.54
9.61
1.94
2.22
14.60
13.93
2.15
2.01
24.12
15.13
12.69
12.51
1.00
14.00
16.00
18.00
20.00
22.00
24.00
26.00

y Resolution
Greatly increases peak capacity over HPLC
Increase the ability of MS and MS/MS exact mass
y Sensitivity
Able to ID low level metabolites
Critical to understanding metabolic pathway
Need to ID bad metabolites (glutathione conjugates)
y Speed
Can run in 10 minutes what previously took 30 min
This is what makes this important to discovery labs
ACQUITY UPLCTM
Systems for
Metabonomics
Biochemical Pathways
Challenges
y Complex Samples
Biofluids like:
Plant extracts
100s -10,000s of endogenous primary and
secondary metabolites
y Ability to see small changes amongst the tall

trees
y Can we go fast?
To some, speed is important
FIA vs HPLC vs UPLC
Resolution
10 minute Run
100
3.72
HPLC
Detected Peaks 1186
2.58
3.55
3.99
2.31
0.57
0.71 1.12
0.46
5.55 5.80
4.10
2.87
5.92
5.13
2.73
4.53 4.93
3.29
2.04
1: TOF MS ES+
TIC
5.26e4
6.23
1.63
100
2.20
UPLCTM
2.86
3.24
Detected Peaks 2246
2.64
0.390.52
1.94 2.03
%
0.33
1.00
0.75
5.125.39
1.00
7.30
5.745.87
6.29
6.53
7.22
4.14
3.97
1.46
6.72
4.41
2.45
3.66
3.10 3.49 3.72
4.30
1.31
1: TOF MS ES+
7.54
TIC
5.26e4
2.00
3.00
4.00
5.00
Female Black Mouse, PM Collection
6.00
7.00
Time
Sensitivity*
Metabonomics XIC m/z = 401.
H031024CSa014
1: TOF MS ES+
401
210
4.80
100
HPLC
%
0.58
3.74
0.73
1.501.80
2.13
2.37 2.62 3.12

3.40
4.61
4.09
4.37
5.06
5.85
5.94
5.64
5.38
6.02
6.09
6.48 6.72 7.06
7.97
7.457.73
0
H040116a_RP_ESI+_010a
1: TOF MS ES+
401
756
3.87
100
UPLCTM
%
0.27
0.73
1.98
2.78 3.28
2.00
3.00
5.35
5.47
6.52
7.16
7.76
8.41 8.628.97
9.51
Time
1.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
HPLC and UPLCTM

Gender Variation in Black Mice
UPLCTM
HPLC
Scores: Component 2 - Component 3
Scores: Component 2 - Component 3

0.400
Female
0.400
0.300
0.200
Component 3
0.200
Component 3
Female
0.000
-0.200
0.100
-0.000
-0.100
-0.200
Male
-0.400
-0.300
Male
-0.400
Component 2
-0.200
0.000
-0.200
0.000
0.200
Loadings: Component 2 - Component 3
Loadings: Component 2 - Component 3

