lc/ms Übersicht agilent technologies · •1290 infinity uhplc •12x0 infinity hplc •7100...

LC/MS Übersicht

Agilent Technologies

Friedbert Ruppel

LC / LCMS Sales Berlin

A G I L E N T 3 9 J A H R E M A S S E N S P E K T R O M E T R I E

5930A

1971 5992A 1976 5970B MSD

1986

5971 - 5975

1988 - heute

LC/MSD Series

1997-2005

Ion Trap Series

2000-heute

ESI-TOF Series

2003-heute

4500, 7500 Series ICP/MS

1994-Present

6000 Series

2006-heute

6100 Single Quad

2006, 2007

6410 Triple Quad

2006, 2007

6510, 6520 Q-TOF

2006, 2007

A G E N D A

Einführung

Hardware

A. Ionenquellen

B. 6100 Single Quad Familie

C. 6200 TOF Familie

D. 6400 Triple Quad Familie

E. 6500 QTOF Familie

Software

MassHunter Plattform und

Applikations Software

• 1290 Infinity UHPLC

• 12x0 Infinity HPLC

• 7100 Capillary Electrophoresis

• HPLC Chip Cube System

Separations

Technology

MS Detector

Technology

$0.3 B 34% of total revenue

Data Analysis /

Application

Software

Solutions

T E C H N O L O G I E N U N D L Ö S U N G E N

• 6000 Series LC/MS/MS

• 7000 Series GC/MS/MS

• 597X GC/MSD

• Varian Ion Trap LC/MS and GC/MS

• MassHunter MS Software Family

• Mass Profiler Professional w/ Pathway Analysis

• Personal Compound AMRT Databases and Libraries

• Pharma Workflows; ADME, Bioanalysis, Confirmation

• Fast Track Solution Kits for LC, GC, LC/MS & GC/MS

• Clinical Application Offer

• More to come

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W O M I T A L L E S B E G I N N T: I O N E N Q U E L L E N

• Größte Flexibilität

– Electrospray (ESI)

– Nanospray

– APCI

– APPI

• Multimode Source

– ESI und APCI in einer Quelle

– Simultaner Operationsmodus

•

• HPLC-Chip/MS

– Nanospray wird routinefähig

– Reproduzierbare Ergebnise

– Probenvorbereitung auf dem Chip

• Austauschbar zwischen allen MS Techniken

A G I L E N T J E T S T R E A M

Off On

A G I L E N T J E T S T R E A M T H E R M A L G R A D I E N T F O C U S I N G T E C H N O L O G Y

Dramtischer Empfindlichkeits Gewinn für SQ, TOF, Q-TOF, und Triple Quad

Thermische Ionenfokussierung

Verbesserung der Ionisierung und der Ionenextraktion

Effizient für viele Analytenklassen, auch für typische APCI

A G E N D A

Einführung

Hardware

A. Ionenquellen


C. 6200 TOF Familie



Software



S Q T E C H N O L O G I E

skimmer

octopole ion guide

0.6 mm I.D.

dielectric

capillary

+ + + +

+ + + +

+

1 x 10-5

Torr

3 Torr

quadrupole

HED detector

lenses

HPLC , CE

or IC inlet

Nebulizer

Nebulizer

gas inlet

heated

N2

M O D E L L E D E R A G I L E N T 6 1 0 0 B S E R I E

• Höchste Produktivität

• Ideal für 1290 Infinity

(U)HPLC Kopplung

• Ideal für Screening und

qualitativen Applicationen

• Höchste Flexibilität

• Ideal für quantitative

Anwendungen

• Einfach zu Nutzen

• Ideal für

Chromatographeur

• Preisgünstig

• Massenbasierende

Fraktionierung

6120B 6130B 6150B

S P E Z I F I K AT I O N E N D E R A G I L E N T 6 1 0 0 S I N G L E Q U A D S

A G E N D A

Einführung

Hardware

A. Ionenquellen


C. 6200 TOF Familie



Software



A G I L E N T 6 2 0 0 S E R I E : T O F L C / M S

DC Quad

Octopole 2

Ion Pulser

Ion Mirror

Detector

Turbo

Massengenauigkeit besser 1 ppm

>20,000 Massenauflösung

105 Dynamischer Bereich

Bis zu 20 Spektren pro Sekunde

W A S M A C H T T O F A U S : M A S S E N G E N A U I G K E I T

Resolution = (m/z)/w1/2

1000/0.1 = 10,000

922/0.087 = 10,597

Mass accuracy (mass error)

