Advanced Detectors For Empower
ELSD, PDA, SQD and TQD
John Van Antwerp
Waters Corporation
©2010 Waters Corporation | COMPANY CONFIDENTIAL
Overview of Role For Detector in an
UPLC/HPLC system
©2010 Waters Corporation | COMPANY CONFIDENTIAL
Commonly Desired
Characteristics of a HPLC Detector
 High sensitivity
 Negligible baseline noise
 Wide linear dynamic range
 R
Response iindependent
d
d t off variations
i ti
iin operating
ti
parameters (pressures, temperature, flow-rate, etc.)
 Response independent of mobile phase
 Low dead volume
 Selective and universal
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3
Common Realities of a HPLC
Detector
 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
— but is not always linear so calibration techniques are designed
to promote this relationship
 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
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Key
y To All Method Development
p
There is no single
g detector that can be employed
p y
for all HPLC
separations. There is no “magic black box” !
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5
Sensitivity
y - Definition
 Ratio of Signal-to-Noise (S/N or S:N)
Two Typical Concerns:
 Limit
Li it off detection
d t ti
(LOD):
(LOD) S/N = 3
S
 Limit of quantitation (LOQ): S/N =10
N
In this case if N=1 and S=6, then the “Sensitivity Ratio”
would be expressed as: “6/1” or “6” or “6:1”
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How to Increase Signal to Noise
Ratio
If start with Signal-to-noise (S/N) of 3:1
Can increase S/N by increasing peak height (6:1)
Can increase S/N by decreasing noise (8:1)
6:1
3:1
8:1
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Selectivity
y
 Visibility can be dependent upon your sensory device
 Invisibility can sometimes be a great benefit
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8
ELSD utility
y and advantages
g
 Used for detection of compounds less volatile than mobile
phase
— Low-temperature ELSD extends use to semi-volatile analytes in
aqueous mobile phases,
phases making this technique suitable for
analysis of small molecules such as pharmaceuticals
 Often referred to as ‘universal’ detector
— Use for compounds without UV chromaphore:
 Transparent to changes in mobile phase composition
 Same chromatographic requirements as LC/MS
— Volatile modifiers
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Now You See Them and Now You
Don’t?
Diode
array
Diode
Array TIC
TIC
Mass Spec to verify that
compound has been
synthesized
ES+TIC
ELSD to monitor all
compounds and determine
purity
p
y levels
ELSD
2
4.5
Min
PDA to monitor UV/Vis
friendly compounds
7
©2010 Waters Corporation | COMPANY CONFIDENTIAL
10
PDA/ELSD/SQD
/
/ Q
PDA
ELSD
SQD
High confident data in one injection !
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11
PDA/ELSD/SQD
Complete
p
Information from One Injection
j
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12
Understanding PDA
Peak Purity
©2010 Waters Corporation | COMPANY CONFIDENTIAL
Spectral
p
Contrast
 The Spectral Contrast measures the shape difference
between two spectra.
spectra

Spectra are baseline corrected by subtracting interpolated baseline spectra
between peak baseline liftoff and baseline touchdown.

p
are converted into a vector in n dimensional space.
p
Spectra

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
Spectrum A
Absorbance
AU at 2
240 nm
Spectrum A
Spectrum B

200.00
240.00
280.00
nm
320.00
Spectrum B
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
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Spectral
p
Contrast
Spectral Contrast
53 Degrees
Abssorbance
Ethylparaben
EthylPaba
200.00
240.00
280.00
320.00
nm
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Spectral
p
Contrast
Spectral Contrast
10 Degrees
Similar spectra
for structurally
related
compounds
Abso
orbance
Theophylline
Dyphylline
230.00
250.00
270.00
290.00
310.00
nm
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Spectral
p
Contrast
Spectral Contrast
0.5 Degrees
Methylparaben
Ethylparaben
Ab
bsorbance
e
Very similar
spectra CH2
spectra,
difference
Spectral
C t t can
Contrast
differentiate
these spectra.
200.00
240.00
280.00
320.00
nm
©2010 Waters Corporation | COMPANY CONFIDENTIAL
18
Threshold Calculations
 The Threshold Angle is comprised of two parts: First,
First The
Detector Noise Angle is calculated from the chromatographic
baseline and is inversely proportional to the peak height.
The Noise Region in gray forms
a constant cylinder of uncertainty
around the vector.
Absorb
bance
Spectrum A
Noise

