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

2010 Waters Corporation | COMPANY CONFIDENTIAL 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

2010 Waters Corporation | COMPANY CONFIDENTIAL 4

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 !

2010 Waters Corporation | COMPANY CONFIDENTIAL 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

In this case if N=1 and S=6, then the Sensitivity Ratio

would be expressed as: 6/1 or 6 or 6:1

2010 Waters Corporation | COMPANY CONFIDENTIAL 6

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

2010 Waters Corporation | COMPANY CONFIDENTIAL 7

 Selectivity
y

Visibility can be dependent upon your sensory device

Invisibility can sometimes be a great benefit

2010 Waters Corporation | COMPANY CONFIDENTIAL 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

2010 Waters Corporation | COMPANY CONFIDENTIAL 9

 Now You See Them and Now You
Dont?

Diode array
Diode Array TIC
PDA to monitor UV/Vis
TIC friendly compounds

ES+TIC Mass Spec to verify that

compound has been
synthesized

ELSD to monitor all

ELSD compounds and determine
purity
p y levels
2
4.5 7
Min

2010 Waters Corporation | COMPANY CONFIDENTIAL 10

PDA/ELSD/SQD
/ / Q

PDA

ELSD

SQD

High confident data in one injection !

2010 Waters Corporation | COMPANY CONFIDENTIAL 11
PDA/ELSD/SQD
Complete
p Information from One Injection
j

2010 Waters Corporation | COMPANY CONFIDENTIAL 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
Spectra are converted into a vector in n dimensional space.
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

Spectrum A Spectrum A
Spectrum B

240 nm
Absorbance

AU at 2
Spectrum B

200.00 240.00 280.00 320.00 AU at 280 nm

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
p Contrast
Spectral Contrast
53 Degrees

Ethylparaben
EthylPaba
Abssorbance

200.00 240.00 280.00 320.00

nm
2010 Waters Corporation | COMPANY CONFIDENTIAL 16
Spectral
p Contrast

Spectral Contrast
10 Degrees
Similar spectra
for structurally
related
Theophylline
compounds
Dyphylline
orbance
Abso

230.00 250.00 270.00 290.00 310.00

nm
2010 Waters Corporation | COMPANY CONFIDENTIAL 17
Spectral
p Contrast

Spectral Contrast
0.5 Degrees

Methylparaben
Ethylparaben
Very similar
spectra CH2
spectra,
bsorbance
e

difference

Spectral
Ab

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.

Spectrum A The Noise Region in gray forms

a constant cylinder of uncertainty
around the vector.
bance

A vector drawn from the origin to

Noise
the edge of the cylinder creates
Absorb

the noise angle.

Spectrum B The shorter the vector (lower
concentration) the larger the
noise
i angle.
l

2010 Waters Corporation | COMPANY CONFIDENTIAL 19

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

2010 Waters Corporation | COMPANY CONFIDENTIAL 21

Spectral
p Resolution
Benzene

230.00 250.00 270.00

nm

Less resolution at UV maxima

3.6 nm vs 1.2 nm shifted

2010 Waters Corporation | COMPANY CONFIDENTIAL 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

2010 Waters Corporation | COMPANY CONFIDENTIAL 25

Multiple
p Pass Peak Purity
y

Second Pass Peak Purity

2010 Waters Corporation | COMPANY CONFIDENTIAL 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

287.1
309.1 309.1
% % %
287.1 311.1
311.1

200 300 400 200 300 400 200 300 400

m/z m/z m/z
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

2010 Waters Corporation | COMPANY CONFIDENTIAL 29

Empower MS SQD method editor
(Scan)
( )

2010 Waters Corporation | COMPANY CONFIDENTIAL 30

UV And MS Data From The Same Injection
j

injection

MS channel
UV channel
h l

2010 Waters Corporation | COMPANY CONFIDENTIAL 31

Data Review:
MS and UV From Same Injection
j

Overlaid UV
and MS
Chromatograms
Background
Corrected
Spectra

2010 Waters Corporation | COMPANY CONFIDENTIAL 32

Data Review:
MS Spectrum
p Index Plot

Spectrum Index
Plot gives quick
and easy
Background
corrected
spectra for all
integrated
peaks

