waters solutions for dmpk and biomarker analysis · asms 2011 denver, colorado stephen mcdonald...

©2011 Waters Corporation 1

Waters Solutions for DMPK and Biomarker Analysis

Waters User MeetingASMS 2011

Denver, Colorado

Stephen McDonald


Candidate Selection &Confirmation

Phase 1 Phase 2 Phase 3

Pre-clinical testing R&D - 18 months Clinical R&D - 5 years

Drug Development Process

Submit NDASubmit

IND





Submit IND

Instrument Needs

Submit NDA

High throughputMedium/High sensitivity assays

Simple validation required

Moderate throughput Highest sensitivity assayFull validation required

High throughput Medium/High sensitivity assay

Full validation required

QTof

Quad


Rationalizing Instrument Deployment

Question?Precisely how much do I

have?

StudyPK/PDADME

InstrumentTriple Quads

BenefitsMRM, High Sensitivity, High

Selectivity

Question?What is it?

StudyMetabolite ID

Biomarker Analysis

InstrumentQ-Tof

BenefitsFull scan acquisition, accurate

mass


Rationalizing Instrument Deployment

Question?What is it and how much of it is there?

StudiesMet ID for MIST

Identifying metabolites in a PK studyBiomarker studies where performing in vivo analysis

Exploit mutual benefits but only where it makes

sense


Drug Metabolism

PK/PD & ADME

Toxicity (non-GLP)


ng/mL-0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0

Respo

nse

-0

20

40

60

80

100

Ideal Scenario for Discovery DMPK

All peaks detected

Quantify with Linear Response

No Method Development

Fast analysis

No need for expert users

Identify all metabolites


The tools that power the discovery DMPK solution


Nefazodone MSE Analysis


Cleavages unique to molecule itself

PotentialBiotransformations

+O +glucuronide+H2O +sulfate+O2 +glutathione+.. +..+.. +..

Data(Fragmentation Spectra)

H60 10 µM, S+ MSe

m/z50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 410 420 430 440 450 460 470 480 490 500

%

0

100 x10 290.1516

218.0939

152.0834

124.0517

188.1090

178.0996

156.0786

209.0862189.1125

262.1190246.1251

219.0956237.1141

272.1407

486.2277

291.1547 488.2251

Need to bring all this knowledge together in a meaningful wayand have a clear way of visualizing this to the user

Using Chemical Intelligence


Applying Chemical Intelligence


Where Is My Hydroxylation?


Automated Localization of Biotransformation


Accurate Mass Quantification of Nefazodone @ 50pg/mL in Plasma

0.2

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

%

0

100260_19_6_004 1: TOF MS ES+

260.16953

6.52

0.48

0.45

0.43

0.27

2.68

1.990.98

0.92

0.65 1.491.24 1.92

2.50

2.35

3.69

2.88

2.953.39

4.783.98

4.354.69

6.236.15

5.835.645.37

7.15

6.83

11.4211.39

8.85

7.53

8.147.80 8.33 8.71

9.43

9.1811.27

10.059.73 10.2310.85

11.48

11.52

11.60

11.71

11.78

11.95


Time3.50 3.60 3.70 3.80 3.90 4.00 4.10 4.20 4.30 4.40 4.50

%

0

100

3.50 3.60 3.70 3.80 3.90 4.00 4.10 4.20 4.30 4.40 4.50

%

0

100 260.163.69

3.49

3.95

4.11 4.21

260.16 0.05Da3.94

Accurate Mass To Detect Peak in Complex matrix

1Da Window

50mDa Window

Nominal Mass

Accurate Mass


Linear Response Over 4 Orders of Magnitude

Sensitivity in the 50pg/mL range

Fast data acquisition for narrow UPLC peaks

Linear response > 4 orders of magnitide

ng/mL-0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0

Res

pons

e

-0

20

40

60

80

100


Fragment Ions: MSE Elevated Energy Data

116.1071

183.0810

260.1651

157.0647

m/z50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250 260 270 280 290 300