(1000.001-1000.000)/1000)x106=1 ppm

Therefore: 1 ppm @ 1000 m/z is 0.001Da (1 mDa)

921.5 922.0 922.5 923.0 923.5 924.0 924.5 m/z, amu

923.0121

W1/2 = 0.087

+TOF MS: 5.474 to 5.547 min from steroid2TOFap42.wiff Agilent, subtracted (4.696 to ... Max. 1.2e5 counts.

120 140 160 180 200 220 240 260 280 300 320 340 360 380 400m/z, amu

0.00

1.00e4

2.00e4

3.00e4

4.00e4

5.00e4

6.00e4

7.00e4

8.00e4

9.00e4

1.00e5

1.10e5

1.16e5

In

te

ns

ity

, c

ou

nts

289.2159

271.2051

269.1893

287.1997

Epitestosterone

Resolution 6890

meas = 289.2159

actual = 289.2162

-1.06 ppm

922.0092

6 10

actual

actual - measured accuracy Mass

x

W A R U M S O L C H E G E N A U I G K E I T ?

Reserpine (C33H40N2O9) exakte Masse: 609.28066 m/z

Single Quad Genauigkeit: +/- 0.1 m/z = 165 ppm

Anzahl möglicher Summenformeln (nur C, H, O & N)

• 165 ppm 209

• 10 ppm 13

• 5 ppm 7

• 3 ppm 4

• 2 ppm 2

Die akkurate Masse reduziert die Anzahl der Kandidaten

und damit das Risiko weiter in ein falsches Molekül zu

investieren.

Tetraconazole

Azoxystrobin (fragment)

I S O B A R I C C O E L U T I N G S P E C I E S : Identify By Differences In Isotopic Pattern

M O L E C U L A R F O R M U L A G E N E R AT I O N

Proposed Formulas

are scored based on

mass error, isotope

abundances and

isotope spacing

User has control over

relative weighting

In this case, best

match for both adducts

is the same

C S V F O R M AT D ATA B A S E – D R U G S O F A B U S E

P E R S O N A L F O R E N S I C S / T O X D B B . 0 2 . 0 1

Version B.02.01

• Accurate mass database of 7500 compounds with 5000 structures

• Drugs of Abuse, Explosives, Clinical Drugs (e.g. Benzodiazepines, local

anesthetics), other toxic compounds (mycotoxins, plant toxins)

• Searchable from MassHunter Qual, Mass Profiler, Mass Profiler Professional

Select

algorithms

Turn Sample

Purity on Monoisotopic mass

(varies in ppm)

Isotope spacing

(varies in ppm)

Isotope distribution

(varies in %)

Scoring based on

1-naphthol

Mass [M+H]+ = 145.064891

Resolution = 24909

0.06 ppm

Fenpyroximate E

Mass [M+H]+ = 422.208059

Resolution = 38636

-0.25ppm

1290/6540 90sec analysis

250 compounds (125pg on-column)

Spinosad A

Mass [M+H]+ = 732.468714

Resolution = 47094

-0.77ppm

A G E N D A

Einführung

Hardware

A. Ionenquellen


C. 6200 TOF Familie



Software



M R M ( M U LT I P L E R E A C T I O N M O N I T O R I N G )

170 210 250 290

210

222

268 280 165

Quad Mass Filter (Q3) Quad Mass Filter (Q1)

Collision Cell

Spectrum with

background

ions (from ESI)

Q1 lets only

target ion 210

pass through

190 210

210

Collision cell

breaks ion 210

apart

150 170 190 210

210 158

191

Q3 monitors only

characteristic

fragments 158

and 191 from ion

210 for quant and

qual.

