ACQUITY System Solutions

ACQUITY UPLCTM

System Solutions

©2004 Waters Corporation

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 today’s laboratory.

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


For Complete Confidence 2004 Sales Launch Workshops

Waters Corporate OfficeWaters Corporate OfficeACQUITY UPLCTM Laboratory Workshop – MassLynx\Empower

ACQUITY UPLCTM System Overview – Hardware

ACQUITY UPLCTM System Overview - Software & Connections® InsightTM

ACQUITY UPLCTM System Solutions

Providence Biltmore HotelProvidence Biltmore HotelWATERS Laboratory Informatics

ACQUITY UPLCTM Column Chemistry

ACQUITY UPLCTM Sales Collateral

ACQUITY UPLCTM Sales Positioning


ACQUITY UPLCTM Can Fit Anywhere

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

Characterization Toxicology & Disease Markers Natural Products 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, Impurs, Contams, Degrads Metabolites Patent Protection/

Product ExtensionMetabolites

Toxicology & Disease Markers Stability

Routine Quan, QA/QC

in vitro ADME

Bioanalysis

Natural Products

Patent Protection/ Product Extension

ADME(T)

in vivo Characterization

CombiChem Lib. Composition & Purity Physicochemical Properties

Ntrl Prods, Impurs, Contams, Degrads

Natural Products

Method Development

Monitoring Synthesis Products

Formulation & Release

Purity in Bulk Drug Analysis Storage Effects

MD for Wide Range of Compounds Optimization Optimization & Validation

Trace AnalysisPurity in Bulk Drug Analysis

Synthesis Products

MD for Wide Range of Compounds Optimization & Validation

Trace Analysis


Immediate Impact Applications

These Application Areas will be Significantly Impacted by ACQUITY UPLCTM

– Peptide Mapping– Tandem Quadrupole Quantification– Metabolite Identification– Metabonomics


Agenda

Customer Challenges

ACQUITY UPLCTM System Solutions– Proof Statements

Competition– There is none for UPLCTM

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

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

At constant L/dp (improving Speed), 1.7 µm particles offer:– Same Resolution– 9x (+900%) improvement in Speed vs. 5 µm particles (3x vs. 3 µm)– 3x (+300%) improvement in Sensitivity vs. 5 µm particles (2x vs. 3 µm)

ACQUITY UPLC™Systems for Peptide

Mapping


Peptide MappingPeptide Mapping

PRODUCTIONPRODUCTION

Basic &Applied

TargetID

LeadIsolation

LeadCharacterization

DEVELOPMENTDEVELOPMENTDISCOVERYDISCOVERY

LeadID

LeadTesting

ClinicalTrials

LeadOptimization

PilotScale

QA &QC

Full ScaleManufacturing

Lots of searching & identifying

Lots of characterizing and optimizing

Lots of analyzing

Peptide Mapping MS/MS

Peptide Mapping MSPeptide Mapping UV


Peptide Mapping Challenges in QC

Samples are complex– Protein digests

Need resolution of 10’s – 100’s peptides– For stability, purity and ID

Need sensitivity– For stability and purity

Require reproducibility– QC means that you get the same answer each time

Speed– Currently 1-2 hr runs– Has not been a feasible option.


High Resolution Peptide Mapping 100 pmol:Influence of Particle Size

AU

0.00

0.02

0.04

0.06

0.08

AU

0.00

0.02

0.04

0.06

0.08

Minutes

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00

5 µm Peaks = 70

Pc = 143

1.7 µm Peaks = 168

Pc = 360

HPLC

UPLCTM


High Resolution Peptide Mapping:Influence of Particle Size (Enhanced Baseline)

1.7 µm Peaks = 168

Pc = 360

5 µm Peaks = 70

Pc = 143

AU

0.000

0.005

0.010

0.015

0.020

AU

0.000

0.005

0.010

0.015

0.020

Minutes0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00

This appears in the flip chart


High Resolution Peptide Mapping:Influence of Particle Size (Enhanced Baseline)

AU

0.000

0.005

0.010

0.015

0.020

AU

0.000

0.005

0.010

0.015

0.020

Minutes20.00 21.00 22.00 23.00 24.00 25.00 26.00 27.00 28.00 29.00 30.00

1.7 µm Peaks = 168

Pc = 360

5 µm Peaks = 70

Pc = 143

A decrease in particle size yields higher resolution


Injection-to-Injection Reproducibility50 pmol Enolase Tryptic Digest

AU

0.000

0.010

0.020

0.030

0.040

0.050

0.060

0.070

Minutes

0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00

Injection 1

Injection 2Injection 3Injection 4

Injection 5

Std Dev of <0.03 minutes over 1 hr


AU

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

Minutes

0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 110.00 120.00

Injection-to-Injection Reproducibility50 pmol Phosphorylase b Tryptic Digest

Std Dev of <0.07 minutes over 2 hrs


Deamidation T15 HPLC separation, 20 Deamidation T15 HPLC separation, 20 pmolpmol:120 min.:120 min.

