welcome to our eseminar! - instituto de qu mica...

For audio, dial 88-840-7698 or International 01-703-871-3889Chairperson: Rita Willis

Welcome to our eSeminar!

Presenter: Jerry Zweigenbaum

Jerry joined Agilent Technologies Life Science and Chemical Analysis business in April, 2002 in the LC and LC/MS marketing group. Before that, he worked at Eastman Kodak for 20 years. Five of those years were spent in an environmental group and the other 15 in research anddevelopment. Early in his career, Jerry worked as a forensic chemist for the Cape May County (New Jersey) Prosecutor for five years. He received a bachelors and masters degree in chemistry from Stockton College and Murray State University, respectively. He earned hisdoctorate at Cornell University in Analytical Toxicology under the advisement of his mentor and friend, Dr. Jack Henion. His many years of varied academic and industrial experience have included both separation science and mass spectrometry.


Agilent 1100 LC/MSD Trap


LC/MS/MS with the Ion Trap

• Sensitive FULL SCAN MS/MS and MSn

• Data Dependent Analysis with Auto MSn

• Complete Fragmentation with SmartFrag


Multipole geometry allows fast nonlinear scanning

Multipole Geometry Ion Trap Design

++

++

++

Low amplitude dipole field (1/3 frequency of the main RF)

Conversion Dynode

Electron Multiplier

FocusEndcap

ShutterOctopole

Endcap

RingMultipole ion trap design based on multipolar ion fields and asymmetrical geometry

Main RF

Lenses

Optimized asymptote angle


Linear vs. Nonlinear Oscillators-Go Non-linear!!!

Other Design Agilent Design

Time42 3 5 61

Ampl

itude

(A) 2

1

3

0

4

Ampl

itude

(A)

Time

12

0

1 54 62 3

Linear OscillatorA = f(time): linear

Nonlinear OscillatorA increases hyperbolically with timeEnergy is taken up very fast in resonance,leading to faster scan speedsCan distinguish between smaller massdifferences, leading to higher resolution


Smart Fragmentation (SmartFrag - Patented)

Definition

• The amplitude of the MSn

fragmentation voltage is ramped during fragmentation to providea range of internal energies to the ions.

Benefit

• More likely that each ion will receive exactly the energy it needs to fragment

• Greater product ion generation (similar to triple quad)

• More structural information from fewer stages of MS

• Highly reproducible MSn spectra


Adjustable Fragmentation Width (AFW)Advanced tuning and acquisition features

Definition

• User can set mass window around MSn precursor ion to fragment product ions.

• Unlike other vendor’s instruments, this mass window is user-definable (1 to 300 u) rather than fixed.

Benefit

• Allows simultaneous fragmentation of precursor and any product ions that form from low energy pathways.

• More structural information from fewer stages of MS.

Agilent Advantage


Adjustable Fragmentation Width (AFW): Optimized Generation of MS/MS Ions

Advanced tuning and acquisition features

Without SmartFrag and AFW, MS/MS spectrum

provides little fragmentation information

Add SmartFrag: fragmentation increases; base peak results from

water loss

Add AFW: peak from water loss is fragmented along with 359 parent ion


Data-Dependent Precursor Ion Selection for Auto MSn:List of Features

Data-dependent MSn

• Threshold (absolute and relative)

• N most abundant precursors

• Static include list

• Static exclude list

• Preferred mass list

• Preferred charge state

• Isotopic exclusion

• Active exclusion (repeat count and exclude time)


Agilent ESI-oa-TOF


Agilent Design Optimizes Ion Transmission and Mass Resolving Power

Two Stage IonMirror

5 Stage Vacuum System

Agilent Orthogonal Spray Source(s)

Optically CoupledIon Detector

Beam cooling and guidance

Effective Flight PathLength of 2.0m

Low CTE InnerFlight Tube


Inlets – Electrospray Ion SourceDual sprayer design for sample and lock mass compound

Reference Sprayer

Analytical Sprayer


Inlets – Calibrant Delivery System

A B

#1 #2 #3 #4 #5

TOFTOF Lock Mass

CDS bottles deliver tune compound & lock masses


Reserpine Confirmation

Consistent isotope ratios

14 double bond equivalents (DBE)


LC/API-oa-TOF

• Simple and routine accurate mass measurement

• Seamless lock mass at low concentrationAutomated recalibration on every spectrumSaved Data ALREADY processed

• Identification and Confirmation of Unknowns


Break Number 1

For questions, at break please dial 1 on your phone, or type in the Question Box at any time during the presentation.


Reaction Scheme for Preparation of Flurazepam

N

N

O

N

F

Cl

CH3

CH3

ONH2

Cl

F

ONH

Cl

F

N

CH3

CH3

N

N

CH3

CH3

O

Cl

F

O

CH2Cl

NaN3


LC/MS TIC of Flurazepam Intermediate 2-Amino-5-fluoro-2’-fluorobenzophenone

0 2 4 6 8 10 12Time [min]0.0

0.5

1.0

1.5

5x10

Impurity


123.0

166.0250.0

0.0

0.5

1.0

1.5

2.0

4x10

Intens.