5.5433
0.600
255.0647
0.400
Component 2
0.200
2.19
297.14
0.60
5.9370
271.06005.6272
0.40
2.48
206.04
0.200
0.000
-0.200
5.5420
5.9385
5.6269
5.9087
256.0706
4.3017
2.5612
2.1682
2.3162
3.7063
272.0650
3.3764
3.7179
6.2174
3.9211
6.6026
5.6319
5.9753
6.2375
5.0797
3.0971
2.2339
3.9378
6.2133
6.5936
5.5791
5.3441
4.2580
6.2410
5.0888
6.2501
6.2189
6.2216
3.7213
2.3884
4.1937
3.4364
3.9037
5.0858
5.1184
5.2054
3.6129
2.9137
2.2160
4.7559
6.8807
6.2258
5.3406
6.3750
3.9625
5.1776
4.4938
5.1086
5.9253
5.2250
5.7963
2.6416
3.1452
2.8536
6.0124
4.0799
3.9459
4.9815
4.3152
5.1245
6.2281
3.5332
2.4477
6.5969
5.3475
2.2406
6.5776
5.1520
3.8661
4.3869
4.2396
5.1139
5.1774
3.9266
6.2146
3.0164
6.6076
4.1767
4.1940
3.9262
6.6053
4.3899
2.1472
3.3675
5.2901
5.3683
2.8932
2.2552
2.2547
5.6206
3.9156
2.3091
3.9176
5.8441
2.2091
5.3851
4.7218
4.8877
2.2462
3.7295
3.1376
4.0957
3.8177
4.9523
3.1838
3.9182
3.9297
6.7685
5.8798
2.9266
6.2243
3.1343
5.8120
2.8265
2.2834
3.9634
2.8741
5.0658
2.4590
2.1783
4.9501
3.7611
6.2296
5.3206
6.2626
2.2150
3.2304
3.7550
5.2021
2.2275
5.7513
4.1027
5.8610
4.3640
2.6388
2.2545
2.7386
286.0811
6.8645
5.9234
6.0532
2.7409
3.6779
6.5636
3.7307
4.1969
3.8524
5.6584
2.1719
6.0604
2.6461
3.0749
3.2460
2.3217
2.2076
6.2189
3.9072
6.2293
3.3625
2.9160
4.2774
4.9529
3.4513
2.8630
6.1039
5.2817
3.9751
6.2123
5.8407
2.7380
3.7442
3.0085
5.9403
6.6160
3.7595
4.2049
6.8845
3.5442
4.7646
2.8597
5.5465
3.1402
4.0586
3.7346
4.7535
5.8700
2.6184
3.7504
5.0643
2.5861
4.0344
5.3580
2.9228
6.1535
2.9184
5.2845
6.1484
3.7824
4.4067
2.8796
4.2337
3.3611
5.1310
3.5500
4.5508
5.2232
5.3773
5.2353
3.9893
6.0428
4.7592
3.2420
5.0323
3.7427
5.3821
5.6675
2.3348
6.3216
2.1070
2.4898
3.5508
7.1736
4.5379
4.0885
3.1632
6.2237
5.3256
2.3045
5.3187
3.8980
2.7390
2.2952
5.6868
4.5409
5.1627
5.6232
2.7382
2.3570
2.2039
5.8387
5.1551
2.9260
6.1656
3.5904
5.2932
3.7666
6.2460
5.0257
2.4744
6.7468
6.7491
4.2435
5.2492
4.0990
4.9661
2.6728
3.7579
4.5878
6.2472
4.3876
5.2261
6.4727
3.9352
3.2921
2.3705
6.1705
2.9783
2.6950
3.0616
3.0144
3.7326
3.7143
4.9783
3.5153
2.4349
3.9072
7.2911
5.7144
5.7836
3.6196
5.2125
5.3384
3.1391
3.6207
5.2488
3.7188
2.4678
4.5422
2.0837
2.8593
6.4075
5.9001
2.6790
5.9901
5.2638
4.5867
4.1628
2.2499
3.9505
3.8674
2.6477
4.4086
4.3768
5.2381
2.2096
3.7252
5.4947
5.9985
2.4230
5.3067
3.8657
5.3332
4.5353
5.2087
5.4622
2.3293
6.0352
4.9588
5.0020
5.2376
5.1040
3.7453
5.2619
3.7659
2.1850
4.0851
5.6740
2.8961
4.8922
2.8761
3.1387
3.1371
4.4390
3.7635
5.4447
2.2073
2.6943
2.8847
2.2417
5.2825
5.2693
2.6699
3.8325
3.2876
5.2582
5.2745
4.4385
4.7792
5.2237
4.4382
2.4691
2.2041
5.1894
5.2950
5.4524
6.4302
4.0268
4.7287
7.0486
6.3758
3.0250
3.7463
5.2344
5.9969
5.8549
3.0471
5.2493
2.1550
5.8519
3.5592
5.0792
4.1748
4.1228
2.8680
4.0905
6.2497
6.1099
5.1461
5.9157
4.2981
5.8424
5.2799
4.7035
2.1883
5.0762
2.5823
4.2838
5.2332
3.1418
5.2832
3.2852
5.8572
3.9932
4.8429
3.7617
2.9294
3.0356
2.2300
3.7600
3.5281
3.3292
2.7356
6.1895
2.8443
5.6754
4.8658
2.1758
3.1448
2.3002
5.0524
5.8521
6.2600
5.9842
5.8484
6.4494
2.1995
5.4480
5.0062
3.0986
5.3299
5.9684
5.0981
3.0234
5.1015
5.3830
2.6665
6.2476
6.3744
2.6431
5.3216
5.2159
5.6598
2.2082
5.8693
5.5855
5.9506
5.0844
5.1985
3.2533
5.9951
4.3166
5.2006
2.5512
5.2783
273.0797
2.2023
4.4044
4.8081
3.0370
3.0355
5.2417
2.4365
2.6048
4.7466
5.9570
3.0866
2.9000
5.8552
4.2322
5.6617
2.5296
2.6793
5.2277
6.0887
2.8262
4.5919
4.8683
5.2528
5.5928
4.9272
2.4842
3.0282
2.8895
5.3439
2.4510
4.6253
4.5017
4.0899
4.8314
3.6909
4.6183
5.9124
6.1804
5.1556
5.8471
2.8592
4.4545
3.7336
5.3218
2.2279
6.0395
5.7205
3.7208
5.8997
4.2338
5.2024
5.7151
4.3540
2.8500
5.2111
4.2383
5.9288
6.2474
6.0202
2.2616
6.1691
2.5638
4.3383
5.2394
5.1499
2.0905
6.2665
3.5534
5.2743
255.2054
3.2862
5.3221
5.9241
4.9402
5.5887
4.6580
2.1541
5.9564
2.9195
5.0155
5.1277
6.3453
2.6865
4.5370
4.6248
5.0581
5.4755
4.8923
298.1478
5.5816
3.5501
2.5305
283.1331
298.1463
5.7197
4.1565
5.7794
3.7759
179.0575
5.1369
3.2823
5.8241
5.7868
3.5563
3.1041
3.0258
3.0281
5.5938
4.4965
301.2110
2.5679
3.2793
4.0794
2.7436
178.0486
5.8495
6.5154
121.0674
5.6597
221.0993
5.1433
4.2939
225.0463
2.8578
287.0973
331.0863
4.0953
6.2863
5.5711
165.0596
2.7433
134.0955
227.1724
421.0888
158.0514
149.0616
223.0651
5.7792
257.0778
108.3563
416.2622
344.1536
5.1251
398.2507
4.3482
247.0876
259.0988
241.0873
207.0486
190.0240
148.1078
315.1547
157.0766
303.1282
405.1703
241.1446
150.0568
4.0992
346.0614
568.2720
145.0343
267.1240
213.0942
108.4247
5.2768
5.9482
6.1933
377.1965
4.1216
134.0799
151.0352
243.0717
191.1123
344.1566
107.0839
266.1398
703.3967
132.0627
230.0526
221.1155
197.1201
209.1130
208.0998
146.0601
249.1093
256.2036
206.0452
199.1046
207.0339
108.5235
137.0471
225.1455
186.0943
176.0797
255.1164
279.1327
215.1235
273.0829
234.1438
242.0592
168.1082
224.0690
192.0962
237.1240
203.0839
227.0309
150.0913
265.0976
302.1944
267.1436
511.2799
234.0611
327.0976
150.0565
204.9856
175.0874
320.2001
204.0783
228.1582
226.1119
850.4274
283.1979
3.0235
343.1861
5.3184
208.0538
327.0912
176.0471
119.0550
236.0973
185.0145
222.0950
432.0957
219.1451
539.2513
195.1047
122.0571
121.0020
151.1092
738.3750
553.2579
401.2705
134.0605
391.1573
627.3162
239.1389
605.2729
625.2918
793.9484
202.0651
166.0611
120.0794
299.1078
310.0472
178.0748
237.1429
395.2049
135.0399
158.0525
179.0915
