introduction to lc-ms to lc-ms dr. michael baynham senior specialist, mass spectrometry applications...

Introduction to LC-MS

Dr. Michael Baynham Senior Specialist, Mass Spectrometry ApplicationsClinical Research Network Coordinator

2 24th April 2006

Applied Biosystems — Advancing Quality Science

© 2006 Applied Biosystems

Overview

● Comparison of LC-MS with more traditional techniques such as LC with DAD detection, GC-MS & immunoassay analysis

● What is the difference between LC-MS and LC-MS/MS?● LC-MS/MS system technology● LC-MS/MS source technology● What effects the way a compound forms an ion?● Summary

Comparison of LC-MS with Traditional Techniques

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Diode Array Detection- not specific enough for differentiation

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Mass Spectrometry Specificity

324308

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Comparison of LC-MS with GC-MS

● GC-MS requires compounds to be volatile to be ionised– traditionally electron impact source is used.

● LC-MS can be used to detect compounds from poly-aromatic (non-polar) to peptide and proteins.

● GC-MS is still able to detect long chain aliphatic compounds (petroleum based anlaytes) and very low mass volatile material better than LC-MS.

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Comparison of LC-MS/MS with Immunoassay

● LC-MS/MS is more specific than immunoassay – LC-MS/MS detection is based on column retention time,

parent mass and structure. Therefore it is compound specific.

● Furthermore, derivitisation is not normally needed to detect the target compound, saving analysis time and money.– Immunoassay is normally based on compound class and

will not be able to determine which compound in a group is detected.

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Comparison of LC-MS/MS with Immunoassay

● LC-MS/MS is generally more sensitive than immunoassay● LC-MS/MS can screen for more than one compound class

with one injection ● Immunoassay measures one compound class per assay

and therefore needs more sample when compound class screening

● Immunoassay is generally more rapid than LC-MS/MS

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General reasons to look at LC-MS(/MS)

● Multi-component analysis– LC-MS(/MS) can detect over 300 compounds of different

classes in just one run using often a low volume of sample– Reduces sample analysis time and allows for multiple

analyses to be done on low volumes of samples with minimal sample pre-treatment

What is the difference between LC-MS and LC-MS/MS?

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LC-MS (first stage of MS/MS)

+ P r e c ( 1 2 2 .1 0 ) : E x p 1 , 6 .8 6 1 m in f r o m S a m p le 1 ( B u s p i r o n e M e ta b o l i t e s ) o f B u s p ir o n e _ P r e 1 2 2 _ 0 1 . . . M a x . 1 .1 e 4 c p s .

2 6 0 2 8 0 3 0 0 3 2 0 3 4 0 3 6 0 3 8 0 4 0 0 4 2 0 4 4 0 4 6 0 4 8 0 5 0 0 5 2 0 5 4 0 5 6 0 5 8 0 6 0 0 6 2 0 6 4 0m /z , a m u

5 %

1 0 %

1 5 %

2 0 %

2 5 %

3 0 %

3 5 %

4 0 %

4 5 %

5 0 %

5 5 %

6 0 %

6 5 %

7 0 %

7 5 %

8 0 %

8 5 %

9 0 %

9 5 %

1 0 0 %

402

Many compounds

enter the MS as ions

Compounds filtered based on

their mass/charge ratio and detected.

Single masses selected when performing

quantitation.

Compounds Ionised

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Many analytesenter the MS

One analyteselected and

broken up by collisions with gas

All the fragment ions from the one analyte detected

+EPI (402.25) Charge (+1): Exp 3, 6.891 min from Sample 1 (Buspirone Metabolites) of Buspirone... Max. 1.6e6 cps.