%

0

100 1.07e260.1645

116.1071

74.060872.0814

56.0506

59.0698

98.0968

86.0968

104.1074

183.0803

157.0647

117.1104153.0702

142.9403118.1137

165.0702

168.0571

182.0732

184.0743

218.1175207.9989185.0771 258.1132242.1552

264.2688

265.2719301.1508

297.2391284.1238


Calibration Lines Using Fragment Ion and Precursor Ion

Compound name: propranolol high energyCorrelation coefficient: r = 0.999660, r^2 = 0.999319Calibration curve: 0.0285934 * x + 12.0348Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

pg/mL-0 20000 40000 60000 80000 100000

Res

pons

e

-0

500

1000

1500

2000

2500

Compound name: propranolol high energyCorrelation coefficient: r = 0.997492, r^2 = 0.994991Calibration curve: 0.0204225 * x + -0.136933Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

pg/mL-0 20000 40000 60000 80000 100000

Resp

onse

-0

200

400

600

800

1000

1200

1400

1600

1800

2000

m/z = 116.107 (10mDa)

m/z = 183.081 (10mDa) LLOQ = 500pg/mL

LLOQ = 500pg/mL


Accessible Quantitative Analysis


ADME Profiling In Drug Discovery

Sample Formatting

• Perform ADME (stability, solubility etc)

Method Development

• Generate LC/MS/MS methods

Run Samples• Run standards and samples

Quantify• Quantify samples

Data Generation

• Calculate data - solubility, stability, protein binding etc.

Report Results• Rank samples and report to project team


ADME with Xevo TQ(S)

• Fast, robust method development to high throughput screening

ACQUITY UPLC®

• MRM sensitivity

Xevo® TQ(S)

• Drive efficiency and Productivity

Software


Fast p450 Screening by UPLC/MS/MS & Xevo TQ-MS

7.8 sec

28 sec

1. acetaminophen2. 1-hydroxybufuralol3. 4-hydroxymephytoin4. 7-hydroxy Coumarin5. 4-hydroxytolbutamide6. 1-hydroxymidazolam

1 2 3 4 5 6

Time 60 min Control

Time 60 min 0.1 µM

Time 60 min 1.0 µM

Time 60 min 10 µM

UPLC and Xevo TQ facilitates rapid analysis with resolution between all the p450 probe

substrates

P450 inhibition and induction testing allows drug – drug interactions and potential

metabolic issues to be identified and eliminated


Fully A

uto

mated

by Q

uan

Optim

ize

High Throughput Quantitative Analysis Xevo TQ and QuanOptimize™

Acquire Quantification Data

Generate Quantification Method

Sample Plate Preparation(ROBOTICS)

Data Process and Generate Report

Build MRM method (inc internal standard)

Optimise MRM transitionCone Voltage and Collision Energy


OpenQuan - Open Access Optimization and Quantitation

LoginUser name, job ID,

e-mail

Select QuanMethod

Set by admin

Submit Optimisation ListImport or cut-and-

paste

Submit Acquisition ListImport or cut-and-

paste

Position SamplesIndicted by Login screen

FinishResults by e-mail or print

Next user


Handling of Multiple Assay Resultswith ProfileLynx

DATA PROCESSING AND REPORT GENERATION

ProfileLynx

Solubility Browser

Microsomal Stability Browser

pH Stability Browser LogP/LogD Browser

Other Assays Supported - Protein Binding (plate or column) - Membrane Permeability(PAMPA, Caco-2, etc.) - Chromatographic Hydro-phobicity Index (CHI) - Immobilized Artificial Membrane

•The ProfileLynx™ Results Browsers contains up to 3 sections:

1.Results Table 2.Chromatogram 3.Calibration Curve

•Pass/Fail indicator column and user selected high-lighted flags allow fast review of the data.

•Chromatogram is interac-tive for manual integra-tion if needed.


ProfileLynx View by Experiment

Single browser to review all experiments


Sample Formatting

• Perform ADME stability, solubility etc.

