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Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation 1 Barcelona, 7 de Barcelona, 7 de Junio Junio de 2012 de 2012

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Page 1: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

Sample Preparation for Sample Preparation for BioanalysisBioanalysis

Evaluation Group; CO R & D ©2011 Waters Corporation 1

Barcelona, 7 de Barcelona, 7 de Junio Junio de 2012de 2012

Page 2: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

Outline

� Introduction

—Challenges in Bioanalytical Method Development

� Sample preparation techniques

—Waters’ solutions

Evaluation Group; CO R & D ©2011 Waters Corporation 2

� Application examples

Page 3: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

Segments Within Bioanalysis

Discovery(Candidate Selection)

>Thousands of cmpds

>Average sensitivity

>Clear/Clean extract

>Easy to use

Development(Pre-clinical)

>Several cmpds

>Better sensitivity

>Clean/Pure extract

>Method development

Late Phase(Phase 1-3 Clinical)

>Targeted cmpds

>Highest sensitivity

>Pure extract

> Reliable and

Evaluation Group; CO R & D ©2011 Waters Corporation 3

>Easy to use

>Reliable

>Non-regulated

>Method development

>Reliable and inter/intra reproduc.

>Regulated

> Reliable and inter/intra reproducibility

>Regulated

Sometimes “methods”

linked

“Methods” always linked

Leads to different testing patterns

Page 4: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

Our Approach to Bioanalytical Method Development

� No “one size fits all”

� Different segments of drug development process

— Scientific and business drivers may be different

— Drivers may be the same but with varying degrees of risk

tolerance

Evaluation Group; CO R & D ©2011 Waters Corporation 4

tolerance

� Use of scientifically appropriate criteria for final method

choice

Page 5: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

Goals of Sample Preparation

Minimize risk

� Minimize matrix effects

— Reduction of ion suppression/enhancement, interferences, background

� Eliminate sample to sample variability

— More reproducible quantitation

— More robust assays

— i.e., Plasma from different subjects or species

Evaluation Group; CO R & D ©2011 Waters Corporation 8

— i.e., Plasma from different subjects or species

� Decrease assay variability

— Pass ISR

— Successful transfer across labs, analysts, sites

Increased sensitivity

� Sample concentration

� Removal of interferences

Cleaner Samples

� Increased instrument uptime

� Improved method robustness

Page 6: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

Different Methods for Different Purposes: Decision Making Process

Evaluation Group; CO R & D ©2011 Waters Corporation 10

Page 7: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

Typical Process

1. The simplest method which meets the assay needs is usually chosen• PPT or LLE are common starting points

2. For challenging assays (low detection limits, closely related endogenous constituents, inhalation products,

Evaluation Group; CO R & D ©2011 Waters Corporation 11

related endogenous constituents, inhalation products, peptides, etc) SPE may be first choice

3. Exact technique chosen will depend on outcome of study and how much risk can be tolerated

Page 8: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

Sample Prep products for bioanalysis

Evaluation Group; CO R & D ©2011 Waters Corporation 12

Page 9: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

Ostro Positioning Statement

� Ostro is a sample preparation device for Bioanalytical

scientists who want to remove phospholipids from their

sample. Unlike alternative techniques such as Liquid

Liquid Extraction (LLE) and Protein Precipitation (PPT),

Ostro uses a combination of filtration and sorbent

interaction to give scientists a cleaner sample in less

Evaluation Group; CO R & D ©2011 Waters Corporation 15

interaction to give scientists a cleaner sample in less

time.

Page 10: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

What is Ostro?

� 96-well plate for phospholipid removal

— Plasma, serum samples (samples tested thus far)

o Bioanalysis, Clinical

� Utilizes in-well protein crash

— Sorbent interaction and filtration

— Silica-based sorbent with C18 bonding in proprietary coverage

Evaluation Group; CO R & D ©2011 Waters Corporation 16

— Silica-based sorbent with C18 bonding in proprietary coverage

that retains phospholipids

Page 11: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

� Methodology

How does Ostro Work?

It is possible to work with lower sample volumes (such as

25µL). When doing so you will need a higher

organic solvent to sample ratio, such as

Place Ostro onto collection plate

Pipette 50-200µL of plasma into wells

Forcefully add 1% formic acid in acetonitrile, 3:1

solvent:plasma (methanol not

Evaluation Group; CO R & D ©2011 Waters Corporation 17

sample ratio, such as 10:1 or 20:1.