Isoaspartic Acid DL Iso VK

Asparagine DLNVK

Aspartic Acid DLDVK

Pc: 320


Deamidation T15 UPLC separation :60 min.Deamidation T15 UPLC separation :60 min.

DLIsoDVK

DLDVK

DLNVK Pc: 500


Deamidation T15 UPLC separation :30 min.Deamidation T15 UPLC separation :30 min.

DLIsoDVK

DLDVK

DLNVK

Pc: 375


Meeting Customer Needs

By utilizing ACQUITY UPLCTM column technology, the ACQUITY UPLCTM System delivers the ultimate in…

Resolution – Will greatly increase peak capacity over HPLC

For stability, purity and ID

Sensitivity– Resolution and peak shape

Pull out possible modifications earlier that will impact activity

Reproducibility– <0.07 minute Std deviation over 2 hrs

Speed– Can run in 30 minutes what previously took 2 hrs

ACQUITY UPLCTM

Systems for Tandem Quadrupole Quantification


Tandem Quad Quantification ChallengesADME– Many samples to analyse– Many different compounds to characterize– May have complex matrices

Pharmacokinetics– Less candidate diversity, more detailed experiments– Complex matrices– Carryover

Regulated Bioanalysis– Many samples, tight deadlines– Complex matrices– Sensitivity– Carryover– Require reproducible methods– Compliance


Experimental Conditions

ACQUITY for UPLCBridged Hybrid C18 column,

– 2.1 x 30 mm, 1.7 µm, 40oC

0.5 ml/minute flow rate5 µL Sample injection Mobile Phase

– A: 10 mM NH4OAc in 20% MeOH– B: 10 mM NH4OAc in 80/20 MeOH/AcN

Gradient– 35%B to 100%B in 1 minute

Alliance HT 2795 for HPLCXterra C18 Column

– 2.1 x 30 mm, 3.5 µm, 40oC

0.3 ml/minute flow rate5 µl Sample injectionMobile Phase

– A: 10 mM NH4OAc in 20% MeOH– B: 10 mM NH4OAc in 80/20 MeOH/AcN

Gradient– 25%B to 100%B in 2.5 minute

Quattro Micro for MS detectionESI+ with MRM data monitoringDwell Time: 100 msInter-scan delay: 20 msInter-channel delay: 20 msSource temperature: 130oCDesolvation temperature: 350oCDesolvation gas flow: 750 L/HrCone gas flow: 50 L/Hr


UPLCTM vs. HPLC*

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

1

100

%

0.66

4

100

%

0.67

UPLCTM

HPLC

0.50 1.00 1.50 2.00 2.50 3.00 3.50Time1

100

%

0.50 1.00 1.50 2.00 2.50 3.00 3.50Time4

100

%

2.59 2.62


HPLC and UPLCTM 309.09>205.13*

0 20 40 60 80 100ppb

Response

r2=0.998

Slope = 88.7

1 ppb, 10 ppb, and 100 ppb

6 Injection each

Response

0 20 40 60 80 100ppb

r2=0.998

Slope = 148.2

1 ppb, 10 ppb, and 100 ppb

6 Injection each

HPLC UPLC™


HPLC and UPLCTM*309.09>205.13

1.60 2.00 2.40 2.80 3.20Time

100

%

2.61 1 ppb, and 10 ppb

6 Injection each

0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20Time2

100

%

0.67Average S/N 1 ppb = 131.5 (4 times over HPLC)

HPLC UPLC™

Average S/N 1 ppb = 32.9

Average RT = 2.62, SD = 0.00428, RSD% = 0.163 Average RT = 0.670, SD = 1.62e-8, RSD% = 1.52e-6