704.3964
598.2809
242.1022
716.1943
327.2051
316.0932
238.0853
241.1842
476.2776
409.0710
319.1257
327.1920
203.1162
100.0769
247.1341
287.1824
630.3097
347.1519
192.0985
211.0773
260.0811
199.0932
150.0643
461.2572
152.0600
417.2630
793.4469
245.1083
257.1253
213.1815
162.6354
308.1480
336.0242
195.1337
495.1407
393.0717
203.0785
274.1034
226.0492
325.0661
245.0438
390.1122
287.0768
375.2577
138.0859
371.2205
194.1870
343.1826
407.0630
177.0641
375.2722
112.0513
327.0691
197.1235
184.9570
467.1621
849.6569
167.0925
523.2543
286.1373
206.0838
505.2298
433.2317
113.9645
245.1785
303.1407
211.0815
330.2150
192.0776
172.0626
106.8332
849.8522
106.9737
169.1183
362.1858
328.2069
151.0430
400.2599
326.0775
134.0972
141.0013
136.0657
177.1206
113.9654
227.0865
269.0928
278.1192
276.0761
184.0751
229.1068
134.0927
137.0629
108.1978
297.1433
400.2698
191.1020
492.1426
278.1462
251.1511
143.0297
490.3329
410.2610
493.1499
279.1328
209.0766
311.1646
428.3042
156.0216
477.2467
169.1194
162.6798
340.0570
248.0957
273.1468
163.0845
246.1470
177.1574
340.1035
160.6943
116.0701
133.0991
133.0677
325.0653
415.0773
499.0067
180.1138
345.1309
240.1719
309.1709
117.0714
110.0565
348.2229
132.1018
315.1519
415.0348
382.1105
317.2103
161.0465
249.1293
359.0933
813.3748
253.1492
355.0530
384.2698
363.2151
152.0715
444.2808
108.4709
204.0701
110.0607
449.1898
160.0397
424.0921
229.0719
367.1494
215.1242
347.2196
405.2257
166.0851
382.2916
427.2692
277.0206
379.2132
326.0888
427.1552
339.0958
196.0950
180.0997
151.1097
416.2565
243.0516
201.0693
402.2850
438.2866
256.1150
848.3350
163.0880
108.0032
497.1381
366.2297
242.2066
848.4288
193.0954
178.1166
155.1032
205.1097
133.0778
366.1506
247.1229
377.1481
511.2493
3.8728
183.0525
456.2055
195.0400
325.1685
397.2101
241.1201
151.9877
211.1317
412.0435
177.1261
365.0713
148.1093
172.0857
275.0336
426.2843
848.3837
306.9974
331.9654
374.1487
290.1733
846.9190
188.0545
327.9623
411.1951
428.1877
442.1008
364.0642
5.9087
170.0621
5.7322
162.2006
313.2052
480.2760
239.1350
240.1512
378.1983
248.0799
205.0806
846.8549
509.2394
593.3277
261.1167
465.1643
144.0681
109.0977
306.0830
198.1851
175.1226
191.0662
171.0777
263.0868
134.0270
276.1049
449.1176
555.3099
444.1619
310.1634
132.0550
131.0673
326.1091
136.0411
158.0296
255.1287
182.1191
356.2660
428.1869
106.8992
325.0899
4.3355
317.1476
413.0476
226.1437
317.1731
397.0566
128.0686
397.0736
381.1814
473.1761
144.0800
462.2750
120.0566
425.2133
212.1259
134.0304
241.0433
262.0408
285.1324
337.2500
413.3033
377.1092
219.1511
519.3132
440.2132
386.2805
216.0705
190.0611
217.1213
108.0941
174.0604
105.0711
122.0601
287.1468
4.6186
380.2249
4.4954
5.1149
127.0394
375.1803
397.3098
254.1027
436.2380
383.2600
289.0387
391.2782
361.1647
362.2047
141.0015
171.0743
330.1888
401.2146
280.1116
235.1353
304.1738
104.0590
245.0658
459.1739
310.1364
207.0658
413.0220
172.0786
208.0787
253.1298
238.1408
145.9959
360.1948
202.0762
343.2476
346.2173
232.1188
139.0088
118.0661
107.0164
331.2187
201.1229
165.0674
177.1549
298.1962
5.2823
130.0649
255.0703
413.1266
189.0653
378.0914
171.1345
276.1770
132.0813
432.1052
332.2051
207.1061
174.1129
461.1110
404.2332
461.2737
3.5419
149.0640
374.2765
135.1134
312.1624
226.1432
316.2111
127.0845
457.1266
235.1363
419.2013
189.0435
437.2292
300.2164
435.1362
179.1357
227.1399
298.1014
365.2313
146.0618
345.2045
265.1193
333.0993
318.1882
228.1592
226.1079
220.1351
398.2541
384.2750
152.0535
132.0443
383.0833
4.1120
347.1857
106.0297
5.9209
153.1272
364.2200
375.2571
346.1916
130.0635
257.1201
220.0643
240.1275
372.2377
288.1630
295.0790
146.0606
275.0699
286.0827
358.2544
314.2269
560.3277
345.1891
305.0830
111.0087
260.1017
384.0743
105.0342
182.0913
272.0663
181.0839
342.2606
237.1503
179.0708
318.1893
137.0598
346.2215
325.1805
5.3172
256.0692
144.0832 3.7203
163.0746
245.1114
202.1076
373.2735
151.1117
382.2590
475.2458
226.1432
136.0614
297.1410
655.2759
245.1162
2.9077
180.0880
439.1955
118.0658
125.0961
315.1544
131.0502
361.2007
347.2207
145.0330
165.0551
379.2105
338.0869
403.2309
165.0644
203.1103
285.0760
4.9392
431.0995
447.0967
5.6056
2.8614
103.0548
5.8525
5.7692
382.0771
3.9912
190.0506
363.2159
188.0362
5.7160 231.1422
5.1286
316.1749
374.2530
343.1827
311.1592
355.2628
135.0844
378.1512
149.0613
271.0620
4.5006
5.7155
5.7800
255.0651
259.0985
181.0866
3.7185
206.0431
Component 3
Component 3
285.0761
0.20
0.00
-0.20
-0.40
2.48
5.01
4.44
2.73
6.04
178.05
2.21
3.64
3.75
374.26
4.85
3.43
2.89
4.42
4.38
3.32
2.45
2.26
2.23
2.88
2.74
2.89
2.18
3.35
131.05
2.76
4.43
2.90 5.43 4.27
3.96
3.74
3.07
3.64
2.49
2.49
4.44
4.97
5.48
3.12
4.28
2.92
2.93
3.13
4.94
2.22
3.03
2.10
190.04
3.14
4.85
3.10
5.04
6.04
3.14
2.19
5.14
3.75
4.44
4.13
4.14
5.23
4.32
2.42
5.60
3.44
2.90
2.74
242.28
2.49
2.89
3.09
4.39
4.38
3.11
3.67
3.27
5.14
5.02
3.07
4.76
3.52
4.70
4.72
3.32
3.12
3.15
3.46
4.70
4.07
3.36
4.68
4.14
3.64
4.97
3.14
3.74
3.35
4.94
2.73
2.21
5.43
5.04
4.42
4.99
5.24
4.28
3.02
3.71
2.14
5.50
5.17
5.24
4.27
2.23
5.19
3.48
2.28
5.46
2.81
3.85
4.70
4.14
4.43
5.13
3.29
3.65
3.69
5.17
5.01
6.22
5.63
5.61
3.43
5.56
2.19
4.63
2.80
3.11
2.18
3.27
4.78
2.18
3.78
3.13
3.86
2.21
3.68
2.45
2.57
4.41
4.09
2.20
5.51
2.31
2.27
5.54
3.94
4.35
4.63
5.75
4.13
4.42
3.86
2.73
2.91
2.89
3.64
5.60
2.87
3.02
4.82
5.64
3.07
4.45
2.26
4.48
2.26
4.28
3.95
3.87
2.70
5.16
5.00
2.99
6.71
4.19
3.46
6.33
