lunch & learn at imsc 2009 - agilent imsc... · agilent’s new 6540 ultra high definition...

Welcome to Agilent’s

Lunch & Learn at IMSC 2009

Agilent Lunch & Learn

Monday 13:00-14:00

Introducing the new Agilent 6540 and

6538 Ultra High Definition (UHD)

Accurate-Mass Q-TOF LC/MS Systems

• 1290 Infinity UHPLC

• Fast, Accurate, High Resolution Q-

TOF

• Using Peak Capacity to Improve

Screening and Complex Sample

Profiling

Ken Imatani & Pat Perkins, Agilent, CA

Agilent Hospitality Suite Monday

Time: 18:15-21:00

Room: Danzig

Agilent Elemental BingoStarts at 19:00

Great prizes to be won!

Drinks and Snacks will be

provided.

Enter & WinWe will be running daily competitions during IMSC

2009, ask a member of staff for more details!

Happy HourDaily from 16:00-18:00

at the Agilent booth (B)

Ultra High Definition

Optimizing all Analytical Dimensions

Signal Response

Mass Spectrum

• Sensitivity

• Dynamic Range

• Linearity

• Mass Accuracy

• Resolving Power

• Acquisition Rate

• Separation Speed

• Peak Capacity

August 2009

IMSC 2009 Infinity LC 6540 QTOF lunch and learn

Agilent’s New 6540 Ultra High Definition

Accurate Mass Q-TOF

• 500 ppb mass accuracy

• femtogram sensitivity

• 5 decades dynamic range

• 40,000 resolving power

• 20 Spectra/sec

• Excellent Linearity and Isotopic

Fidelity

• Supports Agilent Jet Stream and

HPLC-Chip

Exceptional accurate mass, sensitivity,

dynamic range and resolution … perfect

match for 1290 Infinity UHPLC

August 2009


Ion Beam Compression (IBC)* Technology

Drives Higher Resolution

Up to 10-fold ion beam compression

Exit from collision cell

Into slicer and pulser region

Narrowed beam slit enables mass resolution

of 40K

Compressed and cooled ion beam ensures the best sensitivity

performance in high resolution mode

* Patent pending

August 2009


6540

UHD Q-

TOF

6530

Q-TOF

• 1ppm/C Expansion Coefficient for Inner

Flight Tube virtually eliminates calibration

drift due to flight tube elongation.

• 2nd Order Temporal Focusing Ion Mirror

uses high transmission Harp Grid for

maximum sensitivity

Enhanced Ion Flight Tube and Mirror TechnologyStable, Sensitive, High Resolution

DC Quad

Octopole 2

Ion Pulser

Ion Mirror

Detector

Turbo

August 2009


Next Generation – Ultra High Speed Detector


New Bipolar TOF Detector

• New ultra fast and high efficiency scintillator

• New ultra fast response PMT design continues the tradition of high dynamic range and detector lifetime

• Developed by Photonis with Agilent TOF Technology

• Specifically enhances Resolution in 2Ghz Ext. Dynamic Range Mode

Single Ion Response

~800 psec FWHM

2 nsec/div

August 2009

6540 Ultra High Definition QTOF

Maintaining Resolving Power – Across the Mass Range

5x10

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

200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800

922

R=42424622

R=38702

1221

R=43674

1521

R=42750322

R=302182121

R=418251821

R=404052421

R=39332 2721

R=37207

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

Scan Rate Independent


622.00822

623.01059

624.01219 625.01471

m/z 622 and isotopes

August 2009



Mass Accuracy – Repetitive Injections

+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

5x10

0

0.2

0.4

0.6

0.8

1

1.2609.28081

610.28425

611.28650

612.28987


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 calibration and very accurate isotopic ratios

August 2009



Sensitivity – Full Scan MS Mode- 1 picogram

3x10

0

1

2609.280347

(M+H)+

610.283168

611.287790

612.292649


607 608 609 610 611 612 613 614 615 616

4x10

0

1

2

Counts vs. Acquisition Time (min)