Experimental Conditions

Acquity UPLC

Bridged Hybrid C18 Column: 2.1 x 50 mms, 1.7 mm, 40oC

Injection Volume: 5 mL

Flow Rate: 0.6 ml/min – Back Pressure at 10900 psi– No splitting

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

Gradient:– 40% B to 50% A in 0.4 minute– 2 minute total run time

Micromass Quattro Premier

Electrospray

MRM Data Monitoring

Capillary Voltage: 3 kv

Source Temperature: 130oC

Desolvation Temperature: 400oC

Desolvation Gas Flow: 800 L/Hr

Cone Gas Flow: 50 L/Hr

Collision Gas Flow: 0.3 L/Hr

Sample Prep.:– Standards were spiked into crashed

plasma – Rat plasma was crashed by

acetonitrile 2/1 ratio


100 ms vs. 5 ms Dwell TimeAlprazolam in Rat Plasma, 10 ng/mL, 5 ul Injection

100 ms Dwell Time, 10 ms Delay

0

%

0.63

100 105 110 115 120 125 130Scan0

%

0.63Peas Area = 16791

Peak Width = 1.8 s

Points Across Peak = 7

0.25 0.75 1.25 1.75Time

Convert the x axis to scan number

5 ms Dwell Time, 5 ms Delay Peak Area = 16262

Peak Width 1.8 s

0.25 0.75 1.25 1.75Time0

%

0.63

1100 1200 1300 14000

%

0.63Points Across Peak = 60

Convert the x axis to scan number

Scan


Calibration Curves: 100 ms vs 5 msPropranolol, External calibration, 0.05 – 500 ng/mL, 13 points

5 ms Dwell Time, 5 ms Inter-channel Delay 100 ms Dwell Time, 10 ms Inter-channel Delay

0 100 200 300 400 500ng/mL

r2 = 0.995

Slope = 118.6 Slope = 140.5

r2 = 0.993

0 100 200 300 400 500ng/mL


Calibration Curves: 100 ms vs 5 msAlprazolam, Internal calibration, 0.02 – 200 ppb, 13 points

100 ms Dwell Time, 10 ms Inter-channel Delay5 ms Dwell Time, 5 ms Inter-channel Delay

R2 = 0.996

Slope = 0.0109

R2 = 0.998

Slope = 0.0115

0 40 80 120 160 200

ng/mL ng/mL

0 40 80 120 160 200


No Detectable Carryover in Rat PlasmaFirst Blank After 1000 ng/mL Injection

1000 ng/mL, Plasma, 5 ms Plasma Blank

0

100

%

CalP5ms0328058 MRM of 3 Channels ES+ 314.1 > 210.20.63

0.20 0.40 0.60 0.80 1.00 1.20 1.40Time0

100

%

0

100

%

0

100

%

CalNt5ms0328059 MRM of 3 Channels ES+ 314.1 > 210.2

7.00e4


7.00e4


7.00e4

6.91e6

Peak Area = 97963

0.20 0.60 1.00 1.40 Time0

100

%

0

100

%

CalP5ms0328058 MRM of 3 Channels ES+

309.1 > 205.20.63

CalP5ms0328058 MRM of 3 Channels ES+ 260.2 > 116.00.48

4.97e7

6.27e6

Peak Area = 747309

Peak Area = 96015


System Robustness10 ng/mL Standard Spiked in Crashed Rat Plasma

Alprazolam, Internal Calibration, 5 ms Dwell Time, 5 ms Delay

0.50 1.00 1.50Time0

100

%

0

100

%

Robustness03290007 309.1 > 205.2

0.64

Robustness03290983 309.1 > 205.20.64

Retention Time (Min.)

0.4

0.5

0.6

0.7

0.8

Tim

e (M

in) n = 963

Average = 0.65 min, RSD% = 0.26%

Peak Area = 9228

1 150 300 450 600 750 900Peak Area = 9204

Injection No.

Calculated Concentration (ng/mL)

0

5

10

15

20

25

Cal

cula

ted

Con

c. (n

g/m

L)

1 150 300 450 600 750 900

n = 6

Injection No.

R2 = 0.999

0 200 400 600 800 1000ng/mL0.0

Response

n = 963

Average = 10.83 ng/mL, RSD% = 4.43%

AcQuity/QuattroPremier




Resolution – Greatly increases peak capacity over HPLC

Pull away possible interfering endogenous molecules

Sensitivity– Able to quantify low level metabolites

Low dosing of drugImportant metabolites

Speed– Can run in 1 minute and get very good data– Discovery and development labs can benefit

Carryover– Minimal

ACQUITY UPLCTM

Systems for Metabolite ID


Discovery/DevelopmentChallenges

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

Development– Difficult to find some metabolites in the complex matrix– May miss metabolites that are at very low levels– Can’t 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