2.59
3.02
4.12
5.48
5.75
3.67
2.52
3.76
4.73
4.19
3.46
5.28
3.33
5.43
5.26
7.30
5.33
3.27
5.52
4.35
3.27
3.28
5.55
3.31
5.39
5.33
5.75
3.72
5.07
4.36
3.64
2.30
4.35
2.78
3.67
5.41
5.13
5.41
6.06
4.74
3.87
4.56
4.77
5.07
4.70
5.66
3.72
2.49
4.07
5.35
7.33
3.93
5.75
5.55
4.71
2.93
2.87
2.38
5.18
2.65
5.33
3.22
3.63
2.39
5.18
2.44
2.94
4.17
3.70
4.62
4.77
4.71
5.53
3.14
5.75
5.20
2.42
3.01
2.73
2.51
3.15
2.39
4.88
6.30
3.09
4.33
2.43
3.56
2.63
4.27
4.83
3.14
6.21
3.92
5.34
3.03
5.43
4.42
4.76
5.55
6.49
4.94
5.95
6.31
3.24
3.75
2.07
3.73
2.28
5.19
2.17
3.24
5.15
4.38
2.94
3.35
4.45
4.47
6.16
5.30
2.66
3.72
6.82
2.27
2.50
2.64
5.04
4.84
4.28
2.40
3.15
5.53
4.36
4.86
3.71
3.73
3.44
5.13
4.28
4.69
4.32
2.81
2.17
3.65
4.94
2.06
2.10
6.86
7.26
3.54
5.90
2.52
6.51
6.15
6.48
7.21
5.25
3.53
5.33
5.43
4.85
2.30
3.72
4.69
3.63
4.06
5.23
5.76
2.36
5.49
2.85
6.67
6.22
2.20
4.61
4.42
5.06
3.91
5.87
3.34
6.81
5.99
3.70
5.65
3.83
4.69
7.03
5.48
4.72
5.62
4.89
2.71
5.83
5.08
7.17
3.64
5.60
4.76
5.54
6.27
6.08
2.29
6.72
3.45
6.68
5.34
4.14
2.44
3.77
4.18
2.24
3.37
2.89
3.46
3.23
6.54
7.24
2.54
2.83
5.47
3.52
5.41
6.71
4.30
3.69
3.51
4.85
4.15
4.90
3.43
2.67
2.83
6.02
3.50
4.77
2.72
5.78
3.95
5.11
3.40
3.58
6.70
2.79
7.31
3.42
3.07
5.86
2.22
2.25
6.22
3.14
2.92
2.47
2.58
4.62
5.75
6.52
4.13
3.98
3.12
3.52
2.95
5.69
5.08
4.28
3.28
4.73
3.71
4.78
3.84
4.50
5.59
3.74
3.86
2.18
7.25
2.40
3.88
3.28
7.15
5.67
4.29
2.45
2.67
3.21
5.07
7.05
2.49
4.83
7.23
5.01
6.25
5.09
2.93
4.91
6.50
5.00
3.90
2.80
2.48
3.93
3.10
3.47
5.65
3.65
3.29
3.74
4.79
4.78
5.02
2.65
4.37
5.63
4.96
2.09
5.18
3.34
3.70
3.52
3.87
5.12
3.34
4.44
3.14
4.63
5.95
3.25
4.47
5.60
3.13
3.33
7.17
2.90
3.01
6.46
6.45
3.85
4.90
4.96
4.34
3.48
5.00
4.70
5.63
5.00
3.69
5.12
2.47
6.43
4.33
3.70
5.08
3.11
2.90
2.19
3.68
3.03
2.33
2.52
5.54
4.09
3.17
5.05
2.66
4.97
5.89
3.24
6.92
2.37
3.01
2.57
7.02
2.68
5.22
2.20
6.17
6.47
5.04
5.13
6.26
7.12
4.62
5.65
2.43
2.22
5.50
2.30
2.27
4.77
2.99
6.99
2.64
3.28
2.90
6.03
6.87
2.92
3.00
6.53
6.09
4.97
6.45
4.79
298.15
4.28
2.43
6.91
2.61
3.93
4.55
6.32
5.23
3.65
3.68
5.09
2.89
2.93
255.06
285.08
5.41
4.11
2.91
3.44
2.92
6.18
3.75
5.61
7.28
2.82
2.44
3.77
6.31
3.00
2.61
3.66
7.30
3.28
2.90
6.69
2.34
5.35
2.55
4.50
6.59
3.35
2.43
3.87
3.70
5.61
2.18
3.27
2.45
3.28
5.15
2.44
7.22
5.25
3.86
5.12
3.45
3.69
6.49
2.78
6.58
2.13
3.52
4.95
7.31
2.66
4.43
2.30
5.15
4.15
2.18
5.39
3.33
3.70
3.36
4.77
2.29
4.94
5.41
7.04
5.10
3.98
5.48
4.26
2.70
6.08
4.60
3.22
5.18
7.23
4.47
2.84
4.86
5.07
6.46
2.83
6.07
2.62
4.77
3.27
5.03
2.29
2.38
4.63
4.07
3.11
7.24
3.87
5.01
5.75
4.43
2.21
3.98
3.75
5.41
3.27
2.92
4.71
3.26
6.72
6.59
3.66
3.12
379.21
4.34
3.13
3.97
3.74
3.68
4.74
7.24
2.43
6.66
5.15
377.15
3.73
3.74
3.13
7.02
3.28
5.39
2.76
3.27
2.76
3.77
4.97
3.47
4.45
5.35
2.17
2.23
6.72
2.65
2.45
4.15
3.87
3.34
4.34
3.52
2.77
6.54
2.24
5.05
6.73
144.08
5.13
3.69
5.41
2.29
2.40
2.76
3.14
279.13
5.00
2.90
2.68
6.72
465.07
2.43
3.13
287.15
2.90
3.10
2.92
6.70
3.87
6.85
443.09
5.54
7.02
3.87
6.73
144.08
5.13
3.13
2.19
3.87
201.12
226.11
217.10
186.07
162.05
493.15
5.15
2.57
3.87
177.06
305.08
2.99
7.02
2.46
4.77
377.09
5.24
3.27
6.72
271.06
413.04
316.21
2.53
461.12
338.08
431.10
5.39
188.03
207.04
103.06
705.39
361.21
2.49
6.07
7.33
290.01
2.49
6.60
447.10
180.07
248.12
263.13
429.13
255.13
188.07
315.06
134.03
380.21
185.08
255.06
243.29
480.14
500.99
192.09
271.17
398.25
286.07
256.07
135.05
130.06
469.11
105.07
108.12
273.08
3.87
469.00
5.15
229.13
301.21
371.06
2.43
347.07
201.06
397.07
227.13
247.07
288.15
292.02
345.10
422.10
411.12
271.06
377.19
275.00
375.26
347.18
396.05
392.01
335.02
273.09
229.10
146.06
318.19
7.24
523.98
175.12
472.02
2.21
497.10
255.06
233.09
444.11
235.14
262.02
303.04
256.07
632.13
403.14
2.69
435.12
706.39
172.07
506.11
384.28
176.04
363.08
197.12
145.03
397.18
346.05
192.10
403.23
271.06
475.25
157.08
466.08
298.20
331.03
108.20
331.11
371.99
289.07
272.06
329.21
415.05
147.04
494.15
489.13
278.08
363.22
201.12
108.57
448.10
299.14
106.88
261.11
320.20
277.96
401.22
226.02
270.05
327.00
479.15
208.06
465.10
6.72
351.03
330.19
412.20
158.03
133.07
250.06
280.06
107.08
356.24
251.11
312.16
439.23
257.16
243.09
411.20
248.09
319.12
160.04
239.13
255.20
281.11
291.02
202.11
381.13
301.15
216.06
365.23
492.14
332.20
175.09
427.14
386.17
384.11
319.08
179.05
145.08
274.18
3.85
369.06
446.09
113.96
359.17
364.22
134.10
425.10
264.00
243.13
177.16
317.21
197.13
239.13
5.02
553.30
470.11
507.25
467.08
235.10
372.17
373.10
105.03
414.05
432.11
113.96
423.08
142.06
453.15
392.09
161.13
349.18
132.05
247.13
270.18
234.11
299.19
416.27
318.19
542.04
278.17
369.19
378.11
366.11
285.08
298.19
381.08
439.20
164.03
303.15
554.30
118.07
351.06
252.99
443.15
275.10
245.18
439.14
181.06
126.06
362.21
279.12
210.11
382.26
497.13
106.03
462.13
193.09
411.06
237.12
146.06
307.07
243.12
151.04
204.08
213.12
208.05
124.04
464.07
552.06
461.26
3.69
404.24
300.22
219.99
270.17
447.09
402.28
4.43
232.01
187.98
440.07
442.08
380.24
178.04
325.07
311.16
501.21
328.04
361.16
225.00
405.09
327.00
359.04
248.05
359.19
257.10
357.06
455.17
206.00
350.10
441.16
440.20
410.29
443.29
226.07
175.15
190.02
278.07
341.18
329.21
346.19
331.22
150.09
361.19
130.06
298.10
252.12
511.29
150.03
277.01
232.11
267.11
278.13
304.03
352.11
207.03
393.16
549.03
192.03
847.73
342.10
327.20
175.51
421.07
289.11
332.10
405.06
366.07
