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Advanced Detectors For EmpowerAdvanced Detectors For Empower
ELSD, PDA, SQD and TQDELSD, PDA, SQD and TQD
©2010 Waters Corporation | COMPANY CONFIDENTIAL
John Van AntwerpWaters Corporation
Overview of Role For Detector in an Overview of Role For Detector in an UPLC/HPLC system UPLC/HPLC system UPLC/HPLC system UPLC/HPLC system
©2010 Waters Corporation | COMPANY CONFIDENTIAL
Commonly Desired Commonly Desired Characteristics of a HPLC DetectorCharacteristics of a HPLC Detector
High sensitivity
Negligible baseline noise
Wide linear dynamic range
R i d d t f i ti i ti Response independent of variations in operating parameters (pressures, temperature, flow-rate, etc.)
Response independent of mobile phase
Low dead volume
Selective and universal
©2010 Waters Corporation | COMPANY CONFIDENTIAL 3
Common Realities of a HPLC Common Realities of a HPLC DetectorDetector
Registers an output in response to “sample detection”— and other components in mixture
Provides a relationship between response of the detector and concentration of the sample and concentration of the sample — but is not always linear so calibration techniques are designed
to promote this relationship
Stable over long periods of operations Stable over long periods of operations
Can control the detector through software and get a desired separation — however other components of the HPLC system may cause the
detector to perform at lower than optimal levels
©2010 Waters Corporation | COMPANY CONFIDENTIAL 4
Key To All Method DevelopmentKey To All Method Developmenty py p
There is no single detector that can be employed for all HPLC g p yseparations. There is no “magic black box” !
©2010 Waters Corporation | COMPANY CONFIDENTIAL 5
Sensitivity Sensitivity -- DefinitionDefinitionyy
Ratio of Signal-to-Noise (S/N or S:N)
Two Typical Concerns:
Li it f d t ti (LOD) S/N 3 Limit of detection (LOD): S/N = 3
Limit of quantitation (LOQ): S/N =10S
N
©2010 Waters Corporation | COMPANY CONFIDENTIAL 6
In this case if N=1 and S=6, then the “Sensitivity Ratio” would be expressed as: “6/1” or “6” or “6:1”
How to Increase Signal to Noise How to Increase Signal to Noise RatioRatio
If start with Signal-to-noise (S/N) of 3:1
Can increase S/N by increasing peak height (6:1)
6:1Can increase S/N by decreasing noise (8:1)
3:13:1 8:1
©2010 Waters Corporation | COMPANY CONFIDENTIAL 7
SelectivitySelectivityyy
Visibility can be dependent upon your sensory device
Invisibility can sometimes be a great benefit
©2010 Waters Corporation | COMPANY CONFIDENTIAL 8
ELSD utility and advantagesELSD utility and advantagesy gy g
Used for detection of compounds less volatile than mobile Used for detection of compounds less volatile than mobile phase— Low-temperature ELSD extends use to semi-volatile analytes in
aqueous mobile phases making this technique suitable for aqueous mobile phases, making this technique suitable for analysis of small molecules such as pharmaceuticals
Often referred to as ‘universal’ detectorUse for compounds without UV chromaphore: — Use for compounds without UV chromaphore:
Transparent to changes in mobile phase composition
Same chromatographic requirements as LC/MS— Volatile modifiers
©2010 Waters Corporation | COMPANY CONFIDENTIAL 9
Now You See Them and Now You Now You See Them and Now You Don’t?Don’t?
PDA to monitor UV/Vis friendly compounds
Diode Array TICDiode array TIC friendly compounds
Mass Spec to verify that
TIC
Mass Spec to verify that compound has been
synthesized
ES+TIC
ELSD to monitor all compounds and determine
purity levelsELSD
©2010 Waters Corporation | COMPANY CONFIDENTIAL 10
p y
Min
24.5 7
PDA/ELSD/SQDPDA/ELSD/SQD/ / Q/ / Q
PDA
ELSD
SQD
©2010 Waters Corporation | COMPANY CONFIDENTIAL 11
High confident data in one injection !
PDA/ELSD/SQDPDA/ELSD/SQDComplete Information Complete Information fromfrom One InjectionOne Injectionpp jj
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Understanding PDA Understanding PDA Peak PurityPeak Purity
©2010 Waters Corporation | COMPANY CONFIDENTIAL
Spectral ContrastSpectral Contrastpp
The Spectral Contrast measures the shape difference between two spectrabetween two spectra.