NH

OH

O

CH3NH

O-Glucuronide

O

CH3

N

O

O

CH3

NH

OSO3H

O

CH3

60% ~35%

N

O

O

CH3

O

NH2

S OH

Cysteine

Paracetamolm/z = 152

m/z = 328 m/z = 232

m/z = 150

Glucuronide Sulphate

S-CysteineConjugation

m/z = 271


Resolution30 Min TIC

Time2.00 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 30.00

%

0

100

%

0

100H040502a_JG_14 1: TOF MS ES+

TIC2.15e4

1.10

5.02

1.73 4.33

23.33

12.4012.03

10.226.00

9.238.527.62 11.25

21.3113.51

20.7018.6316.4013.9315.67

17.51 19.21

22.04

23.69

24.59 26.6825.8227.49

H042102a_RP_10 1: TOF MS ES+ TIC

3.51e40.67

22.92

0.78

2.09

1.30

11.208.62

2.57

3.55 4.205.615.15 7.336.49 7.93

21.6621.10

20.3117.13

14.5112.71 16.32

19.4318.26

22.23

26.01

23.15

24.40

25.17

26.51

27.34

28.7728.12 29.98

HPLC

UPLCTM


Resolution XIC 30 Minute Run

Sulphates

Time2.00 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

%

0

100H042102a_RP_10 1: TOF MS ES+

232744

2.36

2.05

1.51

0.560.73

6.22

2.68

6.38

6.5923.42

6.8022.5010.39

7.83

24.35

Time2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00

%

0

100H040502a_JG_14 1: TOF MS ES+

2321.11e3

10.28

3.22

9.80

10.37

11.02

11.14

21.58

HPLC

UPLCTM


Speed UPLCTM 10 Minute Run

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

%

0

100H042102a_RP_13 1: TOF MS ES+

TIC4.83e4

0.40

8.71

0.48

5.951.52

0.77

4.95

3.28

2.902.67 4.343.93

5.74

6.326.87

6.74

8.20

8.007.797.547.28

8.54

8.35

9.858.81

9.339.029.48

10.04

10.31


Speed30min HPLC Vs 10min UPLCTM

Time2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00

%

0

100

%

0

100H040502a_JG_14 1: TOF MS ES+

2321.11e3

10.28

3.229.80

10.37

11.02

21.58

H042102a_RP_13 1: TOF MS ES+ 232974

1.65

1.13

1.75

3.65

1.96

3.70

8.483.865.82

8.7710.89

HPLC30 min

UPLCTM

10 min


Sensitivity

Time2.50 5.00 7.50 10.00 12.50 15.00 17.50 20.00 22.50 25.00 27.50 30.00

%

0

100H040502a_JG_14 1: TOF MS ES+

44144.6

31.92

26.896.36

5.190.79

0.90

1.00

5.14

2.152.01

1.945.02

4.692.22

4.073.53

5.25

5.38

6.46

14.716.49

14.66

12.6912.51

10.498.366.61

7.66

9.61

12.46

11.77

14.60

13.93

25.95

22.3717.56

15.13

16.57

15.69

17.66 20.24

18.65

19.25

22.16

20.34

24.12

22.4925.17

31.75

31.71

31.5326.93

27.10 31.34

31.23

31.1127.54

28.19

30.2929.8729.09

31.96

HPLC

Time2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00

%

0

100H042102a_RP_10 1: TOF MS ES+

441187

21.45

20.99

8.83

0.48

1.246.47

6.191.98 5.752.15 3.832.883.24 4.68

5.35

6.517.986.637.47

8.90 20.79

18.6617.87

17.578.9616.3414.5813.1912.1211.569.10

11.06 13.78 15.86

19.47

20.31

25.67

22.69

21.98

24.73

24.1523.06

23.64

26.09

26.46

26.67

UPLCTM

Glutathione Conjugate (sign of tox)




Resolution – Greatly increases peak capacity over HPLC

Increase the ability of MS and MS/MS exact mass

Sensitivity– Able to ID low level metabolites– Critical to understanding metabolic pathway– Need to ID “bad” metabolites (glutathione conjugates)