252.16
443.15
177.16
430.17
163.11
235.15
502.99
383.26
729.43
451.05
222.07
141.96
310.15
348.11
209.05
185.99
435.07
325.17
172.97
153.05
165.06
292.04
342.19
114.07
212.08
543.04
279.96
441.12
181.00
155.98
351.09
365.00
344.15
249.10
464.02
419.21
301.03
348.19
301.24
282.27
369.11
268.17
351.95
445.11
131.06
180.07
287.02
138.08
370.25
373.14
408.13
172.09
150.06
282.06
272.09
379.22
417.27
241.23
295.14
370.07
215.13
422.04
240.03
519.11
309.18
293.07
369.01
278.03
162.06
379.08
313.18
392.01
307.17
501.02
283.20
150.05
245.11
500.01
435.06
671.28
303.12
381.21
431.17
207.04
302.21
283.08
312.14
280.07
221.07
213.18
480.17
354.10
398.19
152.03
274.04
180.11
114.06
302.92
141.11
234.13
156.99
532.39
139.99
186.02
282.92
323.02
172.98
321.04
773.46
118.07
436.12
241.14
310.16
389.25
390.12
334.09
184.01
421.11
429.98
351.09
274.07
354.99
188.07
849.75
357.29
361.02
413.21
295.09
169.98
242.88
429.15
456.09
130.06
294.21
447.12
333.02
163.99
186.03
207.17
130.96
186.98
209.11
624.26
241.07
179.14
229.03
254.07
329.08
485.02
165.99
386.29
347.12
455.22
295.11
122.03
330.23
177.13
397.32
305.11
221.12
262.15
319.08
211.11
319.24
187.07
140.00
175.98
144.98
356.06
309.11
176.11
254.92
112.94
259.05
259.10
286.95
339.09
189.02
261.11
170.11
184.10
413.13
280.12
340.05
192.09
229.14
304.08
169.99
457.12
273.16
350.10
359.00
297.08
414.28
388.05
204.01
263.06
315.09
213.07
132.08
186.07
252.15
287.13
137.09
379.15
431.03
276.07
191.02
265.14
414.16
163.06
165.09
212.06
281.12
402.07
490.34
411.17
159.04
374.00
154.99
258.89
264.06
298.09
146.04
338.05
155.98
327.09
526.11
273.27
385.10
197.13
115.96
250.10
305.09
528.13
474.17
120.07
271.09
471.02
501.01
329.04
311.20
167.06
264.04
430.13
155.07
511.08
162.05
381.04
130.00
415.03
345.19
202.02
282.04
174.06
399.08
346.10
328.17
502.13
341.07
232.15
405.23
366.23
338.07
134.09
285.13
186.11
396.12
262.06
261.06
185.13
227.18
207.02
230.05
523.24
229.23
220.07
135.08
280.17
141.96
335.02
495.15
707.45
387.25
439.08
377.14
242.10
139.99
276.18
424.07
282.11
372.22
275.14
406.27
269.06
267.17
219.16
165.99
189.11
250.01
347.19
389.12
241.88
567.28
143.98
390.09
183.00
426.19
304.16
365.12
433.23
308.03
230.04
138.98
255.07
444.03
112.88
585.28
395.20
351.15
400.10
389.12
375.08
474.15
481.14
386.04
385.17
292.07
455.15
407.18
2.90
450.00
280.04
273.03
371.08
281.05
406.09
441.22
417.12
454.21
431.14
226.06
137.04
113.96
472.16
266.02
196.10
247.07
456.17
276.12
257.04
688.07
233.97
443.14
646.02
158.15
297.02
170.06
228.17
339.98
341.03
263.23
412.04
254.04
277.08
232.14
284.29
363.12
255.03
569.30
276.03
394.21
170.06
426.93
291.11
389.13
292.06
198.01
461.17
326.07
345.10
267.12
374.13
391.14
358.25
186.03
383.08
409.25
336.02
256.03
188.03
290.99
346.22
371.08
349.10
625.26
256.91
114.97
337.08
238.99
355.09
170.99
330.99
365.19
356.11
322.08
323.06
531.32
318.08
349.10
211.05
256.92
361.13
292.02
849.68
415.06
275.03
137.98
202.01
431.11
338.11
301.14
213.89
430.39
444.14
471.17
423.03
217.10
154.99
358.06
270.03
429.17
215.01
213.89
254.05
379.09
194.08
2.49
258.08
208.01
244.22
271.18
391.04
284.33
316.18
363.11
203.97
414.20
234.02
202.01
143.97
261.08
285.12
328.97
189.02
272.01
257.98
343.12
461.18
421.24
348.99
109.71
428.19
551.05
343.17
369.06
316.09
237.15
160.99
211.12
235.02
219.06
206.12
495.14
149.02
458.16
4.44
366.16
363.16
374.12
106.99
449.29
382.02
332.15
506.04
217.11
312.02
193.93
375.08
382.11
595.07
238.10
493.32
338.09
323.07
285.20
205.05
401.28
293.13
254.14
135.08
204.06
259.09
629.17
485.16
207.17
220.12
190.02
432.12
220.02
623.14
343.07
227.10
108.63
381.09
310.13
299.08
300.07
380.08
298.07
349.09
304.30
246.01
237.16
323.12
227.00
207.04
302.14
132.04
457.17
331.99
376.10
476.13
357.06
5.14
531.08
407.14
275.06
384.09
135.11
6.09
260.03
258.05
240.09
471.14
231.05
200.20
171.14
222.02
182.09
374.28
155.05
641.38
239.09
327.05
207.09
419.05
337.01
3.87
272.26
3.86
189.02
295.08
419.11
365.15
106.93
399.26
217.06
357.98
509.29
280.15
235.20
199.08
134.06
186.01
257.27
291.05
422.24
575.37
376.14
397.09
373.09
188.07
267.10
343.18
289.12
437.21
3.27
6.72
318.14
139.99
347.22
478.13
414.03
317.03
237.07
310.04
351.09
372.23
6.47
247.05
321.12
4.99
343.20
2.39
356.27
219.05
309.12
245.98
226.14
846.91
294.04
343.98
3.87
3.86
481.26
437.24
404.03
465.27
299.16
342.07
101.08
685.40
453.21
415.26
113.96
235.18
289.03
153.13
217.01
246.04
232.99
355.09
278.05
333.15
200.13
400.27
207.03
189.04
262.05
319.08
433.12
268.06
399.14
323.11
163.08
253.01
268.03
247.03
179.14
165.06
331.21
597.35
202.00
391.14
395.01
459.17
181.09
335.08
451.03
245.00
300.95
154.99
368.08
113.96
214.05
327.96
217.05
342.98
3.71
231.02
5.12
4.99 3.27 3.86
149.06
233.03
359.24
311.16
519.08
321.12
354.04
355.26
204.14
174.11
204.01
459.17
244.01
382.08
359.15
530.08
3.10
261.13
385.16
256.03
289.16
230.05
239.08
108.05
353.09
275.13
304.12
231.14
302.10
256.26
301.15
311.16
253.14
2.51
371.17
149.06
341.07
2.18
421.24
413.02
427.04
225.20
362.13
3.87
113.96
257.05
477.13
2.91
325.06
242.18
346.10
7.22
489.18
3.15
489.05
214.25
400.13
314.16
245.04
319.12
184.00
374.25
357.97
628.16
402.14
460.13
298.07
296.06
467.01
3.87
313.16 206.04
441.08
189.02
357.14
305.16
226.10
341.97
225.07
5.147.36
149.03
259.10
3.86
401.14
6.72
241.18
Component 2
-0.200
0.000
0.200
0.400
Component 2
-0.200
0.000
0.200
Comparison of Markers Found