Spectrum B
A vector drawn from the origin to
the edge of the cylinder creates
the noise angle.
The shorter the vector (lower
concentration) the larger the
noise
i angle.
l
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Threshold Calculations
 Second, The Solvent Effect corresponds to the constant
portion of the Threshold Angle,
Angle it accounts for solvent
effects and photometric errors.
 The solvent effect can be accurately measured from a
chemically pure standard,
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
p
Resolution
 Spectral Resolution or the ability to differentiate one UV spectrum
from another.
another
 The Waters PDA runs with a fixed 50 micron slit, producing an
optical resolution of 1.2nm.
 For 1.2nm opticall resolution
l
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
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21
Spectral
p
Resolution
Benzene
230.00
Less resolution at
3.6 nm vs 1.2 nm
250.00
nm
270.00
UV maxima
shifted
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22
Peak Impurity and Peak Spectral Homogeneity
 The Peak Purity Algorithm uses Spectral Contrast to
compare all spectra within a peak to the Apex spectrum.
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
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
Limitations
 The UV spectrum of different compounds can be identical.
 The concentration of the impurity may be too low to detect.
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
LC-GC
GC Volume 12 Number 10
October 1994 pages 768-772
©2010 Waters Corporation | COMPANY CONFIDENTIAL
24
Multiple
p Pass Peak Purity
y
Peak Purity
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25
Multiple
p Pass Peak Purity
y
Second Pass Peak Purity
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26
Apparent Pair Of Compounds:
UV Spectra
p
Across First Peak
100
100
100
%
%
%
210
nm
350
210
nm
350
210
nm
350
100
%
10
Time
15
©2010 Waters Corporation | COMPANY CONFIDENTIAL
27
Apparent Pair Of Compounds:
Mass Spectra
p
Across First Peak
100
%
100
309.1
%
100
309.1
287.1
309.1
%
287.1 311.1
311.1
200
300
m/z
400
200
300
m/z
400
200
300
m/z
400
100
%
10
Time
15
©2010 Waters Corporation | COMPANY CONFIDENTIAL
28
Single Mass Chromatograms:
Extracted From MS Spectra
p
Scan ES+
TIC
Scan ES+
309.1
Scan ES+
287 1
287.1
10
Ti
Time
15
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29
Empower MS SQD method editor
(Scan)
(
)
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30
UV And MS Data From The Same Injection
j
injection
MS channel
UV channel
h
l
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31
Data Review:
MS and UV From Same Injection
j
Overlaid UV
and MS
Chromatograms
Background
Corrected
Spectra
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32
Data Review:
MS Spectrum
p
Index Plot
Spectrum Index
Plot gives quick
and easy
Background
corrected
spectra for all
integrated
peaks
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33
Data Review:
Extracting
g Chromatograms
g
From Spectra
p
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34
Reporting:
MS and UV Layouts
y
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35
Difficult Analysis
y
With UV Detection
Expansion of region of 0.03% impurity by UV detection
0.85
0.80
-0.00125
0.75
Lansoprazole
-0.00130
0.70
0.65
UV @ 254nm
-0.00135
-0.00140
0.60
-0.00145
0.50
-0.00150
AU
0.55
AU
0.45
0.40
-0.00155
UV @ 254nm
-0.00160
0.03%
0.35
-0.00165
0.30
S/N = 2
-0.00170
0.25
-0.00175
0.20
-0.00180
0.15
0.10
4.80
5.00
5.20
5.40
Minutes
5.60
5.80
6.00
0.05
0.00
-0.05
0.50 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
Minutes
©2010 Waters Corporation | COMPANY CONFIDENTIAL
36
Enhance Sensitivity And Selectivity
With MS Detection
Expansion of region of 0.03% impurity by MS detection
0.85
0.80
0.75
0.70
0.65
Lansoprazole
UV @ 254nm
SIR @ 298.22 m/z
0.60
0.55
2.0x106
AU
050
0.50
0.45
1.8x106
0.40
1.6x106
0.35
1.4x106
SIR 298
298.22
22
S/N = 870
0.30
Intensity
1.2x10
1
2 106
0.25
1.0x106
0.20
0.15
8.0x105
0.10
6.0x105
005
0.05
4.0x105
0.00
2.0x105
-0.05
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
Minutes
5.50
6.00
6.50
7.00
7.50
8.00
8.50
9.00
9.50
10.00
0.0
4.80
5.00
5.20
5.40
5.60
5.80
6.00
©2010 Waters Corporation
| COMPANY CONFIDENTIAL
Minutes
37
Peak Tracking
In Methods Development
p
100
Scan ES+
TIC
%
33% ACN and 35 mM
Ammonium Formate
100
Scan ES+
314.1+271.1+301.1
%
100
Scan ES+
TIC