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

Complex Samples– Biofluids like:– Plant extracts– 100’s -10,000’s of endogenous primary and

secondary metabolites

Ability to see small changes amongst the tall trees

Can we go fast?– To some, speed is important– FIA vs HPLC vs UPLC™


Resolution10 minute Run

HPLCHPLC

UPLCUPLCTMTM

%

0

1001: TOF MS ES+

TIC5.26e4

3.722.58

2.310.570.46

1.120.712.04

1.63

3.552.87

2.733.29

3.994.10 5.805.55

5.13

4.534.93

5.92

6.23

Detected Peaks 1186

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00

%

0

1001: TOF MS ES+

TIC5.26e4

2.862.20

0.520.39

0.33

2.031.941.31

1.000.751.46

2.64

2.45

7.543.24

3.10

5.124.41

3.663.49

3.724.304.143.97

6.725.39

6.295.875.74 6.53

7.307.22

Detected Peaks 2246

Female Black Mouse, PM Collection


Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

%

0

100

%

0

100H031024CSa014 1: TOF MS ES+

401210

4.80

0.583.74

2.622.370.732.131.501.80

3.123.40

4.614.09

4.37

5.945.85

5.065.64

5.38

6.026.09

6.48 6.727.06 7.977.737.45

H040116a_RP_ESI+_010a 1: TOF MS ES+ 401756

3.87

0.27

2.780.73 1.98 3.285.35 9.51

8.417.165.47 6.52 7.768.628.97

Sensitivity*Metabonomics XIC m/z = 401.

