xevo tq-s for bioanalysis · ©2010 waters corporation . compound name: oh prog correlation...

©2010 Waters Corporation

Xevo TQ-S for Bioanalysis


Key innovations

• High Performance

• Stepwave • RADAR

• Scan Wave

• Versatility

• Ion Sources

• Simplicity of Operation

• Engineered Simplicity


Xevo TQD

Xevo TQD Fast data acquisition

Routine UPLC-MS/MS

RADAR acquisition mode

Universal ion source architecture

Xevo TQ MS

Xevo TQ-S

Frost and Sullivan Product Quality Leadership Award 2011

Xevo TQ-S Highest sensitivity

Fastest data acquisition

ScanWave Enhanced product ion scanning



The Xevo TQ family Built on the same foundations

Best new product, ASMS 2008

Xevo TQ MS More sensitive than TQD

ScanWave Enhanced product ion scanning

Fast data acquisition



Ultra high performance targeted quantification with information rich qualitative supporting data

Routine targeted quantification with information rich qualitative supporting data

High performance targeted quantification with information rich qualitative supporting data


Xevo TQ-S

Ultra high sensitivity for targeted quantification


Time0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40

%

0

100

Time0.50 0.60 0.70 0.80 0.90 1.00 1.10 1.20 1.30 1.40

%

0

Xevo TQ

Xevo TQ-S Enhanced sensitivity 129X increase in peak area 25X increase in signal:noise

An increase in sensitivity? Rel

ativ

e io

n a

bundan

ce

Desompressin (Peptide)

UPLC/MRM, ESI +


An increase in sensitivity?

Fenuron ESI+ 30 7Metamitron ESI+ 32 15Acephate ESI+ 27 7Chlortoluron ESI+ 27 8Aldicarb ESI+ 27 6Demeton S Methyl ESI+ 26 9Phoxim ESI+ 64 19Kresoxim Methyl ESI+ 64 4Azinphos Methyl ESI+ 42 6Azoxystrobin ESI+ 45 4Dimethoate ESI+ 23 10Acetamiprid ESI+ 30 28Fluticasone ESI+ 30 3Formoterol ESI+ 39 4Nefadazone ESI+ 28 3Desmopressin ESI+ 129 25Salmeterol ESI+ 41 8Alprazolam ESI+ 21 13Reserpine ESI+ 25 5Ibuprofen ESI- 13 16Prostaglandin E2 ESI- 30 37

Mean Difference 38 11

Relative Peak Area

Relative S:N

Compound NameIonisation

Mode


Xevo TQ Xevo TQ-S

Larger sampling orifice

How did we increase sensitivity?

Problems associated with a larger sampling orifice:

1)A disperse ion cloud

2) Higher levels of matrix contamination


Ion Block Design

Sampling cone aperture — Increased to 0.8mm diameter

o Approx 5x increase in gas/ion flow

New ion block design

— No supplemental pumping

— All ions (and gas) enter StepWave guide

o Approx 200x increase in gas flow

o Up to 200x increase in ion flux


Ele

ctri

c Fi

eld

Diffuse Ion

Cloud

Designed to deal with problems associated with a larger sampling orifice

Problems associated with a larger sampling orifice:

1)A disperse ion cloud

2) Higher levels of matrix contamination


Ele

ctri

c Fi

eld

Diffuse Ion

Cloud

Maximising signal

Maximising robustness

Designed to deal with problems associated with a larger sampling orifice


SIMION Model

No Electric Field

N2 Flow

1 mbar N2


With Electric Field (25 V between guides)

N2 Flow

E

SIMION Model

1 mbar N2 T-Wave

T-Wave


Ultra High Sensitivity?

Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

1000.83

Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

100

2fg of

Verapamil Time

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75

%

0

100

0.83

Solvent

Blank

0.1fg

6 replicates

RSD<20%

UPLC/MRM of Verapamil (solvent standard) using an

ACQUITY prepared for trace analysis

220 Zeptomoles


Compound name: OH ProgCorrelation coefficient: r = 0.999149, r^2 = 0.998298Calibration curve: 3386.22 * x + 23.6767Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

Conc0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000

Re

sp

on

se

0

500000

1000000

1500000

2000000

2500000

3000000

APCI+, Hydroxprogesterone

Triplicate injections of Standards

50fg to 5,000pg on column

5 orders of magnitude

Correlation coefficient>0.995

Deviation<15%

Linearity of Response?


Assay Robustness

Verapamil, 10pg/µL spiked into supernatant from

2:1 ACN:Plasma protein precipitation.

ACQUITY BEH 2.1x 50mm

1000 on column injections

RSD of peak areas < 5%


10ul loop injection 0.5pg/ul Verapamil in human plasma/ACN_2

Time0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 0.95 1.00 1.05 1.10 1.15 1.20 1.25

%

0

100TC8_100721_1135 MRM of 1 Channel ES+

455 > 165 (Verapamil)2.66e7

0.83

Assay Robustness

Verapamil, 0.5pg/µL spiked

into supernatant from 2:1 ACN:Plasma protein

precipitation.

10µL injections

ACQUITY BDH 2.1x 50mm

>2000 on column injections

RSD of peak areas < 5%

Plasma injection 1

Plasma injection 1000

Plasma injection 2000


Xevo TQ-S

Information rich qualitative supporting

data


Rapid MS to MRM Switching


m/z100 200 300 400 500 600 700 800 900 1000

%

0

100

m/z100 200 300 400 500 600 700 800 900 1000

%

0

100 253.3

123.0

293.3

397.3441.4

619.6485.5

329.3

214.1

100.1149.3 279.3

251.1

415.3

363.4

546.4468.6

600.7

Time0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40

%

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

%

9

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

Targeted compound MRM positive ESI Fluticasone

in plasma

Matrix Profile MS full scan negative ESI

Matrix Profile MS full scan positive ESI

Qu

alita

tive

Qu

an

tita

tive

Rapid MS to MRM Switching


Method Development: Comparison of PPT to SPE Extract

Protein Precipitation

MRM

Full Scan (Extracted

Interference)

Protein precipitation Monitor Alprazolam MRM 309->281 7.03e6

SPE Monitor Alprazolam MRM 309->281 1.56e6

SPE Background ms scan 2.61e7

Protein precipitation Background ms scan 2.44e8


Method Development: Choosing between two gradients

Time0.50 1.00 1.50 2.00 2.50 3.00

%

0

0.50 1.00 1.50 2.00 2.50 3.00

%

1

0.50 1.00 1.50 2.00 2.50 3.00

%

4

0.50 1.00 1.50 2.00 2.50 3.00

%

1

1.95

2.792.32

2.19

1.65 2.36

2.29 2.60 2.962.78

Phospholipids184>184

Radar Plasma Blank

Radar Solvent Blank

Fluticasone501>293

Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25

%

0

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25

%

1

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25

%

4

0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25

%

0

0.97

1.08

1.61

0.831.17 1.29

1.271.17

1.00

1.791.56

1.97 2.23

Phospholipids184>184

Radar Plasma Blank

Radar Solvent Blank

Fluticasone501>293

Gradient method 2 5-95% MeOH in 2.0 minutes

Gradient method 1 5-95% MeOH in 0.70 minutes


Simultaneous MRM and Full Scan Detection of Metabolites

MRM of Drug

Full Scan of Metabolites

Metabolites


RADAR & StepWave

RADAR — Monitor matrix complexity — RADAR enables understanding of matrix complexity — Intelligent decision making when assessing matrix effects — Intelligent decision making when developing methods

StepWave — Extra sensitivity in MRM mode — Reduce matrix effects by simple dilution of complex extracts — Maintain ability to achieve LOQs


Xevo TQ-S

Information rich qualitative supporting

data

ScanWave


ScanWave Technology

Travelling Wave

DC Barrier

RF Barrier

Potential 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


Standard Product Ion Scanning

Continuous output of ions by cell

Most ions filtered out by MS2

Very inefficient


ScanWave Enhanced Product Ion Scanning

Ion accumulation

Mass selective ion ejection

Synchronised with scanning of MS2


StepWave Combined with ScanWave

Time0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00

%

0

100S/N:RMS=60.14

m/z50 100 150 200 250 300 350 400 450 500 550 600 650

%

0

100195

174

105148

236 397365 448

ScanWave enhanced

Product ion spectrum

UPLC/MS/MS

50fg Reserpine

on column

ScanWave enhanced

Product ion scanning

chromatogram

(m/z 195)

A combination providing leading-edge

spectral MS/MS sensitivity


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.