2010 Waters Corporation | COMPANY CONFIDENTIAL 33

Data Review:
Extracting
g Chromatograms
g From Spectra
p

2010 Waters Corporation | COMPANY CONFIDENTIAL 34

Reporting:
MS and UV Layouts
y

2010 Waters Corporation | COMPANY CONFIDENTIAL 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

0.55 -0.00145

0.50 -0.00150

UV @ 254nm

AU
0.45 -0.00155
AU

0.40
-0.00160

0.35
-0.00165
0.03%
0.30

0.25
-0.00170
S/N = 2
-0.00175
0.20
-0.00180
0.15

0.10 4.80 5.00 5.20 5.40 5.60 5.80 6.00

Minutes
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
Lansoprazole
0.75

0.70 UV @ 254nm
0.65 SIR @ 298.22 m/z
0.60

0.55
2.0x106
050
0.50
SIR 298
298.22
22
0.45 1.8x106
S/N = 870
AU

0.40 1.6x106

0.35
1.4x106
0.30
2 106
1.2x10
1

Intensity
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 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00
Minutes 0.0
4.80 5.00 5.20 5.40 5.60 5.80 6.00
2010 Waters Corporation
Minutes| COMPANY CONFIDENTIAL 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 20 25 30
Time 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 Z-
Z-Spray Ionization
Source Provides Reliability
y
Verapamil in ppt human plasma on ACQUITY TQD: 300 injections; % RSD = 2.9

Area Variation over Time

T Human

140000

120000
Verapamil in PPT

100000
Plasma

80000
ount (10pg/L V

60000
RSD% = 2.9
40000
Area Co

20000

0
1 17 33 49 65 81 97 113 129 145 161 177 193 209 225 241 257 273 289
Injection Count

2010 Waters Corporation | COMPANY CONFIDENTIAL 40

Theory of SIR versus Multiple Reaction
Monitoring
g((MRM) ) in Tandem MS

Collision
MS1 Cell MS2
Example of not having
any collisions:

SIR of m/z= 255

Static CID Static

Collision
MS1 Cell MS2 For example:

MRM of m/z= 255 > 209

or

MRM of m/z= 255 > 237

Static CID Static

2010 Waters Corporation | COMPANY CONFIDENTIAL 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 42

Comparing Analysis of Isobaric Compounds
Using
g SIR MS vs MRM MS/MS
/ Modes

MixIso_1G14_022 SIR of 1 Channel ES+

1.31 TIC
Ion Chromatograms
g SIRs of m/z=255 100
5 95e6
5.95e6
From a sample that is
Fenbufen 60 ng/mL Ketoprofen
O
60 ng/mL Fenbufen
%

O OH Ketoprofen Fenbufen

Ketoprofen 0 Time
0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70
O
O
MixIso_1G14_023 SIR of 1 Channel ES+
100 1.31 TIC
OH 6.03e6
From a Sample that is
60 ng/mL Ketoprofen
Both have a MW of 254 6 ng/mL Fenbufen
%

Ketoprofen Fenbufen ??

0 Time
0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 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 SIR of 1 Channel ES+

100
1.31 TIC
6.03e6

From SIR of
% m/ z= 255

0 Time
0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70

MixIso_1G14_024 MRM of 2 Channels ES+

1.31 255.25 > 209.2
Mixture of:
100
1.43e6

60 ng/mL Ketoprofen From MRM of

m/z=
/ 255 > 209 Ketoprofen
6 ng/mL Fenbufen %

0
MixIso_1G14_024 MRM of 2 Channels ES+
1.42 255.25 > 237.2
100
8.06e4

From MRM of Fenbufen

% m/z= 255 > 237

0 Time
0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 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

2010 Waters Corporation | COMPANY CONFIDENTIAL 45

IntelliStart TQD
Q Method Developer
p

2010 Waters Corporation | COMPANY CONFIDENTIAL 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.

2010 Waters Corporation | COMPANY CONFIDENTIAL 47

LC/MS
/ System
y Check Results

2010 Waters Corporation | COMPANY CONFIDENTIAL 48

System Check
Report
p & Notification under Empower
p control

2010 Waters Corporation | COMPANY CONFIDENTIAL 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

2010 Waters Corporation | COMPANY CONFIDENTIAL 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 Dedicated MS Software platform

Embedded relational database Customized Application-managers
Support for regulated laboratory environments Automated System check
Full system suitability reporting QuanOptimize
Method Validation Manager Open Access quantitation

2010 Waters Corporation | COMPANY CONFIDENTIAL 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

2010 Waters Corporation | COMPANY CONFIDENTIAL 52

Thank You For Your Attention

2010 Waters Corporation | COMPANY CONFIDENTIAL 53