0.0 0.5 1 1.5

S/N = 319 RMS

Ratio m/z Diff.Theor. Expt. Theor. Expt. (ppm)100.00 100.00 609.2807 609.2803 -0.5137.24 36.36 610.2839 610.2832 -1.258.59 8.39 611.2867 611.2878 1.771.48 1.45 612.2894 612.2926 5.29

Resolution ~ 33,000

Excellent sensitivity, accuracy and resolution at 1 pg on column

August 2009


Topics

• Example: Pesticides in Foods

• Example: Drug metabolite identification

• Example: Peptide Mapping

August 2009


Pesticides Screening in Foods

• Accurate-Mass Databases for Comprehensive Screening of Pesticide Residues in Food by Fast

Liquid Chromatography Time-of-Flight Mass Spectrometry, Milagros Mezcua, Octavio Malato, Juan F.

García-Reyes, Antonio Molina-Díaz, and Amadeo R. Fernández-Alba, Anal. Chem., 2009, 81 (3), 913-929

DOI: 10.1021/ac801411t

• Method for screening 297 pesticides in foods with Agilent 1100 HPLC, 4.6 x 50 mm XDB-C18, 1.8 µm

column, and TOF-MS detection

• Component identification using accurate mass/retention time (AMRT) matching against a database

• 0.6 mL/min flow rate, 12 minute gradient, approximately 23 minutes run time

• Comparable method performance when tested on 60 commercial produce samples (QuEChERS protocol

sample prep) and compared with results from QQQ [note: 45/60 samples tested positive!]

• Identified several pairs of “isobaric coeluting species” (ICS) which have similar retention times and

accurate mass values for their adduct ions or fragments

August 2009


Focused AMRT Databases

(Personal Compound Databases—PCD)

• Pesticides, Part # (G6854AA):

• 1600 analyte content (formulae & accurate mass)

• Structures

• CAS Numbers & Links to external website (pubchem)

• Forensic & Toxicology, Part # (G6855AA):

• ~7000 analytes content (formulae & accurate mass)

• Structures

• CAS Numbers & Links to external website (pubchem)

August 2009


Starting Conditions for Pesticides Analysis

• Configuration

• 1290 Infinity UHPLC, 6540 QTOF with Agilent jet stream technology

• Eclipse Plus C18 column, 2.1 x 50 mm or 100 mm, 1.8 µm

• Analysis conditions

• A = 0.1% formic acid in water, B = 0.1% formic acid in ACN; various flow

rates (1.2, 1.0, 0.5 mL/min); 60 ºC column temperature

• Gradient = 5% B to 95% B in various times (1.5, 3, 7 , 8.5, 17 minutes)

• 3200 m/z maximum mass, high resolution mode

• MS mode: 100-1000 m/z, various acquisition rates (10, 5, 3, 1

scans/second)

August 2009


RRLCUHPLC

August 2009

0.5 mL/min 2.1 x 100 mm column,

60 ºC, max pressure 410 bar








Mass Accuracy and Mass Resolution and Analysis

Speed Simultaneously


August 2009

-0.60 ppm

R = 30,600-0.37 ppm

R = 31,400

O

O

O

NN

O

N

OCH3 CH3

azoxystrobin

1 spectrum/second

10 spectra/second

Mass Accuracy is Invariant with Acquisition Rate

Pesticides Example

August 2009

-2.00

-1.50

-1.00

-0.50

0.00

0.50

1.00

1.50

2.00

0 1 2 3 4 5 6 7 8 9 10 11 12

Mass E

rro

r (p

pm

)

Acquisition Rate (scans/second)

azoxystrobin 404 m/z

cyproconazole 292 m/z

cyprodinil 226 m/z

diazinon 305 m/z

linuron 249 m/z

paclobutrazol 294 m/z

propazine 229 m/z

tetraconazole 372 m/z

tricyclazole 190 m/z


MS Resolution is Invariant with Acquisition Rate

Pesticides Example

August 2009

0

5000

10000

15000

20000

25000

30000

35000

0 1 2 3 4 5 6 7 8 9 10 11 12

Reso

luti

on

Acquisition Rate (scans/second)

azoxystrobin 404 m/z

cyproconazole 292 m/z

cyprodinil 226 m/z

diazinon 305 m/z

linuron 249 m/z

paclobutrazol 294 m/z

propazine 229 m/z

tetraconazole 372 m/z

tricyclazole 190 m/z


Methanol Mobile Phase for Pesticides Analysis

• Configuration


• Eclipse Plus C18 column, 2.1 x 50 mm, 1.8 µm


• A = 2 mM ammonium formate, 0.01% formic acid in water, B = 2 mM

ammonium formate, 0.01% formic acid in methanol; flow rate 0.8 mL/min,

40 ºC column temperature

• Gradient = 5% B to 95% B in 3 min, stop time 4 min (standard) or 10 min

(sample with matrix)

