Sample Preparation for Sample Preparation for Bioanalysis ... · Sample Preparation for Sample Preparation for Bioanalysis Bioanalysis Evaluation Group; CO R & D ©2011 Waters Corporation
Post on 20-Jun-2020
19 Views
Preview:
Transcript
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
Outline
� Introduction
—Challenges in Bioanalytical Method Development
� Sample preparation techniques
—Waters’ solutions
Evaluation Group; CO R & D ©2011 Waters Corporation 2
� Application examples
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
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
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
Different Methods for Different Purposes: Decision Making Process
Evaluation Group; CO R & D ©2011 Waters Corporation 10
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
Sample Prep products for bioanalysis
Evaluation Group; CO R & D ©2011 Waters Corporation 12
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.
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
� 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
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
Oasis Solid-Phase Extraction (SPE)
Evaluation Group; CO R & D ©2011 Waters Corporation 19
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
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
%
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
Questions?
Evaluation Group; CO R & D ©2011 Waters Corporation 45
APPENDIX
Evaluation Group; CO R & D ©2011 Waters Corporation 46
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
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
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=
top related