Spectra are baseline corrected by subtracting interpolated baseline spectra between peak baseline liftoff and baseline touchdown.
Spectra are converted into a vector in n dimensional space.p p
Vector lengths (concentration) are minimized using least-squares solution.
The vectors are moved into a two dimensional plane and the angle between them is measured.
An angle of 0 degrees means the spectral shape is identical and an angle of 90 degrees indicates no spectral overlap.
©2010 Waters Corporation | COMPANY CONFIDENTIAL 14
Spectral Contrast Spectral Contrast
Spectrum ASpectrum A Spectrum A
Spectrum BSpectrum A
Abso
240
nm
Spectrum B
rbance AU
at 2
nm200.00 240.00 280.00 320.00 AU at 280 nm
The shapes of Spectrum A and Spectrum B are represented by vectors
is the Spectral Contrast Angle which is the difference between spectral shapes
©2010 Waters Corporation | COMPANY CONFIDENTIAL 15
Spectral ContrastSpectral Contrastpp
Spectral Contrast 53 Degrees53 Degrees
EthylparabenEthylPaba
sorb
ance
Abs
©2010 Waters Corporation | COMPANY CONFIDENTIAL 16
200.00 240.00 280.00 320.00nm
Spectral ContrastSpectral Contrastpp
Spectral Contrast10 Degrees
Similar spectra for structurally
TheophyllineDyphylline
related compounds
orba
nce
Abso
©2010 Waters Corporation | COMPANY CONFIDENTIAL 17
230.00 250.00 270.00 290.00 310.00nm
Spectral ContrastSpectral Contrastpp
Spectral Contrast 0.5 Degrees
MethylparabenEthylparaben
e
Very similar spectra CH2
bsor
banc
e spectra, CH2
difference
Spectral C t tAb Contrast can differentiate these spectra.
©2010 Waters Corporation | COMPANY CONFIDENTIAL 18
200.00 240.00 280.00 320.00nm
Threshold CalculationsThreshold Calculations
The Threshold Angle is comprised of two parts: First The The Threshold Angle is comprised of two parts: First, The Detector Noise Angle is calculated from the chromatographic baseline and is inversely proportional to the peak height.
Spectrum A The Noise Region in gray forms
Noise
banc
e
a constant cylinder of uncertainty around the vector.
A vector drawn from the origin to the edge of the cylinder creates
Spectrum BA
bsor
b the edge of the cylinder creates the noise angle.
The shorter the vector (lower concentration) the larger the
i l
©2010 Waters Corporation | COMPANY CONFIDENTIAL 19
noise angle.
Threshold CalculationsThreshold Calculations
Second, The Solvent Effect corresponds to the constant portion of the Threshold Angle it accounts for solvent portion of the Threshold Angle, it accounts for solvent effects and photometric errors.
The solvent effect can be accurately measured from a chemically pure standard running six replicates and taking chemically pure standard, running six replicates and taking the highest obtained purity angle.
Auto threshold will use a look-up table based on peak height for the solvent effect part of the threshold calculation.
©2010 Waters Corporation | COMPANY CONFIDENTIAL 20
Spectral ResolutionSpectral Resolutionpp
Spectral Resolution or the ability to differentiate one UV spectrum from anotherfrom another.
The Waters PDA runs with a fixed 50 micron slit, producing an optical resolution of 1.2nm.
l l For 1.2nm optical resolution a 200nm to 400nm range n = 166. i.e. Spectral Contrast uses 166 dimensions to describe the curve shape.
For 4.0nm optical resolution a 200nm to 400nm range n = 50. i.e. Spectral Contrast uses 50 dimensions to describe the curve shape.p
©2010 Waters Corporation | COMPANY CONFIDENTIAL 21
Spectral ResolutionSpectral Resolutionpp
Benzene
230.00 250.00 270.00nmnm
Less resolution at 3.6 nm vs 1.2 nm
UV maxima shifted
©2010 Waters Corporation | COMPANY CONFIDENTIAL 22
Peak Impurity and Peak Spectral HomogeneityPeak Impurity and Peak Spectral Homogeneity
The Peak Purity Algorithm uses Spectral Contrast to compare all spectra within a peak to the Apex spectrum compare all spectra within a peak to the Apex spectrum. The resulting Purity Angle is a weighted average of all of the calculated angles.