%
50% M
MeOH
OH and
d 15 mM
M
Ammonium Formate
100
Scan ES+
314.1+271.1+301.1
%
0
5
10
15
Time
20
25
30
©2010 Waters Corporation | COMPANY CONFIDENTIAL
38
Enhancing LC/MS Results By Moving
to Tandem Q
Quadrupole
p
Technology
gy
 Ideal for complex matrices
— Physiological
y
g
samples
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
— Precursor Ion Scans
— Neutral Loss Scans
©2010 Waters Corporation | COMPANY CONFIDENTIAL
39
Robustness of ZZ-Spray Ionization
Source Provides Reliability
y
Verapamil in ppt human plasma on ACQUITY TQD: 300 injections; % RSD = 2.9
Area Co
ount (10pg/µL V
Verapamil in PPT
T Human
Plasma
Area Variation over Time
140000
120000
100000
80000
60000
RSD% = 2.9
40000
20000
0
1
17
33
49
65
81
97 113 129 145 161 177 193 209 225 241 257 273 289
Injection Count
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Theory of SIR versus Multiple Reaction
Monitoring
g(
(MRM)
) in Tandem MS
MS1
Collision
Cell
MS2
Example of not having
any collisions:
SIR of m/z= 255
Static
MS1
CID
Collision
Cell
Static
MS2
For example:
MRM of m/z= 255 > 209
or
MRM of m/z= 255 > 237
Static
CID
Static
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41
Multiple Reaction Monitoring (MRM)
Provides Additional Separation
p
Power
 Minimizes matrix interference
 High sensitivity due to additional selectivity
 Ion chemistry and physics makes it the most accurate and
reproducible quantitation
Nominally isobaric
interferences of
chloramphenicol in honey
©2010 Waters Corporation | COMPANY CONFIDENTIAL
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Comparing Analysis of Isobaric Compounds
Using
g SIR MS vs MRM MS/MS
/
Modes
Ion Chromatograms
g
SIR’s of m/z=255
MixIso_1G14_022
100
SIR of 1 Channel ES+
TIC
5 95e6
5.95e6
1.31
From a sample that is
60 ng/mL Ketoprofen
60 ng/mL Fenbufen
Fenbufen
O
%
O
OH
Ketoprofen
Ketoprofen
0
0.80
O
0.90
1.00
1.10
Fenbufen
1.20
1.30
1.40
1.50
1.60
Time
1.70
O
MixIso_1G14_023
OH
SIR of 1 Channel ES+
TIC
6.03e6
1.31
100
From a Sample that is
60 ng/mL Ketoprofen
6 ng/mL Fenbufen
Both have a MW of 254
%
Ketoprofen
0
0.80
0.90
1.00
1.10
Fenbufen ??
1.20
1.30
1.40
1.50
1.60
Time
1.70
©2010 Waters Corporation | COMPANY CONFIDENTIAL
43
Comparing Analysis of Isobaric Compounds
Using
g SIR MS vs MRM MS/MS
/
Modes
MixIso_1G14_023
100
%
0
0.80
Mixture of:
60 ng/mL Ketoprofen
6 ng/mL Fenbufen
From SIR of
m/ z= 255
0.90
1.00
1.10
1.20
MixIso_1G14_024
100
%
1.30
0
0.80
1.40
1.50
From MRM of
m/z=
/
255 > 209
1.42
1.00
1.10
1.20
Time
1.70
Ketoprofen
MRM of 2 Channels ES+
255.25 > 237.2
8.06e4
From MRM of
m/z= 255 > 237
0.90
1.60
MRM of 2 Channels ES+
255.25 > 209.2
1.43e6
1.31
0
MixIso_1G14_024
100
%
SIR of 1 Channel ES+
TIC
6.03e6
1.31
Fenbufen
1.30
1.40
1.50
1.60
Time
1.70
©2010 Waters Corporation | COMPANY CONFIDENTIAL
44
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
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45
IntelliStart™ TQD
Q Method Developer
p
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46
IntelliStart™:
System
y
Performance Check
 IntelliStart™ also features a system performance check
 6 replicate
li
iinjections
j
i
off a k
known compound
d are made
d from
f
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
produced in both (electronic) and printed form.
 Raw data and experimental details are also stored.
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47
LC/MS
/
System
y
Check Results
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48
System Check –
Report
p
& Notification under Empower
p
control
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49
ACQUITY TQD
On--column sensitivity
On
y in matrix
Verapamil in PPT human plasma
2:1 plasma:acetonitrile
250fg
g on column
s/n = 51:1 RMS
No data processing
MRM method automatically
generated by IntelliStart
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50
Software
 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
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51
What Does Sum Of
LC/UV
/
+ MS Provide?
 Everything from the independent techniques
 MS brings more information from a single injection
— Peak purity
— Peak identification
— Sensitivity
 LC improves the quality of MS data
— Easier to interpret
p
and understand the data
— Enhanced sensitivity
— Enhanced ruggedness
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52
Thank You For Your Attention
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53