HPLCHPLC

UPLCUPLCTMTM


HPLC and UPLCTM

Gender Variation in Black Mice

Scores: Component 2 - Component 3

Component 2-0.200 0.000 0.200

Com

pone

nt 3

-0.400

-0.300

-0.200

-0.100

-0.000

0.100

0.200

0.300

0.400

UPLCUPLCTMTMScores: Component 2 - Component 3

Component 2-0.200 0.000 0.200

Com

pone

nt 3

-0.400

-0.200

0.000

0.200

0.400

MaleMale

FemaleFemaleHPLCHPLC

FemaleFemale

MaleMale

Loadings: Component 2 - Component 3

Component 2-0.200 0.000 0.200 0.400

Com

pone

nt 3

-0.200

0.000

0.200

0.400

0.6005.5433

255.0647

5.9370271.0600

5.2823103.0548

4.5006255.0651

5.6272285.0761

2.5612298.14785.3187

400.26982.3045

297.14335.2768131.05025.3184

379.21055.9209

363.2159

5.5816345.18915.9482

361.20075.6597

382.2590

2.2041364.06425.1015

287.1468

5.7160149.06135.1286

271.0620

6.1933347.2207

5.7155259.0985

5.9087403.2309

4.6186285.0760

3.5501305.0830

5.5420256.0706

5.1251118.0658

2.1682283.1331

6.3750377.1965

5.3172231.1422

5.1894170.0621

4.9392374.2530

5.7800181.0866

4.4954431.0995

5.3830380.2249

5.8525355.2628

4.1216145.0330

2.6665127.03945.2528

130.06495.3221

153.12725.1149447.0967

5.2111365.23135.9241

364.22004.4965

256.06925.7322

165.06442.9077316.1749

3.5563179.0708

5.9385272.0650

5.6269286.08112.3162298.14632.2339

421.08886.2501

247.08766.2497

263.08682.2616

318.18825.9506

362.20476.1804

461.27373.0281

346.2215

2.2082289.03875.5887

346.19164.6580

130.0635

6.2281249.10935.1245

146.06013.6909

332.20513.1041

318.1893

6.2626134.06055.3206

401.27055.9969

509.23945.6598

383.26005.7692

135.0844

5.2417172.07865.2743

347.18573.7759

105.0342

3.7595325.06612.2096

243.05162.5823

158.02965.2006

235.13532.5512

304.17386.2665

132.04432.5305

111.00875.8495

373.2735

5.2783104.05905.0155

372.23775.2832

255.12873.2852

182.11915.8572

356.26602.2023

245.06585.3218

135.11345.9951

401.21462.0905

152.05353.6207

110.06075.2488

449.18984.1228

191.06622.8680

171.07775.9124

404.23323.0258

137.0598

3.3764301.21103.9211

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7.22297.02

3.25363.16

3.75374.12

5.54523.24

5.67229.23

4.55421.24

6.32348.99

5.23109.71

5.54374.28

7.04155.05

2.21192.104.94

397.182.73

346.052.83

210.115.64

244.226.03

271.186.87

391.046.87

284.332.99

164.036.71

303.155.15

457.125.07

273.162.54

350.102.83

359.004.42

172.074.82

392.095.64

161.133.07

349.183.70

241.075.65

179.143.83

229.034.69

254.075.15

531.084.35

239.134.85

436.125.26

241.145.89

318.083.63

369.012.27

278.032.50

162.065.04

313.183.07

392.012.64

379.084.84

307.174.28

501.022.30

337.082.42

238.994.70

497.104.07

255.063.72

233.093.36

444.112.28

138.085.09

347.192.91

235.023.46

237.124.35

134.092.55

285.136.33

252.993.40

411.173.36

159.043.52

374.002.26

247.135.48

386.294.72

347.122.93

338.115.13

301.143.00

351.094.33

274.073.63

354.997.02

256.924.63

134.104.36

303.124.86

381.213.71

431.173.73

207.043.44

302.212.67

283.084.55

186.025.05

365.194.45

379.224.47

417.273.09

139.996.16

241.235.30

295.142.44

370.073.45

319.126.09

241.884.88

325.176.30

172.973.09

153.052.42

165.062.18

292.044.33

305.112.43

250.013.56

342.192.63

114.072.27

212.084.27

543.04

2.89186.07

3.07338.082.93

248.123.32

229.104.13

280.043.12

273.033.21

366.165.69

207.027.15

230.053.69

461.183.14

305.092.92

528.132.47

474.172.58

120.074.62

271.094.69

471.022.39

501.013.72

329.045.75

311.206.52

167.064.13

264.044.94

430.133.98

155.073.12

511.083.52

162.052.95

381.045.69

130.002.21

381.082.28

261.114.18

170.112.24

184.103.37

413.135.50

154.996.32

333.025.06

163.993.91

186.035.87

207.173.34

130.965.95

137.982.06

114.062.27

302.922.10

141.114.17

234.136.99

156.996.86

184.017.26

532.393.54

139.995.90

186.022.52

415.066.51

282.926.15

323.026.48

172.987.21

321.045.25

773.462.57

369.062.93

389.127.26

241.884.91

567.286.50

143.985.06

363.165.00

390.093.90

183.002.80

426.192.48

304.164.84

365.123.93

433.233.10

308.033.47

230.047.26

138.982.28

219.062.74

347.074.50

202.015.59

254.923.74

186.116.09

234.024.97

202.017.03

143.976.45

130.004.79

261.086.70

258.892.52

264.062.27

270.182.73

234.117.17

262.153.64

319.085.60

211.114.76

230.055.54

319.243.72

187.072.06

140.006.92

175.986.27

169.986.08

144.982.29

356.064.63

239.136.72

309.113.45

176.116.68

254.923.77

355.092.85

295.095.67

169.986.67

242.882.52

275.034.94

398.192.81

152.032.64

371.082.63

243.122.18

261.066.91

189.023.25

272.012.61

257.983.42

327.093.36

146.042.79

338.057.31

155.983.07

526.116.08

273.275.86

172.972.22

385.102.25

197.136.33

115.966.72

258.085.60

229.133.52

197.123.45

280.122.89

340.053.46

192.092.48

229.143.23

304.086.54

156.997.24

169.992.30

358.062.27

270.033.73

274.076.81

186.985.99

209.116.17

258.893.53

118.073.72

322.083.72

519.112.07

211.116.82

309.184.62

293.072.26

154.996.67

256.914.09

114.975.55

301.246.49

282.274.94

369.115.95

268.176.31

351.953.24