The well volume is 1.9 mL, however in order to mix by aspiration, the

maximum volume is 1.4 mL. This translates to a maximum sample size

of 350µL.

solvent:plasma (methanol not recommended)

Mix thoroughly by aspirating 3x with pipette

Filter samples using vacuum manifold or positive pressure

manifold

Analyze samples

Page 12: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

Hydrophilicmonomer

Lipophilicmonomer

NO

Hydrophilic-Lipophilic Balanced Copolymer

RP SPE:Oasis HLB Sorbent Chemistry

Evaluation Group; CO R & D ©2011 Waters Corporation 18

monomer monomer

Reversed-phase Retention

• Water wettable• Polar retention• Stable across pH 1-14• No silanol interactions• High recoveries for acids, bases and neutrals

Retention of Polars

Page 13: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

Oasis Solid-Phase Extraction (SPE)

Evaluation Group; CO R & D ©2011 Waters Corporation 19

Page 14: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

Comparison of Formats

Traditional Plate µElution PlateCartridges

Evaluation Group; CO R & D ©2011 Waters Corporation 20

� Typical load: 250-1000

µL of undiluted sample

� Minimum elution volume:

typically 200 µL

� Evaporation and

reconstitution necessary

for concentration

� Typical load: 25-375 µL of

undiluted sample (=50-750 µL

diluted sample)

� Minimum elution volume: 25

µL

� No evaporation and

reconstitution necessary

� Typical load: 0,5-1000 mL

of undiluted sample

� Minimum elution volume:

typically 0,5 mL

� Evaporation and

reconstitution necessary

for concentration

Page 15: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

SPE 96-Well Plate Format:Oasis® µElution Plates

� Novel plate technology enables 25 µL SPE elution — Not possible in disk/membrane products

— Allows loading sample volumes from 25 to a maximum of 375 µL

o 50 to 750 µL 1:1 diluted sample, 750 µL is the well volume

— Elution volume in as little as 25 µL

No evaporation means higher throughput and

Evaluation Group; CO R & D ©2011 Waters Corporation 21

� No evaporation means higher throughput and

sensitivity— Sensitive and selective SPE for bioanalytical

clinical samples

— Increased sensitivity: up to 15x

concentration factor (through format change)

� SPE without an evaporation step

Page 16: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

%

100 411.2 > 191.22.17e6

Why Oasis® µElution Format?Sample Enrichment:Up to a 15X Concentration Factor

0.5 ng/mL risperidone

MCX µElution plate

15X concentration

Evaluation Group; CO R & D ©2011 Waters Corporation 22

Time0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40

%

0

100

0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.400

411.2 > 191.22.17e6MCX 10 mg plate

No concentration

Page 17: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

SPE Methodology Oasis® HLB:

Prepare Sample

Condition/Equilibrate

Protocol

Dilution with H3PO4 (final conc 2 %)

200 ul MetOH/200 ul Water

Evaluation Group; CO R & D ©2011 Waters Corporation 23

Condition/Equilibrate

Load Sample

Wash:5 % MetOH in Water

Elute :ACN:MeOH 60:40

200 ul MetOH/200 ul Water

xx ul sample

200 ul

2 x 25 ul

Page 18: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

Simple Straightforward SPE MethodologyOasis® 2x4 Method:

For Bases:pKa 2-10

Use Oasis® MCX

For Strong AcidspKa <1.0

Use Oasis® WAX

For Strong BasespKa >10

Use Oasis® WCX

For AcidspKa 2-8

Use Oasis® MAX

Prepare Sample

Condition/Equilibrate

Protocol 2Prepare Sample

Condition/Equilibrate

Protocol 1

200 ul MetOH/200 ul Water

Evaluation Group; CO R & D ©2011 Waters Corporation 24

Neutrals

Condition/EquilibrateLoad Sample

Wash:5% NH4OH (C)

Elute 1:100% MeOH

Elute 2:2% HCOOH in 60:40 ACN:MeOH (B)

Condition/EquilibrateLoad Sample

Wash:2% Formic acid (A)

Elute 1:100% MeOH

Elute 2:5% NH4OH in 60:40 ACN:MeOH (D)