160

158

190

191

no chemical background

6490 Triple Quadrupole LC/MS System

with iFunnel Technology

23

N E U : 6 4 9 0 T R I P L E Q U A D R U P O L E L C / M S

Zeptomole Emfindlichkeit!

iFunnel Technology • A new paradigm for atmospheric ion sampling

• 10X sensitivity breakthrough for ESI

• Improves assay robustness; simplifies sample prep

Curved, Tapered Collision Cell

• Efficient ion transmission; Reduced noise

• Compact design

New scanning capabilities

• 0.4 m/z precursor isolation reduces interference

• Dynamic MRM

• Data dependent scanning

Agilent Jet Stream

• Thermische Fokusierung des Elektrospray

• Effektivere Desolvati-sierung der Ionen in der Gasphase

• Erzeugt Zone mit vielen Ionen

24

N E W I F U N N E L T E C H N O L O G Y

Hexabore Capillary

• 6 capillary inlets

• 10fache Anzahl der Ionen können transferiert werden

Dual Ion Funnel

• Ionen werden gehalten, Restgas abgepumpt

• Reduktion des Rauschens

• Schont die Turbopumpe

iFunnel Technology nutzt 10fach mehr Ionen

25

B E I S P I E L D E R V E R B E S S E R U N G

Agilent Jet Stream

positive ion

200 400 600 800 1000 1200 1400

m/z

10x

14x

11x

10x 7x

113

302 1034

200 400 600 800 1000 1200 1400

1222

922 622

322

118

m/z

5x

5x

7x

7x

7x 6490

6460

Agilent Jet Stream

negative ion

6 4 9 0 E R R E I C H T Z E P T O M O L A R D E T E K T I O N S L I M I T S

200 zeptomoles

(100 attograms)

Verapamil

Injected on-column

Acquisition Time (min)

+ MRM (455.3 -> 164.9)

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

1 x10

4.2

4.3

4.4

4.5

Mol = 6,02 x 1023 Moleküle

Nano = 10-9

Pico = 10-12

Femto = 10-15

Atto = 10-18

Zepto = 10-21

Ein Zeptomol (zmol = 10-21) sind 602 Moleküle.

U N P R E C E D E N T E D S I X L O G S O F L I N E A R I T Y W I T H 6 4 9 0

Y = 65283 x – 0.390412

R2 = 0.9976

Femtograms

Are

a R

esp

on

se

1

10

100

1000

10000

100000

1000000

10000000

0.1 1 10 100 1000 10000 100000

100 Attogram to 100 Picogram verapmil on column

6 4 9 0 T R I P L E Q U A D S P E Z I F I K AT I O N E N

Specification 6460 6490

S/N 1 pg reserpine 1,000:1 10,000:1

Mass Range (m/z) 3,000 1,400

Mass Resolution 0.7 Da 0.4 Da

Scan Speed, Da/s 5,000 10,000

Polarity Switching 30 msec 30 msec

MRMs/Sec

no cross-talk 200 300

Data Dependent YES YES

Triggered MRM

YES

YES

A G E N D A

Einführung

Hardware

A. Ionenquellen


C. 6200 TOF Familie



Software



A G I L E N T 6 5 0 0 Q T O F T E C H N I K

Ion Pulser

Turbo 2

Octopole 1

DC Quad

Rough Pump

Turbo 1 Turbo 1 Turbo 1

Quad Mass Filter (Q1)

Collision Cell Lens 1 and 2

Octopole 2

A G I L E N T Q - T O F F A M I L I E

6530AA Q-TOF

20K resolution

1 pg sensitivity

5 decades in-scan

6520BA Q-TOF

20K resolution

5 Pg Sensitivity

5 decades in-scan

6540AA Q-TOF

< 40K resolution

2 pg sensitivity

5 decades in-scan

< 0,5 ppm MS

< 2 ppm MS/MS

6 5 4 0 – L E I S T U N G S FÄ H I G K E I T

+ESI EIC(609.28066)

Scan Frag=240.0V Reserpine_40pgms3.d

0.511

Counts vs. Acquisition Time (min)