Method Development

• QuanOptimize

Run Samples• QuanOptimize

Quantify• QuanOptimize

Data Generation

• ProfileLynx

Report Results• ProfileLynx and NuGenesis SDMS

Discovery ADME Workflow Using QuanOptimize, ProfileLynx & SDMS

Op

en

Qu

an

• QuanOptimize


Candidate Selection &Confirmation Phase 1

14C Structure ID

GLP Toxicity 28 days/2 species

Drug-Drug Interaction

Repeated dose toxicity

Metabolism

Single dose toxicity


Micro Sampling

Low Exposure Drugs

Sensitivity Needs Increasing

Inhaled Products

pg/mL sensitivity common place

Low sample volumes from mice / tail bled rats

Dried blood spot samples


Ele

ctri

c Fi

eld

Diffuse Ion

Cloud

Maximising signal

Maximising robustness

Designed to deal with problems associated with a larger sampling orifice


SYNAPT G2-S


Synapt G2-S Quantitation

Compound name: PropanololCorrelation coefficient: r = 0.999771, r^2 = 0.999542Calibration curve: 1.9722 * x + 0.318197Response type: Internal Std ( Ref 1 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

Conc-0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100

Res

pons

e

-0

20

40

60

80

100

120

140

160

180

Blank

2.5pg/mL

2.5pg/mL – 25ng/mL

30x improvement in exact mass sensitivity


G2-S Sensitivity – Product Ion MSE

10µM

1µM

100nM

10nM

Confident Structural elucidation can be performed down to 1000-fold dilutions using non-directed MSE fragmentation spectra


Waters RegulatedBioanalysis System Solution

• Best in class

Sample Preparation Solutions

• Fast, robust method development to high throughput screening

ACQUITY UPLC® I-Class

• Most sensitive MS

Xevo® TQ-S

• Compliant, Interactive workflow-driven data platform

UNIFI


Ultra High Sensitivity?

Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

1000.83

Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

100

2fg of

Verapamil Time

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

100

0.83

Solvent

Blank

0.1fg

6 replicates

RSD<20%

UPLC/MRM of Verapamil (solvent standard) using an

ACQUITY prepared for trace analysis

220 Zeptomoles


10ul loop injection 0.5pg/ul Verapamil in human plasma/ACN_2

Time0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00 1.05 1.10 1.15 1.20 1.25

%

0

100TC8_100721_1135 MRM of 1 Channel ES+

455 > 165 (Verapamil)2.66e7

0.83

Assay Robustness

Verapamil, 0.5pg/µL spiked into supernatant from 2:1

ACN:Plasma protein precipitation.

10µL injections

ACQUITY BDH 2.1x 50

Plasma injection 1

Plasma injection 2000

Plasma injection 4000

>4000 on column injections

RSD of peak areas < 5%


Metabolites In Safety Testing

‘In general, these metabolites are ofinterest if they account for plasma

levels >10% of total systemic exposure to drug related material, measured as area

under the curve (AUC) at steady state.’


Simultaneous MRM and Full Scan Detection

MRM of Parent

Full Scan of Metabolites

Metabolites


List of Potential Metabolites from Text

File

MS Source and Analyzer

Conditions

iMRM Method

Spectra of Parent Automatically Obtained from

MassLynx

Select Metabolites from list of probable

transformations

Identify Product Ions From Parent

Compound

Calculate MRMs and Write MS Method File

MSMS of Parent Compound

Automatic Product Ion Selection

Develop Theoretical MRMs to Investigate


[Benefits]Metabolites detected with

maximum sensitivity

Spectra acquired in the time scale of UPLC peak

Data acquired in one analytical run, no need for confirmatory

experiment

Simple Metabolite Detection and Confirmation

Time0.50 1.00 1.50 2.00 2.50

%

0

100

18_4_022 5: MRM of 1 Channel ES+ 458.195 > 339.845 (AZ1_Hydroxylation)

3.77e5

m/ z1 00 12 0 1 40 1 60 18 0 2 00 2 20 24 0 2 60 28 0 30 0 3 20 34 0 36 0 3 80 40 0 42 0 4 40 46 0

%

0

1 00

18 _4 _0 23 3 ( 2. 07 8) 5: Pro du ct I on s o f 4 58 ES+45 8. 19 5 > 3 39 .84 5 (AZ1_ Hyd ro xyla tion )