Gender Variation
HPLC
UPLCTM
New Endogenous
Metabolites
(m/z)
(m/z)
(m/z)
255
255
241
206
206
401
271
271
313
178
178
441
285
285
325
297
297
382
103
103
377
377
259
259
*Table created using the greatest significance factors
How Fast Should We Go?
Blank
Blank Injection
8.51
8.69
8.98
8.24
H030312_CS_ES+_004
1: TOF MS ES+
TIC
3.31e4
6.28
100
Urine Injection
Urine
H030312_CS_ES+_001
100
9.70 9.86
10.25
6.83
7.70
7.33
8.34 8.56 8.80
1: TOF MS ES+
TIC
3.33e4
9.63 10.20
6.69
6.36
7.76
7.72
6.29
10.45
6.23
6.38
10.54
6.01
5.93
5.82
5.73
10.87
%
4.85
4.49
11.17
10.74
5.58
5.30
11.76
10.84
4.94
10.96
11.26
11.37
25
49
2.00
4.00
6.00
Urine 8.00
2.00
Time
12.00
10.00
4.00
H030312_CS_ES+_003
1: TOF MS ES+
100
Theophylline
6.04
Theophylline
3.69
6.40 7.00 7.16
7.42 7.85 8.36
4.89 5.42 5.77
8.58 8.99
181
10.5510.64
1.39e3
10.75
10.23
11.15 11.78
0
H030312_CS_ES+_003
100
Caffeine
Caffine
6.32 6.59
7.12 7.51
6.04
5.23 5.39
1.33
1: TOF MS ES+
195
2.34e3
7.81 8.15 8.57 9.05 9.41 10.45 10.61 10.85

