advanced detectors for empower - waters corporation · used for detection of compounds less...

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


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


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” !


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


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


SelectivitySelectivityyy

Visibility can be dependent upon your sensory device

Invisibility can sometimes be a great benefit


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


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


p y

Min

24.5 7

PDA/ELSD/SQDPDA/ELSD/SQD/ / Q/ / Q

PDA

ELSD

SQD


High confident data in one injection !

PDA/ELSD/SQDPDA/ELSD/SQDComplete Information Complete Information fromfrom One InjectionOne Injectionpp jj


Understanding PDA Understanding PDA Peak PurityPeak Purity


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.


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



Spectral Contrast 53 Degrees53 Degrees

EthylparabenEthylPaba

sorb

ance

Abs


200.00 240.00 280.00 320.00nm


Spectral Contrast10 Degrees

Similar spectra for structurally

TheophyllineDyphylline

related compounds

orba

nce

Abso


230.00 250.00 270.00 290.00 310.00nm


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.


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


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.


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


Spectral ResolutionSpectral Resolutionpp

Benzene

230.00 250.00 270.00nmnm

Less resolution at 3.6 nm vs 1.2 nm

UV maxima shifted


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.


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


Multiple Pass Peak PurityMultiple Pass Peak Purityp yp y

Peak Purity


Multiple Pass Peak PurityMultiple Pass Peak Purityp yp y

Second Pass Peak Purity


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

%


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


10 15Time

Empower MS SQD method editor Empower MS SQD method editor (Scan)(Scan)( )( )


UV And MS Data From The Same InjectionUV And MS Data From The Same Injectionjj

injection

UV h lMS channel


UV channel

Data Review:Data Review:MS and UV From Same InjectionMS and UV From Same Injectionjj

Overlaid UV and MS ChromatogramsBackground Corrected SpectraSpectra


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


Data Review:Data Review:Extracting Chromatograms From SpectraExtracting Chromatograms From Spectrag g pg g p


Reporting:Reporting:MS and UV LayoutsMS and UV Layoutsyy


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


-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


-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


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


— 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


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


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


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

%


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


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


IntelliStart™ TQD Method DeveloperIntelliStart™ TQD Method DeveloperQ pQ p


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.


LC/MS System Check ResultsLC/MS System Check Results/ y/ y


System Check System Check ––Report & Notification under Empower controlReport & Notification under Empower controlp pp p


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


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


• 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


Thank You For Your AttentionThank You For Your Attention


advanced detectors for empower - waters corporation · used for detection of compounds less...

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