If the Purity Angle is less than the calculated Threshold If the Purity Angle is less than the calculated Threshold Angle, within the noise of the system the peak is spectrally homogeneous.
If the Purity Angle is greater than the calculated Threshold Angle, there is something within the peak that can not be explained by noise. The peak is impure.
©2010 Waters Corporation | COMPANY CONFIDENTIAL 23
UV and Chromatographic UV and Chromatographic LimitationsLimitations
The UV spectrum of different compounds can be identical.
The concentration of the impurity may be too low to detect The concentration of the impurity may be too low to detect.
Each of these three limitations become a trade off to the other two.
Ref: Detecting Coeluted Impurities by Spectral Comparison, Marc V.Gorenstein et al LC-GC Volume 12 Number 10 Marc V.Gorenstein et al LC GC Volume 12 Number 10 October 1994 pages 768-772
©2010 Waters Corporation | COMPANY CONFIDENTIAL 24
Multiple Pass Peak PurityMultiple Pass Peak Purityp yp y
Peak Purity
©2010 Waters Corporation | COMPANY CONFIDENTIAL 25
Multiple Pass Peak PurityMultiple Pass Peak Purityp yp y
Second Pass Peak Purity
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Apparent Pair Of Compounds:Apparent Pair Of Compounds:UV Spectra Across First PeakUV Spectra Across First Peak
100 100 100
pp
210 350nm
%
210 350nm
%
210 350nm
%
100
%
©2010 Waters Corporation | COMPANY CONFIDENTIAL 27
10 15Time
Apparent Pair Of Compounds:Apparent Pair Of Compounds:Mass Spectra Across First PeakMass Spectra Across First Peakpp
100 100 309.1 100
% 309.1
311.1
% 311.1287.1 % 309.1287.1
100
200 300 400m/z
200 300 400m/z
200 300 400m/z
%%
©2010 Waters Corporation | COMPANY CONFIDENTIAL 2810 15Time
Single Mass Chromatograms:Single Mass Chromatograms:Extracted From MS SpectraExtracted From MS Spectrapp
Scan ES+ TIC
Scan ES+ 309.1
Scan ES+ 287 1
10 15Ti
287.1
©2010 Waters Corporation | COMPANY CONFIDENTIAL 29
10 15Time
Empower MS SQD method editor Empower MS SQD method editor (Scan)(Scan)( )( )
©2010 Waters Corporation | COMPANY CONFIDENTIAL 30
UV And MS Data From The Same InjectionUV And MS Data From The Same Injectionjj
injection
UV h lMS channel
©2010 Waters Corporation | COMPANY CONFIDENTIAL 31
UV channel
Data Review:Data Review:MS and UV From Same InjectionMS and UV From Same Injectionjj
Overlaid UV and MS ChromatogramsBackground Corrected SpectraSpectra
©2010 Waters Corporation | COMPANY CONFIDENTIAL 32
Data Review:Data Review:MS Spectrum Index PlotMS Spectrum Index Plotpp
Spectrum IndexSpectrum Index Plot gives quick and easy Background correctedcorrected spectra for all integratedpeaks
©2010 Waters Corporation | COMPANY CONFIDENTIAL 33
Data Review:Data Review:Extracting Chromatograms From SpectraExtracting Chromatograms From Spectrag g pg g p
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Reporting:Reporting:MS and UV LayoutsMS and UV Layoutsyy
©2010 Waters Corporation | COMPANY CONFIDENTIAL 35
Difficult Analysis With UV Detection Difficult Analysis With UV Detection yy
Expansion of region of 0.03% impurity by UV detection
0.70
0.75
0.80
0.85
-0.00135
-0.00130
-0.00125
LansoprazoleUV @ 254nm
0.45
0.50
0.55
0.60
0.65
AU -0.00155
-0.00150
-0.00145
-0.00140
UV @ 254nm
UV @ 254nm
AU
0.25
0.30
0.35
0.40
-0.00175
-0.00170
-0.00165
-0.00160
0.03%
S/N = 2
@
0.00
0.05
0.10
0.15
0.20
-0.00180
Minutes4.80 5.00 5.20 5.40 5.60 5.80 6.00
©2010 Waters Corporation | COMPANY CONFIDENTIAL 36
-0.05
Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00
