meeting the challenges of bioanalysis...key challenges in routine bioanalysis operation sensitivity...
Post on 20-Feb-2021
2 Views
Preview:
TRANSCRIPT
-
©2009 Waters Corporation 1
Meeting the Challenges of Bioanalysis
Impact of Recent Advances in UPLC and Mass Spectrometry
Dr. Jing Lin
-
©2009 Waters Corporation 2
Outline
Introduction
Meeting the key challenges in bioanalysis
—Increasing sensitivity
—Enhancing specificity
—Improving productivity
Summary
-
©2009 Waters Corporation 3
Introduction
Bioanalysis plays an important role in drug development
Quantitative analysis of compounds and metabolites in
biological matrices is normally achieved by LC/MS/MS or
GC/MS(MS)
Bioanalysis forms part of the regulatory submissions for new
drug application, IND etc. in GLP studies
Bioanalytical laboratories are facing many common challenges
— Increasingly stringent regulations
— Higher business pressure and tougher competition
— New and evolving technical/scientific challenges
-
©2009 Waters Corporation Company Confidential 4
Matrix Effects
New guidance on the determination and acceptability of matrix
effects as a result of sample preparation
Should be assessed by comparison in six different sources of
matrix if using analog internal standard (IS)
— Variability (%CV) across 6 lots
-
©2009 Waters Corporation Company Confidential 5
Incurred Sample Reanalysis (ISR)
Require a % of patient samples be re-analyzed and compared to
original
— 5% to 10% of the study samples
Various acceptance criteria were discussed and a consensus
proposed
— 4/6/20 rule‗: 2/3 of the reanalysed samples have to show
< 20% variation of the original (?) value
Must have SOP for ISR in place
Direct impact on productivity and cost of analysis
Regulatory Changes in Bioanalysis (2)
-
©2009 Waters Corporation | COMPANY CONFIDENTIAL 6
Regulatory Changes in Bioanalysis (3)
Metabolite In Safety Testing (MIST)
Newest guideline published in June 2009
— ICH Topic M 3 (R2)
— http://www.emea.europa.eu/pdfs/human/ich/028695en.pdf
Simplified and less stringent guideline than initially discussed
Nonclinical characterization of a human metabolite(s) is only
warranted when that metabolite(s) is observed at exposures
greater than 10% of total drug-related exposure and at
significantly greater levels in humans than the maximum
exposure seen in the toxicity studies.
Such studies should be conducted to support Phase III clinical
trials.
http://www.emea.europa.eu/pdfs/human/ich/028695en.pdf
-
©2009 Waters Corporation | COMPANY CONFIDENTIAL 7
Key Challenges in Routine Bioanalysis Operation
Sensitivity
— Ever increasing demand
— New highly potent drugs, low level of metabolites
— Innovative formulation: controlled-release, inhalation…
— Specific population studies: pediatric …
— Implementation of Dried Blood Spot
Specificity
— Monitoring & Minimizing matrix interference: specific FDA guideline
— Detecting and quantifying metabolites: MIST guideline
Productivity
— Analyze more samples
— Maximizing system up-time
— Easy access to non specialist users
— Efficient workflow
-
©2009 Waters Corporation 8
Xevo QTof
-
©2009 Waters Corporation 9
Increasing The Sensitivity
-
©2009 Waters Corporation 10
Optimizing ESI Desolvation
High temperature heater
— High temperature alloy
element