BasesStrong Acids

Strong Bases

Acids

xx ul sample( 1:1 diluted with E)

200 ul

25 ul + 25 ul

25 ul + 25 ul

1/1 dilution with water

Page 19: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

Example 1: Amitriptyline and Imipramine

N

NCH3

CH3

ImipramineMW 280.4pKa = 9.5

Assay UseRoutine sample analysis, patient screening

IS: AmitriptylineMW 277.4pKa = 9.4

NCH3

CH3

Evaluation Group; CO R & D ©2011 Waters Corporation 27

Routine sample analysis, patient screening

Assay Requirements

� Very high throughput

� LLOQ 50pg/mL

� Lab is concerned about system robustness and build up of phospholipids on

LC columns and in MS source

� Direct injection to speed up workflow

� Simplest sample prep possible

Page 20: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

Ostro:Results obtained

Compound name: ImipramineCorrelation coefficient: r = 0.999854, r^2 = 0.999707Calibration curve: 0.337053 * x + 0.00290389Response type: Internal Std ( Ref 2 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

Re

spo

nse

10.0

15.0

ng/mL

Re

sid

ua

l

-15.0

-10.0

-5.0

0.0

5.0

%

100

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

0

100

MRM of 5 Channels ES+ 281.3 > 85.99 (Imipramine)

8.40e41.79

0.320.29 0.81 0.932.12

2.14

MRM of 5 Channels ES+ 281.3 > 85.99 (Imipramine)

8.40e4

%

100

%

100

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

0

100

MRM of 5 Channels ES+ 281.3 > 85.99 (Imipramine)

8.40e4

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

0

100

MRM of 5 Channels ES+ 281.3 > 85.99 (Imipramine)

8.40e41.79

0.320.29 0.81 0.932.12

2.14

MRM of 5 Channels ES+ 281.3 > 85.99 (Imipramine)

8.40e4

1.79

0.320.29 0.81 0.932.12

2.14

MRM of 5 Channels ES+ 281.3 > 85.99 (Imipramine)

8.40e4

Blank plasma

50 pg/mL Imipramine

in extracted plasma

Recoveries > 85%, M.E. < 30%

Evaluation Group; CO R & D ©2011 Waters Corporation 28

ng/mL0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0 47.5 50.0

Re

spo

nse

0.0

5.0

Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

0

2.112.090.33

0.281.790.800.38 1.95

2.162.52 2.86

2.57

Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

0 Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

0

2.112.090.33

0.281.790.800.38 1.95

2.162.112.09

0.330.28

1.790.800.38 1.952.16

2.52 2.862.57

Standard Imipramine IS Calc. conc.conc. ng/mL Area Area Response ng/mL %Dev

Standard 0.05 2497.6 129705.5 0.019 0.049 -3Standard 0.1 4962.0 131220.5 0.038 0.104 3.6Standard 0.5 22382.3 127023.4 0.176 0.514 2.8Standard 1 44244.4 123398.3 0.359 1.055 5.5Standard 5 229420.2 128530.1 1.785 5.287 5.7Standard 10 456192.0 135164.7 3.375 10.005 0Standard 50 2283448.0 134905.7 16.926 50.210 0.4

QC 0.15 6288.6 115481.8 0.054 0.153 2QC 1.5 65502.4 123519.1 0.530 1.565 4.3QC 15 427702.9 84416.0 5.067 15.023 0.2

QC 0.15 6320.3 119670.9 0.053 0.148 -1.3QC 1.5 62000.8 117743.2 0.527 1.554 3.6QC 15 612827.1 119457.4 5.130 15.212 1.4

Page 21: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

Choice of Technique: Lipid Levels

LLE w/ 5% NH4OH

LLE%

0

100

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

0

1001.91e8

2.161.92

1.38

1.96 2.27 2.882.782.642.582.43

1.91e82.181.90

2.33 2.822.622.562.45 2.72

MRM 184 -> 184

Evaluation Group; CO R & D ©2011 Waters Corporation 29

PLR plate

RP SPE

PPT

Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

0

100

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

0

100

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

0

100

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.800

1.91e8

1.901.771.96

1.91e8

1.90

1.77

2.191.96

1.91e81.90

1.421.38

1.281.61

2.161.94

2.212.86

Page 22: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

Lipid Level Comparison Between PPT and PL Removal Plate

%

100

0_75_Sirocco_083010_001b_FS 1: MRM of 6 Channels ES+ 184.4 > 184.4 (Lipid 184)