• 1700 m/z maximum mass, high resolution mode

• MS mode: 100-1000 m/z, 3 scans/second)

August 2009


Chamberlain, P., Hall, T., Zavitsanos, P. Agilent Technologies application note 5989-1688EN, January 2005.

Better Response with Methanol Mobile Phase


August 2009

0

100000

200000

300000

400000

500000

600000

700000

800000

Res

po

nse

(are

a c

ou

nts

)

MeOH gradient, 2 mM ammonium formate, 0.01% formic acid ACN gradient, 0.1% formic acid

Chromatographic Observations using Standards

Same Instrument and Column

August 2009


3.0642.726

Acquisition Time (min)

1.8 2 2.2 2.4 2.6 2.8 3 3.2 3.4 3.6 3.8 4 4.2

2 mM ammonium

formate + 0.01%

formic acid

pH ~ 3.5

2 mM ammonium

acetate

pH ~ 5.0

2 mM ammonium

acetate + 0.02%

acetic acid

pH ~ 4.0

Thiabendazole, pKa 4.73

N

NH

N

S

4.564

4.4743.543

Acquisition Time (min)

3.4 3.6 3.8 4 4.2 4.4 4.6 4.8

OH

CH3

N

NN

Cl

HOH

H

N

NN

Cl

CH3

Cyproconazole

ACN gradient

0.1% formic acid

Methanol gradient

2 mM ammonium

formate + 0.01%

formic acid

37Cl isotopomer

Isobaric Co-eluting Species

Example: 63 ppm Mass Difference (18 mDa)


August 2009

Isobaric Co-eluting Pair m/z ppm

Difference

Differentiate by

RT No fragment m/z

Tricyclazole

Propazine

190.0433

190.0667

123 X

(1.3 min)

X

Methiocarb

Diethofencarb fragment

226.0896

226.1074

79 X X

Diethofencarb fragment

Cyprodinil

226.1074

226.1339

117 X

(0.53 min)

X X

Cyproconazole -37Cl

Paclobutrazol

294.1182

294.1368

63 X

Clofentezine-37Cl

Diazinon

305.0618

305.1083

300 X

Chlorfenvinphos

Triflumuron

358.9768

359.0405

177 X

Tetraconazole

Azoxystrobin fragment

372.0288

372.0979

186 X X

294.13690

294.11840

Mass-to-Charge (m/z)

294.08294.10

294.12294.14

294.16294.18

Paclobutrazol

0.44 ppm

R=29,600

Cyproconazole-37

Cl

0.66 ppm

R=30,500

Identify These Pesticides By Isotopic Pattern And

Mass

cyproconazole

paclobutrazol

August 2009


Pesticides in Pepper Matrix, 10 ppb

3 Scans/sec Acquisition Rate


August 2009

163.05348(M+H)+

164.05652(M+H)+ 165.04954

(M+H)+

163 164 165

Methomyl

-0.90 ppm

R =24,200

258.05655

(M+H)+

257.06237

(M+H)+259.05936

(M+H)+

(M+H)+

256 257 258 259 260

256.05959(M+H)+

260.06128

Imidacloprid

-0.06 ppm

R =28,900

(M+H)+

269.15754(M+H)+

(M+H)+

268 269 270

268.15425

270.13975

Diethofencarb

-0.33 ppm

R =29,100

(M+H)+

361.03783(M+H)+

360.04448(M+H)+

362.04095(M+H)+

(M+H)+

359 360 361 362 363

363.03121

359.04026

Triflumuron

-0.60 ppm

R =31,700

489.04371(M+H)+

491.04126(M+H)+

490.04738(M+H)+ 492.04627

(M+H)+

489 490 491 492

Flufenoxuron

-0.34 ppm

R =34,500

2.872 3.162

1.048

1.2131.232

1.318

1.331

1.5221.897

2.069

2.233

2.233 2.470

2.483

2.589

2.661

2.780

2.8392.8651.700

Acquisition Time (min)1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3 3.1 3.2 3.3