0.0 0.25 0.5 0.75 1

5 x10

0

0.2

0.4

0.6

0.8

1

1.2 609.28081

610.28425

611.28650

612.28987

Counts vs. Mass-to-Charge (m/z)

609 610 611 612 613 614

613.29210

Isotope Obs % Calc % Obs m/z Calc m/z Diff (ppm)

1 100 100 609.28081 609.28066 0.25

2 35.87 37.24 610.28425 610.28393 0.52

3 8.58 8.59 611.28650 611.28671 -0.34

4 1.41 1.48 612.28987 612.28941 0.75

5 0.14 0.21 613.29210 613.29203 0.11

Run

Error (ppm)

1 0.96

2 -0.17

3 0.25

4 0.02

5 0.39

6 0.13

7 0.01

8 0.52

9 0.04

10 0.30

Mean 0.25

Std. Dev 0.32

40pg reserpine on-column, 10 injections

250 ppb mass accuracy with and accurate isotopic ratios

Exzellente Massengenauigkeit und “Isotopic Fidelity”

M A S S H U N T E R M A S S P R O F I L E R Differential analysis for two samples or two sample groups

• Support for METLIN database with 8000 lipids for lipidomics

• MFG performance improvements (x10) and absolute scoring

• Compatibility with Mass Profiler Professional and MassHunter Qual

=> Very well accepted easy-to-use entry level software

Features increased

by x fold

Features

decreased by x fold

M A S S P R O F I L E R P R O F E S S I O N A L B . 0 2 . 0 0 NEW optional integrated Pathway Analysis software

=> Once markers are ID’d, determine the biological context!

Agilent is only MS vendor with integrated PA software!

Metabolomics

A G E N D A

Einführung

Hardware

A. Ionenquellen


C. 6200 TOF Familie



Software



M A S S H U N T E R W O R K S TAT I O N

• Control of Agilent 6000 Series QQQ, TOF and Q-TOF

• Control of most 1100, 1200 and 1290 HPLC modules incl. Chip-LCMS

• LC/MS Data Processing for QQQ, TOF, Q-TOF, SQ and Trap

• Control for GC/MS/MS & data processing for GC/MS and GC/MS/MS

• Control & data processing for ICP/MS

LC/MS GC/MS ICP-MS

Eine Softwareplattform für alle Agilent Massenspektrometer

M A S S H U N T E R W O R K S TAT I O N

• Workflows werden durch Applikationssoftware abgebildet

Die z.Z. beliebteste und neueste MS Software am Markt

• ~ 5000 Kopien seit Sep-2006 !

• Unterstützt LC/MS, GC/MS + ICP-MS

• Innovative Algorithmen für höchste Produktivität

C:/Documents and Settings/jfjeldsted/Trident/Triple (QqQ) Acquisition Method.wrf

Ü B E R S I C H T D E R A G I L E N T L C - M S S Y S T E M E

SQ TOF QQQ Q-TOF

Anwendung Synthesekontrolle

Reinheitskontrolle

Massenbasierte

Fraktionierung

Quantifizierung UV

inaktiver Substanzen

Studium und

Ausbildung

General Unknown

Screening

Synthesekontrolle

Biomarker Discovery

Forensik

Strukturaufklärung

Quantifizierung

Strukturaufklärung

General Unknown

Screening

Biomarker Discovery

Metabolomics

Forensik

Vorteile Einfach

Preis

einfache

Methodenentwicklung

(generischer Gradient)

alle Informationen sind in

den Daten (Forensik /

Doping / Pharmaforschung)

Empfindlichkeit

einfach in der Routine

einfache

Methodenentwicklung

alle Informationen sind in

den Daten (Forensik /

Doping /

Pharmaforschung)

umfassende

Softwarepakete (Profiler /

Metlin / Metabolid ID)

Nachteile geringere Empfindlichkeit

als QQQ

große Datenmengen

Methodenentwicklung

aufwendig

Nur Targetanalyse

geringere Empfindlichkeit

als QQQ

große Datenmengen