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460m/z, amu

5%

10%

15%

20%

25%

30%

35%

40%

45%

50%

55%

60%

65%

70%

75%

80%

85%

90%

95%

100% 402.3

122.1

219.2150.1

238.2

178.2

98.1

196.2 281.2

N

NN N

N

O

O122

281238

196

OH

LC-MS/MS - Product Ion Scan

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LC-MS/MS - Multiple Reaction Monitoring (MRM)


Only one of the fragment ions from the one analytefiltered off and detected



XIC for Prazepam (325/271)500 fg on column (in urine)

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Why use LC-MS/MS rather than LC-MS

● Specificity and Sensitivity– LC-MS/MS is often 20 -100 times more sensitive compared

to LC-MS in a multi-targeted screening approach.– LC-MS/MS is much more specific than LC-MS as there is a

second filtering process. ○ Can use shorter columns allowing quicker run times as there is

less background interference from the sample.

– Allows the generation of library searchable spectra

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FROM MS TO MS/MS

1.12e9 cpsTIC of +Q1: from Full Scan 100 - 800

% In

tens

ity

0.5 1.0 1.5 2.0 2.5 3.0

Time, min

0.120.41

0.88 1.111.34

1.521.81

2.10 2.28

2.45 2.74

5101520253035404550556065707580859095

10 ng of drug injected on-column

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FROM MS TO MS/MS

5.20e5 cpsXIC of +Q1: from 482.0 amu from Full Scan

0.06 0.35 0.53 0.700.93 1.11

1.28

1.64

1.81 2.10

2.57

2.803.04

0.5 1.0 1.5 2.0 2.5 3.0

Time, min

5101520253035404550556065707580859095

% In

tens

ity

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FROM MS TO MS/MS

6.14e5 cpsTIC of +Q1 SIM (482): from SIM

0.09 0.240.36 0.610.74 0.921.03 1.28 1.45 1.701.87 2.02 2.242.37

2.57

2.762.883.01

0.5 1.0 1.5 2.0 2.5 3.0

Time, min

5101520253035404550556065707580859095

% In

tens

ity

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FROM MS TO MS/MS

4.20e4 cpsMRM from 482 - 258

0.18 0.54 0.77 1.00 1.241.36 1.57 1.76 2.00 2.20 2.37

2.64

2.94

0.5 1.0 1.5 2.0 2.5 3.0

Time, min

5101520253035404550556065707580859095

% In

tens

ity

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LC-MS/MS Systems for Small Molecule Analysis

● Iontrap (traditional spherical traps)● Quadrupole Time of Flight (QqTOF) systems● Triple quadrupole (QQQ) systems.● Q TRAP® systems.

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Similarities between these systems?

● They all separate ions based on their mass/charge (m/z) ratio

● All are capable of LC-MS/MS analysis● In clinical samples the charge (z) is normally 1

Triple Quadrupole (QQQ) Systems

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QQQ systems

● Qualitative analysis (spectral data acquisition)– Relative low sensitivity in scan mode due to lower duty

cycle.– Low mass accuracy and resolution versus QqTOF systems.

● Quantitative analysis (multi-targeted applications)– MRM possible – High sensitivity and linearity– Simultaneous multi component analysis possible.

Spherical (3-dimensional) Ion Traps

ions

Heated transfer capillary

Ion transfer 3D Trap

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Spherical Ion Traps Product Ion Operation

● MS/MS in time, not in space– Ion processing (trapping, isolating, excitation

(fragmentation) and scanning take place at the same location (inside the trap cavity).

● Can do MSn

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One mass isolated and then

fragmented by excitation.

All the fragment ions trapped in the spherical trap are then scanned out based on their mass/charge

Spherical Ion Trap Product Ion Product Ion SScancan

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Spherical Ion Traps● Qualitative analysis (spectral data acquisition)

– High sensitivity in full scan and product ion scan mode – Low mass accuracy and resolution versus QqTOF systems.– Space charge effects possible (Resolution loss, mass shifts,

isotope ratio shift, loss of dynamic range and accuracy).– Low mass cut off.

● Quantitative analysis (multi-targeted applications)– Lower sensitivity and linearity.– No real MRM possible.– No simultaneous multi component analysis (relative low scan

speed compared to MRM on triple).