1.34e81.931.841.60

1.53

1.18

1.111.07

0.140.34

1.25

1.69

2.02

PPT

%

100

0_75_Sirocco_083010_001b_FS 1: MRM of 6 Channels ES+ 496.4 > 184.4 (Lipid 496)

5.63e71.69

1.64

PPT

Evaluation Group; CO R & D ©2011 Waters Corporation 30

184 -> 184

Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40

%

0

100

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.400

0.34

0_75_Ostro_083010_001b_FS1 1: MRM of 6 Channels ES+ 184.4 > 184.4 (Lipid 184)

1.34e8

2.02

PL Removal Plate

496 -> 184

Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40

%

0

100

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.400

0_75_Ostro_083010_001b_FS1 1: MRM of 6 Channels ES+ 496.4 > 184.4 (Lipid 496)

5.63e7

PL Removal Plate

Page 23: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

Example 2: Oxycodone and D6 IS in Plasma

OxycodoneMW 315.4pKa = 8.5

Assay Use

O

O

CH3

O

N

CH3

OH

IS:D-6 Oxycodone

MW 321.4pKa = 8.5

Evaluation Group; CO R & D ©2011 Waters Corporation 31

Assay UseRoutine analysis or screening of patient samples, GLP or clinical lab

Assay Requirements

� LLOQ 50 pg/mL

� Simple method

� Method must work for urine too

� Must transfer across lab with varying levels of expertise

� Metabolites and related compounds need to be cleaned up and

quantitated also

Page 24: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

Oasis HLBResults obtained

Compound name: OxycodoneCorrelation coefficient: r = 0.997957, r^2 = 0.995918Calibration curve: 0.126844 * x + 0.00151723Response type: Internal Std ( Ref 2 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: 1/x^2, Axis trans: None

ng/mL

Re

sid

ua

l

-5.0

0.0

5.0

Standard Oxycodone IS Calc. conc.conc. ng/mL Area Area Response ng/mL %Dev

Standard 0.05 154.3 20459.4 0.008 0.047 -5Standard 0.1 271.3 17595.4 0.015 0.110 9.6Standard 0.5 1131.9 17532.5 0.065 0.497 -0.6Standard 1 2440.3 18100.7 0.135 1.051 5.1Standard 5 12431.3 18628.3 0.667 5.249 5

Recoveries > 85% and M.E. < 25% (2 matrices, 2 analytes)

Evaluation Group; CO R & D ©2011 Waters Corporation 32

ng/mL0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500

Re

spo

nse

0.0

20.0

40.0

60.0

All standards and QC samples easily meet regulatory criteria

Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

0

100

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

0

100

MRM of 2 Channels ES+ 316.287 > 241.009 (Oxycodone)

8.93e31.62

1.241.160.75 0.88 1.071.04 1.441.35 1.46

1.652.041.79 2.16

MRM of 2 Channels ES+ 316.287 > 241.009 (Oxycodone)

8.93e3

1.160.740.85

0.92 1.11 1.25 1.34 1.631.541.36 1.831.81 2.00 2.242.10Time

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

0

100

Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

0

100

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

0

100

MRM of 2 Channels ES+ 316.287 > 241.009 (Oxycodone)

8.93e3

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80

%

0

100

MRM of 2 Channels ES+ 316.287 > 241.009 (Oxycodone)

8.93e31.62

1.241.160.75 0.88 1.071.04 1.441.35 1.46

1.652.041.79 2.16

MRM of 2 Channels ES+ 316.287 > 241.009 (Oxycodone)

8.93e3

1.62

1.241.160.75 0.88 1.071.04 1.441.35 1.46

1.652.041.79 2.16

MRM of 2 Channels ES+ 316.287 > 241.009 (Oxycodone)

8.93e3

1.160.740.85

0.92 1.11 1.25 1.34 1.631.541.36 1.831.81 2.00 2.242.10

Blank plasma

50 pg/mL oxycodone

in extracted plasma

Standard 10 24624.5 19441.6 1.267 9.973 -0.3Standard 50 115082.9 17982.5 6.400 50.441 0.9Standard 100 214974.1 18094.1 11.881 93.653 -6.3Standard 500 828731.8 14250.0 58.157 458.478 -8.3