1.9 2e 5326

315

340

354

m/ z1 00 12 0 14 0 16 0 18 0 2 00 2 20 2 40 26 0 28 0 30 0 3 20 3 40 3 60 38 0 40 0 42 0 44 0 4 60

%

0

1 00

18 _4 _0 25 5 ( 2. 14 0) 3: Pro du ct I on s o f 4 42 ES+44 2.2 > 32 5. 84 7 ( AZ1_ Par en t)

8.9 8e 6326

313

340

425

Time0.50 1.00 1.50 2.00 2.50

%

0

100

18_4_025 3: MRM of 1 Channel ES+ 442.2 > 325.847 (AZ1_Parent)

1.08e8

Time0.50 1.00 1.50 2.00 2.50

%

0

100

18_4_022 1: MRM of 1 Channel ES+ 416.148 > 325.847 (AZ1_Dealkylation)

5.66e6

m/ z1 00 1 20 14 0 16 0 1 80 20 0 22 0 2 40 2 60 28 0 30 0 3 20 340 36 0 3 80 4 00 42 0

%

0

1 00

18 _4 _0 23 4 ( 1. 97 6) 1: Pro du ct I on s o f 4 16 ES+4 16 .1 48 > 32 5. 84 7 ( AZ1 _D ea lkyla tion )

5.5 6e 5326

313298

340

Time0.50 1.00 1.50 2.00 2.50

%

0

100

18_4_022 2: MRM of 1 Channel ES+ 428.184 > 325.829 (AZ1_Demethylation)

3.58e6

m /z10 0 12 0 140 1 60 1 80 20 0 22 0 24 0 260 2 80 3 00 3 20 34 0 36 0 380 4 00 4 20

%

0

1 00

18_ 4_ 023 3 (1 .87 1) 2 : Pro du ct I ons of 428 ES+42 8.1 84 > 3 25 .82 9 ( AZ1 _De me thy la tio n)

3. 92 e4326

147235

176

340

Dealkylation Demethylation

Dosed Compound

MRM 442.2>326

HydroxylatedMetabolite

MRM 458.2>340

PIC Spectrum

PIC Spectrum

PIC Spectrum

PIC Spectrum


Work-flow Oriented System Solutions

Increasing Regulatory Demands

Quality and Consistency of Results

Evolving Analytical Demands


Single Platform

Multiple Techniques

Elegant &Simple

Secure &Compliant

FutureProof

Unites our end-to-end system

solution

One software forChromatography

& MS

Workflow based design adjusts to roles and capabilities of

users

Compliant and validatedfor your

environment

Will grow with your needs

UNIFI:Scientific Information System


Supporting StudiesCandidate Selection &Confirmation


Submit IND

Submit NDA

Supporting Studies

Pharmacology, Biomarker



Metabolism


Ion Mobility Separation


An added dimension of separation with Synapt HDMSE


Metabolomic Biomarker Analysis

Targeted Chemspider search with >22 million compounds


Global Lipid Profiling


0

5

10

15

20

25

30

35

40

45

50

Nor

mal

ized

Con

c (n

g pe

r sa

mpl

e)x

1000

0

MTC

MTln3

Absolute quantitation of TCA Cycle intermediates with internal standards

Targeted Metabolite Biomarker Analysis


Peptide Biomarker Analysis

Amyloid β0.1 ng/mLOasis (MCXµ-elution),

UPLC,Xevo TQ-S

Sensitive,Robust, meets

assay requirements

Evolving analytical demands


Drug Development Process

Xevo TQ(S) MS

Synapt G2-S

Xevo G2 QTof

Drug Metabolism




PK/PD & ADME

Toxicity (non-GLP)

14C Structure ID

GLP Toxicity 28 days/2 species

Drug-Drug Interaction


Submit IND

Pharmacology, Biomarker

Dose Ranging Study

PK/PD

Metabolism

Proof of Concept


Submit NDA


Questions?

Thank You

waters solutions for dmpk and biomarker analysis · asms 2011 denver, colorado stephen mcdonald...

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