QqTOF Technology

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QqTOF Product Ion MS/MS Operation

Scan precursors Detect all productsCAD

Q1 LINAC® Q2Collision Cell

product ionspectrum

Q1 transmits a given mass, sending a single precursor into Q2 Ions are fragmented in Q2 to produce product ions TOF scans selected mass range to detect product ions TOF

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QqTOF systems

● Qualitative analysis (spectral data acquisition)– High sensitivity in full scan and product ion scan mode – High mass accuracy and resolution versus iontraps and

QQQ systems (but mass accuracy does not help distinguish samples where they have have the same molecular formulae but different structures).

● Quantitative analysis (multi-targeted applications)– Lower sensitivity and linearity– No real MRM possible– No simultaneous multi component analysis (relative low

scan speed compared to MRM on triple)

Hybrid Linear Ion Trap Technology (Q TRAP® System)

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Location of Linear Ion Trap

Q0

N2 CAD Gas

linear ion trap3x10-5 Torr

Exit lens

Aux AC

LINACTM

Collision Cell

Q1 Q2 Q3

The ion trap is in the Q3 region of a triple quadrupole.

Because the trap is now a quadrupole it can hold more ions than a conventional 3D trap which reduces space charge effects. This leads to better spectra, giving you more confidence in the results.

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Many analytesenter the MS and are separated by a quadrupolemass filter



All the fragment ions trapped in the Linear trap and then scanned out based on their mass/charge

Q TRAP® System - Enhanced Product Ion Scan

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Q TRAP® Systems● Qualitative analysis (spectral data acquisition)

– High sensitivity in full scan and product ion scan mode – Higher mass accuracy and resolution versus QQQ systems

but less than QqTOF systems.– Reduced potential of space charge effects.– No low mass cut off.

● Quantitative analysis (multi-targeted applications)– High sensitivity and linearity.– MRM possible.– Simultaneous multi component analysis possible.

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LC-MS/MS Source Technology

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Ionization techniques for small molecule applications

● Electrospray Ionisation● Atmospheric Pressure Chemical Ionisation

Electrospray Ionisation

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Electrospray Source Principle

Ion Spray: Electrospray Ionisation (ESI)softest ionisation techniquepolar to ionic substances

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Atmospheric Pressure Chemical Ionization

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Heated Nebulizer (APCI)

Heated Nebulizer:Atmospheric Pressure Chemical Ionisation (APCI)corona discharge needlepolar to unpolar and thermally stable compounds

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Antiretroviral agents AntidepressantsAmino acids

ImmunosuppressantsMMA

Vitamins

Electrospray

AnalyteAnalyte

Ionisation Methods of Choice

What effects the way compounds form an ions?

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Acid or base (pH)?

● For compounds containing Nitrogen, a low pH or high concentration of H+ ions will increase the amount of compound forming the ions (typically add formic or acetic acid to the LC system).

● For compounds containing an acid group, a higher pH or lower concentration of H+ ions will increase the amount of compound forming the ions (typically add ammonia to the LC system).

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Can Standard LC conditions be Used?

● Yes, but some LC Buffers may cause problems.● Typically methanol and acetonitrile systems buffered with

ammonia acetate or formate are used in LC-MS.● Flow rates for nL/min to mL/min● One example of a troublesome LC-MS buffer is

Phosphate buffer and the use of a non-volatile ion pairing agent (e.g. SDS) which can can cause severe suppression and complex spectra.

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LC/MS/MS Summary● LC-MS/MS offers several advantages over more traditional

analytical techniques including higher sensitivity and selectivity.

● There are many different types of LC-MS/MS systems which vary functionality. It is important to understand the type of analyses intended the technology.

● There are different types of source available. Having an idea of the polarity of the compound will guide you to the most appropriate source, but generally Electrospray is the most flexible.

● Take care when transferring methods to LC-MS from LC with UV detection as the original buffer may not be ideal.