QC 0.25 594.1 18144.6 0.033 0.246 -1.5QC 0.75 1714.8 17840.9 0.096 0.746 -0.6QC 7.5 17403.2 17705.8 0.983 7.737 3.2QC 75 159931.8 16837.3 9.499 74.872 -0.2

QC 0.25 587.429 18774.037 0.031 0.245 -2QC 0.75 1727.213 18001.096 0.096 0.777 3.6QC 7.5 17599.27 18394.02 0.957 7.859 4.8QC 75 159529.25 17495.1 9.119 75 0

QC 0.25 719.038 20666.336 0.035 0.266 6.3QC 0.75 1914.771 19666.842 0.097 0.757 0.9QC 7.5 18367.924 19519.512 0.941 7.373 -1.7QC 75 168356.422 18418.785 9.14 71.683 -4.4

Page 25: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

Example 3: Ropinirole and IS

NH

O

NCH3

CH3 O

N

N

F

CH3

CH3

Ropinirole Citalopram (IS)

Evaluation Group; CO R & D ©2011 Waters Corporation 33

Assay UseRegulated analysis of patient samples

Assay Requirements

� LLOQ 5 pg/mL

� Highest selectivity method possible

� Free from closely related endogenous interferences

� Prefer to concentrate without evaporation

CH3

Page 26: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

Oasis MCXResults obtained

%

MRM of 2 Channels ES+ 261.2 > 113.8

1.29e41.76

0.55

%

MRM of 2 Channels ES+ 261.2 > 113.8

1.29e4

%

MRM of 2 Channels ES+ 261.2 > 113.8

1.29e41.76

0.55

1.76

0.55

5 pg/mL Ropinirole

S/N 125

>10X level in blank

ng/mL

Re

sid

ua

l

-5.0

0.0

Recoveries higher than 90% and M.E. lower than 5%

Evaluation Group; CO R & D ©2011 Waters Corporation 34

Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00

%

0

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.000

0.250.44

0.33 1.360.83 1.120.97 1.27 1.48 1.64 2.231.97 2.12 2.772.41 2.57 2.87

MRM of 2 Channels ES+ 261.2 > 113.8

1.29e4

0.550.25

0.441.78

1.080.84 0.98 1.23 1.541.352.02

1.85 2.15 2.42Time

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00

%

0 Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00

%

0

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.000

0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.000

0.250.44

0.33 1.360.83 1.120.97 1.27 1.48 1.64 2.231.97 2.12 2.772.41 2.57 2.87

MRM of 2 Channels ES+ 261.2 > 113.8

1.29e4

0.250.44

0.33 1.360.83 1.120.97 1.27 1.48 1.64 2.231.97 2.12 2.772.41 2.57 2.87

MRM of 2 Channels ES+ 261.2 > 113.8

1.29e4

0.550.25

0.441.78

1.080.84 0.98 1.23 1.541.352.02

1.85 2.15 2.42

Blank Plasma

Basic Mixed-mode SPE Extraction MethodEasily Meets LLOQ of 5 pg/mL

ng/mL0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0

Re

spo

nse

0.0

10.0

20.0

ng/mL

Correlation Coefficient: r = 0.999054, r2 = 0.998108

Calibration Curve: 2.20969 * x + 0.00746661

Curve Type: Linear, Origin: Exclude, Weighting: 1/x2, Axis trans: None

Page 27: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

Example 4: Peptides in Plasma

DesmopressinMW 1069pI = 8.6

Assay Use

Evaluation Group; CO R & D ©2011 Waters Corporation 35

Assay UseRegulated analysis of patient samples

Assay Requirements

� LLOQ 1-5 pg/mL

� Highest selectivity method possible

� Free from closely related endogenous interferences

� Challenging detection limits

� Need to concentrate without evaporation

Page 28: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

Path to Peptide SPE Screening Protocol

Oasis® WCXµElution

Oasis® MAXµElution

Dilute plasma with 4% H3PO4

Protocol

Oasis® WCXµElution

Oasis® MAXµElution

Dilute plasma with 4% H3PO4

Protocol

Original Protocol Optimized Protocol

Evaluation Group; CO R & D ©2011 Waters Corporation 36

4% H3PO4

Condition MeOH/Equilibrate H2O