445.113.75

131.062.07

180.075.54

409.252.19

184.105.33

270.172.91

265.143.27

447.092.49

424.072.80

282.114.83

372.223.02

275.147.23

406.272.94

269.065.01

267.176.25

219.166.26

165.992.19

189.114.11

160.994.73

346.103.91

328.176.53

414.206.71

241.882.83

204.014.30

263.063.69

281.053.73

315.092.58

213.073.51

132.082.31

186.074.85

252.154.15

287.133.43

134.104.90

137.093.43

379.152.67

431.032.83

276.076.02

191.023.50

265.144.77

414.162.72

163.065.78

165.093.90

212.063.95

281.122.58

402.074.42

506.115.13

330.234.89

177.132.71

189.026.26

213.892.36

361.025.49

413.215.22

849.684.14

303.042.31

264.004.32

281.054.37

406.095.63

441.224.96

417.123.72

454.212.52

151.045.28

204.083.33

213.122.47

208.052.51

124.047.25

154.992.40

185.132.59

275.103.02

245.184.12

439.142.52

181.062.26

126.062.07

298.095.00

299.194.14

259.052.63

259.104.42

467.082.66

235.105.76

357.292.20

359.172.80

150.054.70

245.112.66

330.993.86

386.175.15

150.064.38

282.062.94

272.093.35

261.062.27

248.094.28

222.074.09

141.963.01

310.152.73

348.112.51

209.053.15

185.99

2.74162.053.13

263.132.52

135.054.44

130.062.73

146.063.14

158.032.30

393.162.38

549.034.13

192.035.18

847.732.65

230.046.22

395.203.15

444.035.33

112.883.01

585.285.65

351.153.65

400.103.29

389.123.74

375.084.79

474.154.14

481.143.52

386.044.78

385.174.14

292.075.48

155.983.67

497.132.52

106.033.76

462.133.71

502.132.83

341.075.76

554.305.47

414.283.52

388.055.41

219.162.64

254.055.63

455.225.62

295.112.94

122.035.95

403.144.13

470.114.73

507.254.06

188.075.23

849.754.28

361.132.68

292.023.43

312.145.13

280.074.28

221.074.97

155.982.73

356.114.95

207.042.72

290.993.43

239.134.63

255.206.21

155.983.28

351.093.92

365.005.34

344.156.22

249.104.33

374.133.45

380.086.22

431.142.94

226.062.09

137.044.42

464.073.88

269.063.28

227.183.93

343.126.22

250.103.87

369.194.42

378.112.70

366.115.16

285.085.00

298.194.09

189.024.97

706.394.42

435.125.60

113.962.87

423.083.02

142.063.68

453.155.41

294.214.42

447.125.54

177.163.94

317.214.94

249.103.68

179.052.45

145.085.62

274.182.66

240.032.33

371.083.14

472.024.13

381.134.78

301.153.27

216.063.03

464.025.61

257.163.43

243.095.56

411.204.77

430.174.71

163.115.53

235.153.14

502.992.45

432.124.14

133.075.02

250.063.69

280.065.17

107.083.70

485.164.14

208.064.43

465.103.93

277.015.02

455.152.65

407.183.44

206.122.92

495.145.43

271.065.04

475.254.14

396.125.56

443.153.58

154.994.77

240.094.71

343.205.34

112.944.62

444.145.65

471.173.86

372.172.73

373.106.22

429.152.45

376.104.43

476.135.56

213.184.69

480.173.52

227.003.68

336.024.13

469.113.15

523.982.49

160.043.14

279.963.86

310.135.63

291.115.00

389.133.65

412.203.35

135.082.43

204.062.49

298.104.07

252.122.83

134.063.33

341.035.07

280.152.61

382.113.67

422.105.24

108.573.52

225.003.87

196.103.68

247.075.12

456.173.34

276.124.44

257.043.28

688.074.77

332.152.44

506.043.77

295.084.60

419.112.49

201.064.99

298.204.42

466.083.68

552.063.44

461.263.28

439.082.93

316.094.28

174.063.28

399.083.33

203.975.04

297.084.45

200.13

4.45132.05

5.04431.11

4.15407.14

5.75553.30

5.12457.17

2.66331.99

4.41446.09

4.09113.96

4.14262.02

6.22427.14

3.24172.09

2.19188.03

5.01139.99

5.25108.63

6.22251.11

2.49312.16

5.63439.23

6.54179.14

4.70292.06

4.71278.13

2.93304.035.55

267.114.90

255.034.50

259.095.00

154.99

2.23279.13

2.23226.114.14

480.145.19

106.885.13

441.165.60

228.173.66

238.103.27

411.124.36

405.093.34

163.083.70

472.163.52

266.025.13

458.162.45

433.123.26

309.123.28

194.082.29

231.052.49302.102.49

346.10

5.13384.28

3.14176.045.13391.14

2.18384.09

4.42465.074.43

377.094.97

705.393.03

188.073.27

425.103.27

354.104.43

478.13

2.49188.034.14

105.075.23

108.123.14

181.004.83

441.125.12

300.075.12

299.084.07

343.183.70

443.154.62

177.165.12

461.174.63

267.103.87

629.174.77

376.143.27

397.093.52

268.03

3.14500.993.14

277.964.77

341.183.87

311.16

5.04255.06

3.67350.103.68

404.034.63

443.143.14

233.975.18

106.93

3.96316.215.24

108.203.28

331.113.28

442.083.14

187.983.27

440.075.18

106.993.87

359.153.87

399.144.29

220.073.65

428.193.68

551.052.23

189.043.87

385.16

4.77429.17

4.97278.05

5.24108.05

3.87371.17

4.44103.063.87174.11

5.02113.96

4.77239.08

2.49207.04

3.87357.14

2.49311.16

3.14134.034.15268.064.44489.183.86

314.163.86

402.143.87

628.16

3.66226.142.17

207.033.27

353.093.71460.13

4.99296.06

4.43477.13

3.87400.13

5.12298.07

2.45144.085.13

459.17 3.27441.08

2.19382.08 2.18

206.043.86

313.16

3.86401.14


Comparison of Markers FoundGender Variation

HPLCHPLC((m/zm/z))

UPLCUPLCTMTM

((m/zm/z))

New Endogenous New Endogenous MetabolitesMetabolites

((m/zm/z))

255 255206271178285297103377259

241206 401271 313178 441285 325297 382103377259*Table created using the greatest significance factors


How Fast Should We Go?