— 650oC required for optimal ESI
operation
Optimal zone of desolvation
— Balanced gas distribution
— Vertical nebulizer positioning
-
©2009 Waters Corporation 11
Optimizing Gas Flow Dynamics
Shape of chamber
— Smooth, curved surface
— Reduces turbulence
— Excellent beam stability
Size of chamber
— Efficient desolvation
— Excellent linear response
Temperature of chamber
— Thermally regulated
— Prevents solvent deposition
Tangential exhaust
— Gas flows modeled to efficiently
remove non-ionized materials from
source chamber
-
©2009 Waters Corporation | COMPANY CONFIDENTIAL 12
Meeting the Sensitivity Challenge with Xevo™ TQ MS
Fluticasone Dipropionate is a
corticosteroid used in the
treatment of asthma
Inhaled delivery results in low
circulating plasma levels
Assay requirements are for
low pg/mL sensitivity
Xevo TQ MS delivers pg/mL
sensitivity
Fluticasone Propionate 3 pg/mL in Plasma
O
OH
COSCHF
OCOCH
Time1.10 1.20 1.30 1.40 1.50 1.60
%
0
100
090309_HLB_QUAN_029 Sm (Mn, 2x1) MRM of 3 Channels ES+ 501.3 > 293.2 (Fluticasone)
3.76e3
1.19
1.15
1.25
1.23
1.561.33
-
©2009 Waters Corporation 14
Improving Assay Sensitivity with UPLC®
Assay sensitivity is dependent upon peak intensity
Extra efficiency of UPLC® produces sharper,
narrower peaks
Higher linear velocities of UPLC and shorter
columns also give sharper peaks
UPLC = Faster, sharper, more intense peaks
-
©2009 Waters Corporation 15
5-fold increase in sensitivity as
a result of UPLC
UPLC analysis allowed the
quantification of the 24 hr
samples
New data indicated a different
elimination phase of the
pharmacokinetics significantly
affecting the oral bioavailability
Samuele Pedraglio ∗, Marco Giulio Rozio, Paola Misiano,Veronica Reali, Giulio Dondio, Chiara Bigogno; J Pharm. Biomed Anal 44 (2007) 665–673
The impact of UPLC and raised sensitivity on a PK profile
Solving Bioanalytical Challenges with
UPLC® Technology: Sensitivity
-
©2009 Waters Corporation 16
Enhancing The Specificity
- Minimizing matrix effect
- Confirming peak identity
- Detecting & Quantifying metabolites
-
©2009 Waters Corporation 17
Separation of
metabolite
critical pairs is
essential for accurate
quantitation and is
becoming more
important in order to
satisfy increasing
Regulatory
expectations
(MIST)
Ref: Donegan M,BMS, www.cosmoscience.org/archive_2008.htm
Impact of UPLC® on Resolution
-
©2007 Waters Corporation 18
Simple (but dirty) sample preparation by protein
precipitation (PPT)
Phospholipids are the main remaining matrices
after sample clean-up
Evaluate impact of UPLC on matrix effect vs
HPLC
Quantification Of TerfenadineIn Human Plasma
-
©2007 Waters Corporation 19
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00
%
0
MRM of 2 Channels ES+
2.17e4
Terfenadine
0.50 1.00 1.500
472.1 > 436.1
1.37e4
1.09S/N = 81PhospholipidsTerfenadine
0.1 ng/mL Terfenadine
PPT
High pH
S/N 81
>30% Suppression
Resolution and Sensitivity:HPLC, High pH
-
©2007 Waters Corporation 20
0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00
%
0
MRM of 2 Channels ES+
2.17e4
Terfenadine
0.50 1.00 1.500
472.1 > 436.1
1.37e4
1.09
S/N= 482
Minutes
PhospholipidsTerfenadine
0.1 ng/mL Terfenadine
PPT
High pH
S/N 482 (6X HPLC)
-
©2007 Waters Corporation 21
Are matrix effects measured in HPLC and UPLC the same or
statistically different?