2.549

2.865

2.464

2.4642.457

Acquisition Time (min)1 2 3 4 5 6 7 8 9

TIC

Matrix Suppression Effect

Response of Standard = 1


August 2009

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.80

Atr

azin

e

Azo

xys

tro

bin

Ca

rba

ryl

Ca

rbe

nd

azim

Ca

rbo

fura

n

Ch

lorf

en

vin

ph

os

Clo

fen

tezin

e

Cyp

roc

on

azo

le

Cyp

rod

inil

De

iso

pro

pyla

tra

zin

e

Dia

zin

on

Die

tho

fen

ca

rb

Flu

dio

xo

nil

Flu

fen

ox

uro

n

Ima

za

lil

Ima

za

pyr

Imid

ac

lop

rid

Lin

uro

n

Me

thio

ca

rb

Me

tho

myl

Mo

lin

ate

Mo

nu

ron

Pa

clo

bu

tra

zo

l

Pro

ch

lora

z

Pro

pa

zin

e

Te

tra

co

na

zo

le

Th

iab

en

da

zo

le

Tri

cycla

zo

le

Tri

flu

mu

ronR

es

po

ns

e r

ela

tive

to

sta

nd

ard

spiked pepper matrix spiked tap water matrix

Metabolite ID

August 2009


Experimental Conditions for Verapamil Metabolite

ID Analysis

• Configuration


• Eclipse Plus C18 column, 2.1 x 50 mm, 1.8 µm


• A = 0.1% formic acid in water, B = 0.1% formic acid in ACN; 1.2 mL/min;

60 ºC column temperature

• Gradient = 5% B to 95% B in 1.0 minute, stoprun 1.5 minutes, postrun

0.5 minutes

• 1700 m/z maximum mass, extended dynamic range mode

• MS mode: 100-1000 m/z, 3 scans/second

August 2009


Verapamil: Major and Minor Metabolites (Phase I)

August 2009

desmethyl

Isopropyl

dealkylation

monohydroxy

dihydroxy

TICverapamil


6x10

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

2.6

2.8 455.290321

(M+H)+

441.274704

471.284287487.279822

(M+H)+

Counts vs. Mass-to-Charge (m/z)435 440 445 450 455 460 465 470 475 480 485 490 495 500 505

Coeluting Metabolites with Parent Drug: Need

Wide Dynamic RangeFive decades of response in a

single scan

2.68 million

counts

25 counts

verapamil dihydroxy metabolite of verapamil

desmethyl

metabolite

monohydroxy

metabolite

400

counts

August 2009


Metabolite ID Results

August 2009


Rapid Peptide Mapping

August 2009


6530 vs. 6540: Comparison of Peptide Mass

Spectra

CCTKPESERMPCTEDYLSLILNR

+4

+4

+3

+3

+2

+2

6530 Q-TOF

6540 Q-TOF

6530 Q-TOF

6540 Q-TOF

6530 Q-TOF

6540 Q-TOF

August 2009


Peptide Mapping: Serotransferrin

Peak width 1.1 sec

27 data points

0.68 ppm

August 2009


Summary

• Unmatched chromatographic peak capacity and resolving power—can

even resolve diastereomers when parameters are adjusted appropriately

• Superb MS mass accuracy and resolution—invariant with acquisition rate

• Sophisticated identification software and algorithms—handle complex

samples, overlapping isotopic patterns

• Sum (product?) is greater than the parts

• Run faster chromatography for higher throughput

– MS can keep up without sacrificing resolution

– MS can keep up without sacrificing mass accuracy

– MS has the dynamic range to handle coeluting peaks

– Software has the power to identify components of complex

chromatograms and spectra

– Result: more information and results per analysis and per unit time

lunch & learn at imsc 2009 - agilent imsc... · agilent’s new 6540 ultra high definition...

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