9.82
11.24 11.83
0
H030312_CS_ES+_003
100
6.59
Nortriptylline
Nortriptyline
6.00
7.00 7.51 7.85 7.91
8.52 8.92 9.33
1: TOF MS ES+
264
1.57e4
9.94 10.14
6.36
6.09
5.51 5.96
10.39
10.64
11.07 11.64
10
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
11.96
Time
12.00
8.00
10.00
Time
12.00
Speed
Infusion vs. Chromatography
RAT URINE
1: TOF MS ES+
TIC
3.74e4
100
Infusion of Mouse Urine

0.54 0.91 1.14
0.17
0
H040120b_RP_ESI+_027a
1: TOF MS ES+
TIC
3.74e4
0.48
100
0.36
0.56
UPLCTM 1 minute gradient

1.23
%
1.02
0
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
Time
10.00
Mass Spectra
Combined Spectra Across UPLCTM Run
255.0702
206.0477
297.1439
100
1: TOF MS ES+
8.06e3
178.0551
553.3046
298.1455
509.2752
428.1962
345.1637
465.2274
385.2008
597.3319
685.3845
641.3597
729.4149
773.4482
4,8-Dihydroxyquinoline-2-carboxylic acid
206.0406
100
297.1440
817.4836
849.5232
1: TOF MS ES+
4.82e3
Mystery Metabolite
178.0480
%
311.1653
220.1168
166.0841
127.0370
312.1604 382.0857
413.1523440.1879
474.1742 511.1755
605.4402 671.1874
568.2643
541.2761
100
0
100
Combined Spectra Infusion
Where are they?