10 analytes run on both HPLC and UPLC
— With different pHs, gradients and sample concentrations
Data were paired according to identical conditions
—An HPLC data point with a UPLC data point under the same
conditions
43 sets of paired data subjected to paired t-test
A p value less than 0.05 indicates 2 populations are different
Paired t-test returned a ―p‖ value of 0.0006
—There is a difference between matrix effects measured in HPLC
versus UPLC™
Therefore, the reduction in matrix effects with UPLC™ is
statistically significant
Statistical Comparison of Matrix Effects
-
©2007 Waters Corporation 22
Assessment of Matrix Effects and Metabolites with MS
Require acquisition of qualitative (spectral) data
Targeted and untargeted approaches can be used
Parent ion scan for detection of targeted/expected
matrix/metabolites
— Detection of lipid interference or metabolites with common
fragment ions
Full-scan MS for detection of untargeted /unexpected
matrix/metabolites
MS/MS spectra for confirmation of peak identity
-
©2007 Waters Corporation 23
Conventional Tandem Quadrupole vsXevo TQ MS
Standard tandem quadrupole MS
— Very sensitive in MRM mode
— Significantly less sensitive when acquiring in spectral mode
o Full scan MS, MS/MS or parent ion scan
— Switching between MS and MS/MS is nearly impossible
Xevo TQ MS
— Provides outstanding MRM sensitivity
— Incorporates ScanWave collision cell technology
o Innovative way of using existing travelling wave technology
— Significantly enhances sensitivity in spectral mode
— Designed for rapid MS to MS/MS switching
— Collects more useful information from a single experiment
o High quality Quantitative & Qualitative data simultaneously
-
©2009 Waters Corporation 24
Standard Product Ion Scanning
Continuous output of ions by cell
Most ions filtered out by MS2
Very inefficient
-
©2009 Waters Corporation 25
ScanWave EnhancedProduct Ion Scanning
Ion accumulation
Mass selective ion ejection
Synchronised with scanning of MS2
-
©2009 Waters Corporation 26
ScanWave Enhanced sensitivity full scan MS/MS
Travelling Wave
DC Barrier RF BarrierPotential Energy
Low m/z Ion
High m/z Ion
Intermediate m/z Ion
Storage Region ScanWave Region
Travelling Wave
Travelling Wave
Accumulation
Transfer
High m/z Ejection
Medium m/z Ejection
-
©2009 Waters Corporation 27
Standard
Data
ScanWave
Enhanced
Data
ScanWave …enhanced sensitivity full scan MS/MS
Scan speed = 5,000 amu/second
Conventional
product ion spectrum
ScanWave enhanced
product ion spectrum
-
©2009 Waters Corporation 29
PIC Scan
Acquisition
MRM
Acquisition
PIC SCAN:
MRM data is used as a specific
trigger for the acquisition of a
ScanWave enhanced product
ion spectrum.
Rapid Mode Switching Product Ion Confirmation Scan
-
©2009 Waters Corporation 30
UPLC/MS/MS Analysis of Fluticasone Propionate in Plasma
MRM data
Which peak is the
Fluticasone?
-
©2009 Waters Corporation 31
PICSComparison in TargetLynx
Reference Spectrum from Quanpedia
-
©2009 Waters Corporation 32
Dual Scan-MRM
How does it work?
Rapidly alternates between MRM and
MS full scan acquisition modes.
5ms switch time
No compromise to MRM or MS full scan
How is MS data acquired on Xevo TQ?
Collision cell remains pressurised in both modes
MS2 scans to produce fast MS spectra
T-wave ensures MS ions transit efficiently.
-
©2009 Waters Corporation 33
Dual Scan-MRM
15 points Across1.5 Sec Peak
Full Scan MS
(Matrix)
MRM
(Analyte)
Excellent MRM peak definition
+
Full Scan data acquisition
Faster LC/MS/MS method
development and matrix
monitoring
Simpler detection and
confirmation of active
compounds and associated
metabolites.