Load Diluted Plasma

Wash 1:5% NH4OH

Wash 2:100% MeOH

Elution:2% FA in 60/40 ACN/MeOH

Dilute:0.1 % TFA

4% H3PO4

Condition MeOH/Equilibrate H2O

Load Diluted Plasma

Wash 1:5% NH4OH

Wash 2:20% ACN

Elution:1% TFA in 75/25 ACN/H2O

Dilute:H2O

Page 29: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

Oasis® PST SPE Protocol for Peptides

Oasis® WCXµElution

Oasis® MAXµElution

Dilute plasma with 4% H3PO4

Condition MeOH/Equilibrate H O

Protocol

Evaluation Group; CO R & D ©2011 Waters Corporation 37

Condition MeOH/Equilibrate H2O

Load Diluted Plasma

Wash 1:5% NH4OH

Wash 2:20% ACN

Elution:1% TFA in 75/25 ACN/H2O

Dilute:H2O

Page 30: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

SPE Recoveries Using Peptide Screening Protocol

% S

PE

Reco

very

60

80

100

120

Oasis MAX

Oasis WCX

Evaluation Group; CO R & D ©2011 Waters Corporation 38

Great results for diverse peptides:Screening protocol results in method for 75% of peptides!

% S

PE

Reco

very

0

20

40

Page 31: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

Final SPE Results after BNP, Enfuvirtide and Somatostatin Methods Optimized

% S

PE

Reco

very

40

60

80

100

120

Screening Protocol

Modified Protocol

Evaluation Group; CO R & D ©2011 Waters Corporation 39

% S

PE

Reco

very

Minor, compound specific, modifications for 3 peptidesresult in excellent recovery for all peptides

0

20

Page 32: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

Desmopressin: Oasis WCX

Baseline

Magnification

Factor 25x Compound name: Desmopressin (1)Correlation coefficient: r = 0.999622, r^2 = 0.999244Calibration curve: 1.5469 * x + 0.000270265

Response type: Internal Std ( Ref 2 ), Area * ( IS Conc. / IS Area )Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

Res

pons

e

6.0

8.0

10.0

12.0

14.0

5pg/mL in plasma

1 pg/mL

0.50 1.00 1.50 2.00

%

0

100 x25

1.04

765

Recovery higher than 90% and M.E. lower thant 5%

Evaluation Group; CO R & D ©2011 Waters Corporation 40

Baseline

Magnification

Factor 5x

Sample Name Std. Conc Area IS Area Calc. Conc. %DevBlank human plasma 2.024 20334 0.00030.001 ng/mL 0.001 5.015 17062 0.0010 2.70.002 ng/mL 0.002 9.138 17886 0.0018 -90.005 ng/mL 0.005 22.187 16283 0.0049 -1.40.01 ng/mL 0.01 45.187 17035 0.0096 -3.60.02 ng/mL 0.02 113.447 17912 0.0231 15.40.05 ng/mL 0.05 240.559 18804 0.0467 -6.60.1 ng/mL 0.1 490.062 18654 0.0959 -4.11 ng/mL 1 4365.578 15747 1.0125 1.35 ng/mL 5 30420.492 20869 5.3239 6.510 ng/mL 10 48969.102 17701 10.1042 120 ng/mL 20 104231.141 19458 19.5643 -2.2

DesmopressinDesmopressin 11--10000pg/10000pg/mLmL

Conc0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0

0.0

2.0

4.0

1pg/mL in plasma

Blank

Time0.50 1.00 1.50 2.00

%

0

100

0.50 1.00 1.50 2.00

%

0

100 x5

1.04

138

1.050.83

Page 33: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

Angiotensin I: Oasis MAX Basic Starting LC and SPE Protocols

350 µL human plasma

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

4

MRM of 1 Channel ES+ 433.1 > 513.2 (AngiotensinI)

1.54e3Area

0.8626

MRM of 1 Channel ES+ 433.1 > 513.2 (AngiotensinI)

1.54e30.8519

10 pg/mL

5 pg/mL

Evaluation Group; CO R & D ©2011 Waters Corporation 41

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

%

4

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

4

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

4

Area19

MRM of 1 Channel ES+ 433.1 > 513.2 (AngiotensinI)