Eliminates the need for 2 analytical runs for confirmation

Rapid Mode Switching Precursor Ion Confirmation Scan


ScanWave Technology

MRM

MRM ScanWave DS

A product ion confirmation (PIC) scan is a useful tool for method development, or when there may be two peaks within a single MRM transition


An Alternate Approach to Full Scan Profiling of Metabolites

Screen for selected panel of metabolites — Commonly reported metabolites

— User definable

Predicts metabolite MRMs based upon Parent Compound MSMS

Generates MANY MRMs — 1 msec dwell

With RADAR, full scan MS may be added to the experiment


( ) +

P

F1

F2 F3 ( )

P M

F1

F2+M F3

P+M > F1 P+M > F1+M P+M > F2 P+M > F2+M P+M > F3 P+M > F3+M

iMRMs :


List of Potential Metabolites from Text

File

MS Source and Analyzer

Conditions

iMRM Method

Spectra of Parent Automatically Obtained from

MassLynx

Select Metabolites from list of probable

transformations

Identify Product Ions From Parent Compound

Calculate MRMs and Write MS Method

File

MSMS of Parent Compound

Automatic Product Ion Selection

Develop Theoretical MRMs to Investigate


iMRM method


[Benefits] — Metabolites detected with

maximum sensitivity

— Spectra acquired in the time scale of UPLC peak

— Data acquired in one analytical run, no need for confirmatory experiment

Simple Metabolite Detection and Confirmation

Time0.50 1.00 1.50 2.00 2.50

%

0

100

18_4_022 5: MRM of 1 Channel ES+ 458.195 > 339.845 (AZ1_Hydroxylation)

3.77e5

m/ z1 00 12 0 1 40 1 60 18 0 2 00 2 20 24 0 2 60 28 0 30 0 3 20 34 0 36 0 3 80 40 0 42 0 4 40 46 0

%

0

1 00

18 _4 _0 23 3 ( 2. 07 8) 5: Pro du c t I on s o f 4 58 ES+45 8. 19 5 > 3 39 .84 5 (AZ1_ Hy dro x y la tion )

1.9 2e 5326

315

340

354

m/ z1 00 12 0 14 0 16 0 18 0 2 00 2 20 2 40 26 0 28 0 30 0 3 20 3 40 3 60 38 0 40 0 42 0 44 0 4 60

%

0

1 00

18 _4 _0 25 5 ( 2. 14 0) 3: Pro du c t I on s o f 4 42 ES+44 2.2 > 32 5. 84 7 ( AZ1_ Par en t)

8.9 8e 6326

313

340

425

Time0.50 1.00 1.50 2.00 2.50

%

0

100

18_4_025 3: MRM of 1 Channel ES+ 442.2 > 325.847 (AZ1_Parent)

1.08e8

Time0.50 1.00 1.50 2.00 2.50

%

0

100

18_4_022 1: MRM of 1 Channel ES+ 416.148 > 325.847 (AZ1_Dealkylation)

5.66e6

m/ z1 00 1 20 14 0 16 0 1 80 20 0 22 0 2 40 2 60 28 0 30 0 3 20 340 36 0 3 80 4 00 42 0

%

0

1 00

18 _4 _0 23 4 ( 1. 97 6) 1: Pro du c t I on s o f 4 16 ES+4 16 .1 48 > 32 5. 84 7 ( AZ1 _D ea lk y la tion )