Enhance Sensitivity And SelectivityEnhance Sensitivity And SelectivityWith MS DetectionWith MS Detection
Expansion of region of 0.03% impurity by MS detection
0.75
0.80
0.85
Lansoprazole
UV @ 254nm
050
0.55
0.60
0.65
0.70 UV @ 254nmSIR @ 298.22 m/z
2.0x106
SIR 298 22
AU
0.30
0.35
0.40
0.45
0.50
1 2 106
1.4x106
1.6x106
1.8x106SIR 298.22S/N = 870
005
0.10
0.15
0.20
0.25
Inte
nsity
6.0x105
8.0x105
1.0x106
1.2x106
©2010 Waters Corporation | COMPANY CONFIDENTIAL 37
-0.05
0.00
0.05
Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00
0.0
2.0x105
4.0x105
Minutes4.80 5.00 5.20 5.40 5.60 5.80 6.00
Peak Tracking Peak Tracking In Methods DevelopmentIn Methods Development
100
pp
%
33% ACN and 35 mM Ammonium Formate
Scan ES+ TIC
100
%Scan ES+ 314.1+271.1+301.1
100
%
50% M OH d 15 M
Scan ES+ TIC
100
%
50% MeOH and 15 mM Ammonium Formate
Scan ES+ 314.1+271.1+301.1
©2010 Waters Corporation | COMPANY CONFIDENTIAL 38
0 5 10 15 20 25 30Time
Enhancing LC/MS Results By Moving Enhancing LC/MS Results By Moving to Tandem Quadrupole Technology to Tandem Quadrupole Technology Q p gyQ p gy
Ideal for complex matrices— Physiological samplesy g p— Food matrix— Environmental samples
Need to reduce analysis time— Need selectivity of Tandem MS to remove interferences
Need increased sensitivity— Remove chemical noise
Additional experiments— Product Ion Scans
©2010 Waters Corporation | COMPANY CONFIDENTIAL 39
— Precursor Ion Scans— Neutral Loss Scans
Robustness of ZRobustness of Z--Spray Ionization Spray Ionization Source Provides ReliabilitySource Provides Reliabilityyy
Area Variation over Time
Verapamil in ppt human plasma on ACQUITY TQD: 300 injections; % RSD = 2.9
120000
140000
T H
uman
80000
100000
120000
Vera
pam
il in
PPT
sma
40000
60000
ount
(10p
g/µL
V Pla
RSD% = 2.9
0
20000
1 17 33 49 65 81 97 113 129 145 161 177 193 209 225 241 257 273 289
Injection Count
Are
a C
o
©2010 Waters Corporation | COMPANY CONFIDENTIAL 40
Injection Count
Theory of SIR versus Multiple Reaction Theory of SIR versus Multiple Reaction Monitoring (MRM) in Tandem MSMonitoring (MRM) in Tandem MSg ( )g ( )
MS1Collision
Cell MS2Example of not having
any collisions:
SIR of m/z= 255
StaticCIDStatic
MS1Collision
Cell MS2 For example:MS1 Cell MS2 For example:
MRM of m/z= 255 > 209
or
©2010 Waters Corporation | COMPANY CONFIDENTIAL 41
StaticCIDStatic
MRM of m/z= 255 > 237
Multiple Reaction Monitoring (MRM)Multiple Reaction Monitoring (MRM)Provides Additional Separation PowerProvides Additional Separation Powerpp
Minimizes matrix interference
High sensitivity due to additional selectivityHigh sensitivity due to additional selectivity
Ion chemistry and physics makes it the most accurate and reproducible quantitation
Nominally isobaricinterferences of interferences of
chloramphenicol in honey
©2010 Waters Corporation | COMPANY CONFIDENTIAL 42
Comparing Analysis of Isobaric Compounds Comparing Analysis of Isobaric Compounds Using SIR MS vs MRM MS/MS ModesUsing SIR MS vs MRM MS/MS Modesg /g /
Ion Chromatograms SIR’s of m/z=255 100MixIso_1G14_022 SIR of 1 Channel ES+
TIC5 95e6
1.31g
%
5.95e6
OFenbufen
From a sample that is60 ng/mL Ketoprofen60 ng/mL Fenbufen
0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70Time0
OHO
OKetoprofen
FenbufenKetoprofen
O
OH
O
From a Sample that is60 ng/mL Ketoprofen
100MixIso_1G14_023 SIR of 1 Channel ES+
TIC6.03e6
1.31
Both have a MW of 254
Fenbufen ??Ketoprofen
60 ng/mL Ketoprofen6 ng/mL Fenbufen
%
©2010 Waters Corporation | COMPANY CONFIDENTIAL 43
0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70Time0