-
©2009 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 3.20 3.40 3.60 3.80
%
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 3.00 3.20 3.40 3.60 3.80
%
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 3.00 3.20 3.40 3.60 3.80
%
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 3.00 3.20 3.40 3.60 3.80
%
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 3.00 3.20 3.40 3.60 3.80
%
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 3.00 3.20 3.40 3.60 3.80
%
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 3.00 3.20 3.40 3.60 3.80
%
3
220109_SW_DS_133 5: MS2 ES- TIC
1.72e9
2.31;282.81.77;278.90.33;400.7 1.48
168.81.42;488.7
1.21;523.40.96;748.6
2.17;885.4 2.46;851.3
2.68;792.0 3.03791.9
3.19833.3
220109_SW_DS_133 4: Parents of 184ES+ TIC
7.54e9
2.54;757.52.15519.5
1.98541.4
0.13757.8
2.26;523.2
220109_SW_DS_133 3: Parents of 227ES+ TIC
1.26e7
1.55;324.3
0.17348.6
0.67270.80.26;270.3
1.44294.1
1.19320.3
0.98294.4
0.79306.0
1.69;326.0 2.24242.0
1.97348.0
2.12226.2
2.36241.1
2.52269.0
3.13296.0
2.70225.1
3.07;286.6 3.19;331.8 3.71293.9
3.62;225.3
220109_SW_DS_133 2: MS2 ES+ TIC
1.15e10
2.16;183.61.41;125.50.26119.7
1.98181.0
1.61313.7
2.39;148.5 2.54;758.4
3.03369.0
3.32180.6
220109_SW_DS_133 1: MRM of 3 Channels ES+ 325 > 297 (Alprazolam Metabolite)
7.86e6
1.56;325.0
220109_SW_DS_133 1: MRM of 3 Channels ES+ 314.2 > 286.1 (AlprazolamD5)
5.18e6
1.60;309.2
220109_SW_DS_133 1: MRM of 3 Channels ES+ 309.2 > 281 (Alprazolam)
8.32e6
1.60;309.2
Quantify MRM
Quantify MRM of Internal Std
Quantify MRM of Metabolite
Matrix Full Scan +ve
Monitor Analyte Common Fragment
Monitor Lipid Common Fragment
PIC Spectrum
Matrix Full Scan -ve
Detect, Quantify, Confirm All in One Run
-
©2009 Waters Corporation 35
Improving The Productivity
- Reducing analysis time
- Maximizing system up-time
- Easy access
- Efficient workflow
-
©2009 Waters Corporation 36
Reducing Analysis Time with ACQUITY UPLC
HPLC-MS/MS
%
Time2.0 2.5 3.01.50.5 1.00
UPLC-MS/MS
Reduced run timeMore sensitive Better resolution
To benefit from the advantages of UPLC the MS must be designed to be fully compatible
-
©2009 Waters Corporation 37
MS Acquisition Speed
The MS must be capable of acquiring at data rates
compatible with narrow UPLC peaks without loss in
performance
Good quality quantification requires:
— 10-15 MRM data points per chromatographic peak
— No loss in signal
— No increase in cross talk
Latest MS design provides
— 6ms per MRM data point (167 data points per second)
— 20 ms to switch between + and – modes
— 20 ms to switch between ESI and APCI (ESCi)
-
©2009 Waters Corporation 38
T-Wave For Speed
Traditional hexapolereplaced with
Stacked ring electrodes
T-Wave used to control the transit time of ionsRapidly clearing between channels
•(eliminates cross talk)Rapidly refilling
•(no signal loss)
-
©2009 Waters Corporation 39
Engineered Simplicity
Best performance can only be
routinely achieved if the
instrument is designed to be
simple to operate and service
Innovative ion source
IntelliStart (ready to run)
technology
-
©2009 Waters Corporation 40
New API Source DesignPerformance, Usability & Serviceability
ZSpray™
— A time proven geometry
(many years of experience
& 1000s in use daily)
— Robust performance in the face of
complex biological samples
Designed to be compatible with
high flow HPLC and UPLC®
applications
Innovative, Ergonomic Engineering
— Tool free maintenance
— Novel tool free source exchange
— Integrated gases
— Plug and play probes
-
©2009 Waters Corporation 41
Integrated UPLC/MS by Design
Column Optimally
positioned to eliminate
band broadening
Built-in Fluidics
Automated delivery of
reference solutions
Automated flow
switching
-
©2009 Waters Corporation | COMPANY CONFIDENTIAL 42
IntelliStart
Automated system
calibration
Automated sample
tuning and method
development
Automated, pre-
analysis system
performance check
-
©2009 Waters Corporation | COMPANY CONFIDENTIAL 43
XXSystem Suitability Check
LC/MS System check: Run 1
LC/MS System check: Run 6
%
0
100
Time
%
0
100
Time
Peak area = 4351.6
Peak area = 4435.2
LC/MS System Check
• System suitability check automatically queued
• Instrument Pass/Fail calculated based on user-defined parameters
• Results stored to ELN database, or email to supervisor
• Instrument status displayed on instrument interface
-
©2009 Waters Corporation Company Confidential 44
Optimization of MS Conditions
Line from infusion
pump, contains
analyte
Line from LC pump,
contains mobile phase
Probe may need to
be changed
Infusion Setup Xevo Setup
-
©2009 Waters Corporation Company Confidential 45
Developing an MRM Method:Automated Parent & Fragment Optimization
Step 1
IntelliStart™
— Input the compound
name
— Input the parent
mass or formula
— Select the adduct
— Click Start and get a
coffee
-
©2009 Waters Corporation Company Confidential 46
Developing an MRM Method:Reporting
IntelliStart™ Results Reporting:
Ionization mode
Best MRM transition(s)
Cone voltages and Collision energies
Generation of a report showing how parameters were selected
All parameters automatically populated into the method set
-
©2009 Waters Corporation Company Confidential 47
Comparison of Manual vs. IntelliStart MRM Method Development: 5 Analytes
Manual Development Time IntelliStart™ and ESCi®
Plumb for infusion(time taken to find all the bits needed….)