5.5 6e 5326

313298

340

Time0.50 1.00 1.50 2.00 2.50

%

0

100

18_4_022 2: MRM of 1 Channel ES+ 428.184 > 325.829 (AZ1_Demethylation)

3.58e6

m /z10 0 12 0 140 1 60 1 80 20 0 22 0 24 0 260 2 80 3 00 3 20 34 0 36 0 380 4 00 4 20

%

0

1 00

18_ 4_ 023 3 (1 .87 1) 2 : Pro du ct I ons of 428 ES+42 8.1 84 > 3 25 .82 9 ( AZ1 _De me thy la tio n)

3. 92 e4326

147235

176

340

Dealkylation Demethylation

Dosed Compound

MRM 442.2>326

HydroxylatedMetabolite

MRM 458.2>340

PIC Spectrum

PIC Spectrum

PIC Spectrum

PIC Spectrum


ScanWave & StepWave

ScanWave — Enhanced product ion spectra — Confirm target compound quantitative data with PICS — Indicate presence of related metabolites

StepWave — Enhances signals in all ionisation modes — Highest quality of spectra MS/MS data for structural

confirmation


Xevo TQ-S

Versatility

&

Simplicity of operation


IntelliStart™ Automated MS Resolution &

Calibration Checks

IntelliStart™ Automated MRM

Method Developer

IntelliStart™ Automated LC/MS

System Check

IntelliStart™ Automated System

Monitoring.

Automatically ensure the system is ready

to run

Quanpedia Automated MRM Time Window &

Data Rate Scheduling

Simplicity of Operation


Solvent + Analyte and Internal Standard

Extracted Matrix + Analyte and Internal Standard

A= 100

A= 90

A= 95

A= 85

XICAnalyte XICInternal Standard

MF = Amatrix/Asolvent

MF = 90/100 = .90

MF = Amatrix/Asolvent

MF = 85/95 = .89

IS Normalized MF = MFAN/MFIS

IS Normalized MF = .90/.89 = 1.01

Spiked Method Calculating a Simple Matrix Factor


Alternate Approach for Calculating Matrix Factors

AN and ISinjected through the column

2) Inject Solvent Blank

3) Inject Extracted Matrix Blank

LC Column

1) Determine Analyte Profile

Solvent Blank injected with post column infusion of AN and IS

Extracted Blank injected with post column infusion of AN and IS

MRM Signal

AN and IS infused post-column

Analyte = AN Internal Standard = IS


Automating Matrix Factor Calculations: Integrated Intellistart Fluidics


Time0.40 0.60 0.80 1.00 1.20 1.40

%

1 Time1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60

%

1

Matrix Factor Calculation Results (MassLynx 4.1 SCN 737


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Source versatility


A common ion source platform

Designed to be compatible with HPLC, UPLC and GC applications

Designed for performance, usability & serviceability.

XEVO Family Ion Sources


Source versatility

DART DESI

ASAP

APGC TRIZAIC™ Source with nanoTile


Simple and tool-free Allowing you to change between ion source options in minutes.

Universal source architecture Enables the widest range of ionization techniques to be utilized

ESI, APCI, ESCi

Nano-flow, TRIZAIC

APPI, ASAP, APGC

Compatible with third party sources (DESI, DART, LDTD, etc)

Simplicity of Operation & Versatility


Xevo TQ-S

Summary


Xevo TQ-S Key MS Technologies

StepWave — Large increase in sensitivity (all acquisition modes)

— Designed to be robust to sample matrix contamination

RADAR — Targeted MRM at the same time as full scan MS

ScanWave — Enhanced product ion spectra


Xevo TQ-S Designed to Meet Customer Needs

Ultra high sensitivity to make:

— Detection of banned substances easier

— Monitoring of low exposure drugs easier

— Blood spot analysis easier

— It possible to reduce matrix effects by simple sample dilution

— It possible to work with small sample volumes

Information rich supporting data


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Thank You

Xevo TQ-S

Consider Quantitative Assays Previously Beyond Your Reach

xevo tq-s for bioanalysis · ©2010 waters corporation . compound name: oh prog correlation...

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