1.54e3Area0.87

12

MRM of 1 Channel ES+ 433.1 > 513.2 (AngiotensinI)

1.54e3Area0.86

3

1 pg/mL

5 pg/mL

Blank plasma

Page 34: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

Angiotensin II: Oasis WCX Basic Starting LC and SPE Protocols

350 µL human plasma

5 pg/mL

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

-2

98

MRM of 2 Channels ES+ 349.8 > 263 (AngiotensinII)

3.64e3Area

0.7980

Evaluation Group; CO R & D ©2011 Waters Corporation 42

1 pg/mL

Blank plasma

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

%

-2

98

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

-2

98

MRM of 2 Channels ES+ 349.8 > 263 (AngiotensinII)

3.64e3Area

0.7810

MRM of 2 Channels ES+ 349.8 > 263 (AngiotensinII)

3.64e3Area

0.799

Page 35: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

Bioanalysis of peptides

� In-house seminars

� Peptides’ video:

www.waters.com/pepDVD

Evaluation Group; CO R & D ©2011 Waters Corporation 43

www.waters.com/pepDVD

� Peptides’ day

� … and more

Page 36: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

Conclusions

� Methods are not one-size fits all

� Depending on the complexity of the particular assay

Waters offers different solutions: PPT, PLR and SPE

Evaluation Group; CO R & D ©2011 Waters Corporation 44

� Mixed-mode SPE facilitates routine achievement of low

pg/mL LLOQ’s for both large and small molecules

Page 37: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

Questions?

Evaluation Group; CO R & D ©2011 Waters Corporation 45

Page 38: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

APPENDIX

Evaluation Group; CO R & D ©2011 Waters Corporation 46

Page 39: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

Recent Regulatory Discussions:Incurred Sample Reanalysis (ISR)

� Incurred Sample Reanalysis (ISR) discussions at recent Crystal City

Meeting, February, 2008; followed by CVG (Canada), EBF (Europe)

— ISR will be required

— Must have SOP for ISR in place

— A % of study samples will need to be reanalyzed

Evaluation Group; CO R & D ©2011 Waters Corporation 47

— Various acceptance criteria were discussed and a consensus proposed

— AAPS Journal paper published describing one such approach to ISR and

determination of criteria3

o One particular proposal for acceptance criteria that was suggested by

various AAPS representatives is the use of the 4/6/20 rule4

o Two thirds of repeats must be within 20% of the original value

1AAPS Journal 2007: 9 (1) Article 42AAPS Journal 2007: 9 (1) Article 113AAPS Journal 2007: 9 (3) Article 404CVG White Paper from 2cnd Workshop on Recent Issues in GLP Bioanalysis, April 2008

Page 40: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

SPE Extraction Procedure: Calculating Recovery

Sample Matrix

RESPONSE

Spike Standards into Blank Matrix

Blank Sample Matrix(No analyte(s))

Recovery of the Extraction Procedure (RE)*(or, SPE Recovery)

Evaluation Group; CO R & D ©2011 Waters Corporation 48

Extracted Sample(with analyte(s))

(with analyte(s))

RESPONSEExtracted Sample(with analyte(s))

RESPONSEPost-Extracted SPIKED Sample

% RE = 100 x

*Matuszewski, B.K., Constanzer, M.L., Chavez-Eng, C.M. Anal. Chem. 2003, 75,

3019-3030.

Post-Extracted SPIKED Sample

Spike Standards into Extracted Matrix

Both extracted samples should be in the same solution

Page 41: Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation

Quantitative Assessment of Matrix Effects:Post-Extracted Spiked Sample

Blank Sample Matrix(No analyte(s))

)Response

Response((MF)Factor Matrix Matrix of Presence=

Standard Solution(Analyte(s))

Evaluation Group; CO R & D ©2011 Waters Corporation 49

� Both samples should be in the same

composition solution

� MF Value <1, negative % ME = suppression

� MF Value >1, positive % ME = enhancementPost-Extracted SPIKED Sample

Spike Standards into Extracted Matrix

Response Matrix of Absence

100*1)-)Response

Response(((ME) EffectsMatrix%

StandardSolvent

Sample Spiked Extracted-Post=