196.9222
229.1954
144.1182
256.0699
150
200
250
1: TOF MS ES+
1.67e4
447.6763
446.7061
212.8847
786.2729813.1944
445.5121 448.6691
381.4590 444.6927
347.0142
449.4927
300
350
400
450
500
542.6557
550
586.0293
629.5122
600
650
673.6452
704.3780 740.3656
700
750
800
m/z
850

y Resolution
More peak capacity allows for the ID of more complete set of
markers
Critical to identifying population differences due to toxic effects,
efficacy etc.
y Sensitivity
Resolution and peak shape add in identifying low level
endogenous metabolites
y Speed
Can get throughput of FIA without the ion suppression
Theoretical Run Time Compatibility Chart

Instrument
Separation Time (min)

1
10
>30
(Pw 1-2 sec)
(Pw 5-7 sec)
(Pw 7-9 sec)
(Pw 10-12 sec)
Full
scan
SIR or
MRM
Full
scan
SIR or
MRM
Full
scan
SIR
or
MRM
Full scan
SIR or
MRM
ZQ
Quattro micro
Quattro Premier
LCT-Premier
Qtof Family
Summary
y ACQUITY UPLCTM Systems provide over HPLC unmatched
Speed
Same information in shorter amount of time
Sensitivity
Because of more efficient chromatography yielding sharper peaks and
better resolution
Resolution
More information in same amount of time
Achieved using small particles, optimized delay volumes and the ability to
access linear velocities appropriate for small particles.
y Whether UV or MS
y Whether Discovery or Development
y ACQUITY UPLCTM will enable our customers
To enhance productivity
To have Complete Confidence in their data
ACQUITY UPLCTM
INNOVATION

ACQUITY System Solutions

Загружено:

Сведения о документе

Авторское право

Доступные форматы

Поделиться этим документом

Поделиться или встроить документ

Параметры публикации

Этот документ был вам полезен?

Это неприемлемый материал?

Авторское право:

Доступные форматы

ACQUITY System Solutions

Загружено:

Авторское право:

Доступные форматы

ACQUITY UPLCTM

Ultra Performance LCTM is a new category of separation science

Waters is leading the way into a new era of Separation Science

For Complete Confidence

Waters Corporate Office

Providence Biltmore Hotel

2004 Waters Corporation

ACQUITY UPLCTM Can Fit Anywhere

Toxicology & Disease

Delivery, Formulation &

Routine Quan, QA/QC

2004 Waters Corporation

MD for Wide Range of

Optimization & Validation

Immediate Impact Applications

2004 Waters Corporation

y Meeting Customer Need

2004 Waters Corporation

2004 Waters Corporation

Particle Size Evolution

2004 Waters Corporation

Properly designed small particles allow us to achieve

Summary of Theoretical Trade-offs

y At constant L/dp (improving Speed), 1.7 m particles offer:

2004 Waters Corporation

2004 Waters Corporation

Peptide Mapping Challenges in QC

y Need resolution of 10s 100s peptides

High Resolution Peptide Mapping 100 pmol:

2004 Waters Corporation

High Resolution Peptide Mapping:

This appears in the flip chart

2004 Waters Corporation

High Resolution Peptide Mapping:

A decrease in particle size yields higher resolution

Std Dev of <0.03 minutes over 1 hr

2004 Waters Corporation

Std Dev of <0.07 minutes over 2 hrs

2004 Waters Corporation

Deamidation T15 HPLC separation, 20 pmol

2004 Waters Corporation

Deamidation T15 UPLC separation :60 min.

2004 Waters Corporation

Deamidation T15 UPLC separation :30 min.

2004 Waters Corporation

Meeting Customer Needs

Tandem Quad Quantification Challenges

Many samples, tight deadlines

2004 Waters Corporation

y Quattro Micro for MS detection

y Bridged Hybrid C18 column,

y ESI+ with MRM data monitoring

2.1 x 30 mm, 1.7 m, 40oC

y 0.5 ml/minute flow rate

A: 10 mM NH4OAc in 20% MeOH

y Alliance HT 2795 for HPLC

2.1 x 30 mm, 3.5 m, 40oC

y 0.3 ml/minute flow rate

A: 10 mM NH4OAc in 20% MeOH

2004 Waters Corporation

y Dwell Time: 100 ms

UPLCTM vs. HPLC*

Alprazolam, 10 ppb, 309.09 > 205.13

2004 Waters Corporation