50 100 150 200 250 300 350 400 450 m/z

O

F

NH

Cl

Auto MS/MS of Impurity on Agilent 1100 LC/MSD Ion Trap and Proposed Structure


LC/ESI-ToF MS of 2-Amino-5-chloro-2’fluorobenzophenone impurity

+TOF MS: Period 1, 8.705 to 8.739 min from ACFBP_3.wiff Agilent Max. 2.7e5 counts.

250.0 250.5 251.0 251.5 252.0 252.5 253.0 253.5m/z, amu

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.4e5

2.6e5

Intensity, counts

250.0434

252.0406

251.0473


Table of Possible Empirical Formulas for m/z 250.0434 < 3 ppm Error

Formula Calculated m/z mDa error error (ppm) DBEC10 H13 N2 F Cl2 250.0434 -0.0336 -0.1346 4

C9 H13 N2 O F P Cl 250.0432 0.141 0.564 4C5 H12 N5 F2 Cl2 250.0432 0.1661 0.6642 0.5

C5 H16 N3 O F P2 Cl 250.0435 -0.1715 -0.6858 -0.5C3 H7 N10 O2 Cl 250.0436 -0.2487 -0.9949 5C4 H13 N3 O7 Cl 250.0436 -0.2541 -1.0165 -0.5

C4 H12 N5 O F2 P Cl 250.043 0.3407 1.3629 0.5C6 H16 N3 F P Cl2 250.0437 -0.3461 -1.3845 -0.5C10 H17 O P2 Cl 250.0437 -0.3712 -1.4848 3

C13 H10 N O F Cl 250.0429 0.4535 1.8138 8.5C11 H17 P Cl2 250.0439 -0.5459 -2.1834 3

C8 H9 N4 O F2 Cl 250.0427 0.6533 2.6128 5C10 H11 N O2 F2 Cl 250.044 -0.6893 -2.757 4.5

H10 N11 O Cl2 250.0441 -0.736 -2.9435 0.5


Break Number 1

For questions, at break please dial 1 on your phone, or type in the Question Box at any time during the presentation.


Reaction Scheme for Preparation of Enalapril

CDI

ONHONa

NH

O

O

CH3

OH

O

CH3N

N

NH

O

O

CH3

O

O

O

CH3

N

NH

O

O

CH3

O

O

O

O

ONaCH3


LC/MS TIC of Enalaprile IntermediateN-(1-ethoxycarbonyl-3-phenyl propyl) alanine

0 2 4 6 8 10 Time [min]0.0

0.5

1.0

1.5

6x10Intens.

Impurity


Auto MS/MS of Impurity on Agilent 1100 LC/MSD Ion Trap and Proposed Structure

117.1

134.1 160.1174.0

206.1

220.0

All MSn, 4.1-4.2min (#337-#348), Background Subtracted

0

1

2

3

4x10Intens.

50 100 150 200 250 300 350 400 450 m/z

NH

O

O

OH

O

CH3

CH3


LC-ESI/ToF MS of Impurity in N-(1-ethoxycarbonyl-3-phenyl propyl) alanine

+TOF MS: Period 1, 4.236 to 4.322 min from EPPA_3.wiff Agilent Max. 1.4e5 counts.

170 175 180 185 190 195 200 205 210 215 220 225 230 235 240 245 250 255 260 265 270m/z, amu

0.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

7.0e4

8.0e4

9.0e4

1.0e5

1.1e5

1.2e5

1.3e51.4e5

Intensity,counts

266.1391

174.0754

267.1423206.1165220.1324175.0791


Accurate mass measurement of [M+H]+ and Fragment Ions of Impurity

Formula calculated

m/z error (mDa) error (ppm) DBEStructure

C14 H20 N O4

266.1386

0.4152

1.5601

5.5

NH

O

O

OH

O

CH3

CH3

C13 H18 N O2

220.1332

-0.8054

-3.6588

5.5

NH

O

O

CH3

CH3

C12 H16 N O2 206.1175 -1.0553 -5.1201 5.5

NH

OH

O

CH3

C7 H12 N O4

174.076

-0.6844

-3.932

2.5

CH 3

N H

O

O

O H

O

C H 3

C H 3


Accurate Mass Difference –Precursor to Product Ion The POWER of Neutral Loss

Product Ion

Neutral Loss

Empirical Formula

error (ppm)

number of possibilities Loss of

220.1332 46.0054 CH2O2 -1.725 1 Formic acid

206.1175 60.0211 C2H4O2 -0.4904 1 Methyl formate

174.076 92.0626 C7H8 -0.0034 1 Toluene


Conclusions

The 1100 LC/MSD Trap provides --

• Sensitive Full Scan MS/MS that can lead to proposed structures for trace impurities

• MS/MS can be automated and with ramped energies only need be analyzed once

• The 1100 LC/MSD TOF provides accurate mass measurement for identification and confirmation of unknowns.

• Separately they both provide powerful tools for identification of unknowns -- together the power is staggering!

welcome to our eseminar! - instituto de qu mica...

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