Urine

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00Time10

100

%

0

100

%

0

100

%

H030312_CS_ES+_003 1: TOF MS ES+ 181

1.39e37.167.006.406.04

5.775.424.893.69

7.42 10.6410.557.85 8.368.58 8.99 10.23 10.75

11.15 11.78

H030312_CS_ES+_003 1: TOF MS ES+ 195

2.34e37.517.126.596.32

6.045.395.231.33

10.617.81 10.458.15 8.579.05 9.419.82

10.85

11.24 11.83

H030312_CS_ES+_003 1: TOF MS ES+ 264

1.57e49.949.338.527.917.857.517.006.59

6.366.095.965.51

8.92 10.14

10.39

10.6411.0711.6411.96

Theophylline

Caffine

Nortriptylline

Blank

2.00 4.00 6.00 8.00 10.00 12.00Time49

100

%

H030312_CS_ES+_001 1: TOF MS ES+ TIC

3.31e46.28

6.01

5.93

5.825.73

5.585.30

4.944.854.49

8.698.51

8.247.767.72

6.38

8.989.70 9.86

10.25

10.54

10.74

10.84

10.96

11.26

11.37

Blank Injection Urine InjectionUrine

2.00 4.00 6.00 8.00 10.00 12.00Time25

100

%

H030312_CS_ES+_004 1: TOF MS ES+ TIC

3.33e48.568.34

7.707.336.83

6.696.36

6.296.23

8.80 9.63 10.20

10.45

10.87

11.17

11.76

Theophylline

Caffeine

Nortriptyline


SpeedInfusion vs. Chromatography

Time0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00

%

0

100

%

0

100RAT URINE 1: TOF MS ES+

TIC3.74e4

0.910.540.17

1.14

H040120b_RP_ESI+_027a 1: TOF MS ES+ TIC

3.74e40.48

0.36 0.56

1.23

1.02

Infusion of Mouse UrineInfusion of Mouse Urine

UPLCUPLCTMTM 1 minute gradient1 minute gradient


Mass Spectra

Combined Spectra Across UPLCCombined Spectra Across UPLCTM TM RunRun

Mystery MetaboliteMystery Metabolite

Combined Spectra InfusionCombined Spectra InfusionWhere are they?

4,8-Dihydroxyquinoline-2-carboxylic acid

m/z100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850

%

0

100

%

0

100

%

0

1001: TOF MS ES+

8.06e3255.0702206.0477

178.0551

297.1439

597.3319553.3046298.1455509.2752428.1962345.1637385.2008 465.2274

685.3845641.3597 729.4149773.4482 817.4836

849.5232

1: TOF MS ES+ 4.82e3206.0406

178.0480

166.0841127.0370

297.1440

220.1168 311.1653

312.1604 382.0857413.1523440.1879474.1742 511.1755 605.4402568.2643541.2761 671.1874 786.2729813.1944

1: TOF MS ES+ 1.67e4447.6763

212.8847

196.9222

144.1182

446.7061445.5121229.1954444.6927381.4590

256.0699 347.0142

448.6691

449.4927 542.6557 586.0293629.5122 673.6452704.3780 740.3656




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.

Sensitivity– Resolution and peak shape add in identifying low level

endogenous metabolites

Speed– Can get throughput of FIA without the ion suppression


Theoretical Run Time Compatibility Chart

Instrument Separation Time (min)

1(Pw 1-2 sec)

5(Pw 5-7 sec)

10(Pw 7-9 sec)

>30(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 X √ √ √ √ √ √ √Quattro micro X √ √ √ √ √ √ √Quattro Premier X √ √ √ √ √ √ √LCT-Premier √ √ √ √Qtof Family X √ √ √


Summary

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

– ResolutionMore information in same amount of time

– Achieved using small particles, optimized delay volumes and the ability to access linear velocities appropriate for small particles.

Whether UV or MS…

Whether Discovery or Development

ACQUITY UPLCTM will enable our customers– To enhance productivity– To have Complete Confidence in their data


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