Comparing Analysis of Isobaric Compounds Comparing Analysis of Isobaric Compounds Using SIR MS vs MRM MS/MS ModesUsing SIR MS vs MRM MS/MS Modes
100MixIso_1G14_023 SIR of 1 Channel ES+
TIC6.03e6
1.31
From SIR of
g /g /
0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70Time0
%
From SIR of m/ z= 255
Mixture of:60 ng/mL Ketoprofen6 ng/mL Fenbufen
100MixIso_1G14_024 MRM of 2 Channels ES+
255.25 > 209.21.43e6
1.31
KetoprofenFrom MRM of
m/z= 255 > 2096 ng/mL Fenbufen
100
0
%
MixIso_1G14_024 MRM of 2 Channels ES+ 255.25 > 237.2
8.06e41.42
Ketoprofen/
%FenbufenFrom MRM of
m/z= 255 > 237
©2010 Waters Corporation | COMPANY CONFIDENTIAL 44
0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70Time0
Do you need a high MRM acquisition rate?Do you need a high MRM acquisition rate?
Travelling Wave Ion Transport The effect of MRM acquisition rate on signal intensity
100 data points per second
©2010 Waters Corporation | COMPANY CONFIDENTIAL 45
IntelliStart™ TQD Method DeveloperIntelliStart™ TQD Method DeveloperQ pQ p
©2010 Waters Corporation | COMPANY CONFIDENTIAL 46
IntelliStart™: IntelliStart™: System Performance CheckSystem Performance Checkyy
IntelliStart™ also features a system performance check
6 li i j i f k d d f 6 replicate injections of a known compound are made from the LC system with known chromatographic retention time
Data quality measurements are made by System Suitability processing to produce a pass/fail report
Users may define tolerances for pass criteria
Results are logged in the System Console and reports are Results are logged in the System Console and reports are produced in both (electronic) and printed form.
Raw data and experimental details are also stored.
©2010 Waters Corporation | COMPANY CONFIDENTIAL 47
LC/MS System Check ResultsLC/MS System Check Results/ y/ y
©2010 Waters Corporation | COMPANY CONFIDENTIAL 48
System Check System Check ––Report & Notification under Empower controlReport & Notification under Empower controlp pp p
©2010 Waters Corporation | COMPANY CONFIDENTIAL 49
ACQUITY TQD ACQUITY TQD OnOn--column sensitivity in matrixcolumn sensitivity in matrixyy
Verapamil in PPT human plasma2:1 plasma:acetonitrile
250fg on columngs/n = 51:1 RMS
No data processing
MRM method automaticallyMRM method automatically generated by IntelliStart
©2010 Waters Corporation | COMPANY CONFIDENTIAL 50
SoftwareSoftware
For the first time, a Tandem Quadrupole MS is available on For the first time, a Tandem Quadrupole MS is available on both MassLynx and Empower platforms. (Both SQD and TQD)
• Scalable, networked CDS Solution• Embedded relational database• Support for regulated laboratory environments• Full system suitability reporting• Method Validation Manager
• Dedicated MS Software platform• Customized Application-managers• Automated System check• QuanOptimize• Open Access quantitation
©2010 Waters Corporation | COMPANY CONFIDENTIAL 51
• Method Validation Manager • Open Access quantitation
What Does Sum Of What Does Sum Of LC/UV + MS Provide?LC/UV + MS Provide?//
Everything from the independent techniques
MS brings more information from a single injection MS brings more information from a single injection— Peak purity
— Peak identification
— Sensitivity
LC improves the quality of MS data — Easier to interpret and understand the datap
— Enhanced sensitivity
— Enhanced ruggedness
©2010 Waters Corporation | COMPANY CONFIDENTIAL 52
Thank You For Your AttentionThank You For Your Attention
©2010 Waters Corporation | COMPANY CONFIDENTIAL 53