2-5 min Enter file name, mass or formula and adduct into IntelliStartPress StartCompound is automatically infused
Change probes for different ionization
2 min ESCi does this automatically
Check cone voltages 2 min IntelliStart does this automatically
Check collision energies
2 min IntelliStart does this automatically
Optimize gas flow 2 min IntelliStart does this automatically
Optimize temperature 2 min IntelliStart does this automatically
Adjust parameters for optimum conditions
2 min IntelliStart does this automatically
Total time:15 min X 5 = at least 75 mins
Total time (while you were away…..)4 min X 5 = 20 min, AUTOMATED
-
©2009 Waters Corporation | COMPANY CONFIDENTIAL 48
Select compounds
from database
Ready to
Acquire &
Process
Create
LC/MS
methods
Use Predefined Analysis
Use Predefined Compounds
QuanPedia™
A central LC/MS/MS data base based
on compound name
Allowing methods to be stored,
controlled, shared, and downloaded
across multiple instruments and sites
-
©2009 Waters Corporation | COMPANY CONFIDENTIAL 49
QCMonitor: Automated Monitoring of Assay Performance
Real-time, proactive monitoring of:
— Blanks
— Standards
— Quality control
Action on failure:
— Flag error
— Terminate sample run
Prevent repeated analysis
Prevents loss of precious samples
— No injection if the LC/MS system is not performing appropriately.
-
©2009 Waters Corporation | COMPANY CONFIDENTIAL 50
Data Processing with TargetLynx™
Advanced software package for quantitative analysis
— ApexTrack™ enabling accurate and consistent integration
— A full range of automatic quality control checks
— Simple rapid data review with intuitive browser
— Data re-processing directly from the browser
Fully compliant data processing
— Full audit trail recorded for data processing activities
— Electronic signature and secure data management tools
Data export function
— Allows for generation of partial or full sets of digital information, in
ASCII or XML, for import into other systems including statistical
analysis packages and LIMS software
http://www.waters.com/watersdivision/images/products/3quan.htm
-
©2009 Waters Corporation | COMPANY CONFIDENTIAL 52
TrendPlot
A tool to monitor long-term data
trends/system performance
Monitor LOD/LOQ performance
Track internal standard response
Track QC response
Plot intra- and inter-batch data
from TargetLynx
-
©2009 Waters Corporation | COMPANY CONFIDENTIAL 53
Setup instrumentation
Create LC/MS/MS methods
Monitor data proactively
Process, qualify & report
Plot trends in results
Efficient Bioanalysis Workflow
IntelliStart™
QuanPedia™
QCMonitor™
TargetLynx™
TrendPlot
-
©2009 Waters Corporation 54
Summary
-
©2009 Waters Corporation 55
Impact of Recent Advances in UPLC & MS Technology
Increasing Sensitivity
— Contribution of UPLC is real and proven
— New source design allowing more efficient ionization
Enhancing Specificity
— UPLC providing better resolution and minimizing co-elutions
— ScanWave increasing available acquisition options, allowing
quantification, confirmation and detection of drug and
metabolites simultaneously
Improving Productivity
— UPLC reducing analysis time
— Fully integrated UPLC MS system designs
— Engineered simplicity
— Efficient workflow : from method development to data processing
-
©2009 Waters Corporation 56
Acknowledgements
Erin Chambers
Diane Diehl
Rob Plumb
Joanne Mather
Paul Rainville
Geneen Baynham
Jessalynn Wheaton
David Little
Gary Harland
top related