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Tandem Mass Spectrometry SystemsFor Clinical Research

API 2000™system

Entry level system

API 4000™ system

High performance, high sensitivity

systemfor applications such as targeted steroid analysis

API 5000™ System

Highestperformance,

ultimate sensitivity

system

API 3200™ System

Economichigh performance

systemIdeal for

TDM / IEM / Steroids / etc.

Increased Sensitivity

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Use of TMS in Clinical Research ApplicationsTDM

ImmunosuppressantAntiretroviral

AntipsychoticsAnticonvulsive

AntiviralBenzodiazepine

3200

NBS

30-40 DiseasesFatty Acid

Oxidation DisorderAmino AcidDisorders

Organic AcidDisorders

Other Diseases

3200

Biomarker

SteroidsT3/T4

Vitamin DHomocysteine

Orotic AcidVLCFA

Bile AcidCancer

Etc.3200 to 5000

Clin Tox

General unknown Screening (GUS)

Drugs Of Abuse (DOA)

Triple / QTRAP

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LC/MS/MS for Research on

Therapeutic Drug Monitoring

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Why adopt Tandem Mass Spectrometry for TDM?● Cost advantage vs Immunoassays

● Time To Result – Increased speed and throughput

● Flexibility adding new drugs to the panel

● Simultaneous detection of all drugs

● More accurate quantitation due to less cross reactivity with metabolites – Increased selectivity and specificity

● LC/MS/MS shows excellent reproducibility and dynamic range

● Very robust assay

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● Immunosuppressants–Cyclosporin A–Tacrolimus–Sirolimus–Everolimus–Mycophenolic Acid (MPA)

● LC/MS/MS, the ideal alternative for immunoassays● Simultaneous detection and quantitation● Excellent sensitivity and very good signal-to-noise ratio

Sirolimus

Clinical Research: Therapeutic Drug Monitoring

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Instrument configuration: modified setting to include MPA

API 3200 system

AB POROS® R1/20 Perfusion Column

API 3200 built-in switching valve.

Autosampler

Pump B

Pump A

Phenomenex Column Luna Phenyl-Hexyl

Column oven at 60 d.C

Methanol 10% +0.1 % Acetic Ac

Methanol 97% + 0.1 % Acetic Ac. + 10 mM CH3COONH4

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XIC of +MRM (14 pairs): 931.6/864.8 amu from Sample 11 (E 4) of 030806 Patienten Merc Ph... Max. 2700.0 cps.

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.019 38 57 76 95 114 133 152 171 190 209 228 247 266 286

Time, min

0.0

2000.0

4000.0

6000.0

8000.0

1.0e4

1.2e4

1.4e4

1.6e4

1.8e4

2.0e4

2.2e4

2.4e4

2.6e4

2.8e4

3.0e4

3.2e4

1.20

0.19 0.28 0.660.48 2.201.570.78 2.51

XIC of +MRM (14 pairs): 931.6/864.8 amu from Sample 10 (E4) of 030807 Patienten Bruno Ca... Max. 6800.0 cps.

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.510 19 29 38 48 57 67 76 86 95 105 114 124 133 143 152 162 171 181 190 200 209 219 228 238

Time, min

0.00

1.00e4

2.00e4

3.00e4

4.00e4

5.00e4

6.00e4

7.00e4

8.00e4

9.00e4

1.00e5

1.10e5

1.17e5

2.04

New 2 D LC-MS/MS methodology >

< Previous LC-MS/MSmethodology

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Fast Separation and Detection of Multiple Immunosuppressants and Internal Standards

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Calibration Curves with Blood Calibrators

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Dynamic Range and Linearity for EVEROLIMUS

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0 250 500 750 1000

0

250

500

750

1000

1250

Y = 1.06X + 8.404

CsA Co nc ent r at io n b y L C/ MS/ MS ( ng / m L)

CsA Co nc .b y EMIT( ng / mL)

CyclosporinA: EMIT Immunoassay vs. LC/MS/MS

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Cyclosporin A Tacrolimus Sirolimus Everolimus (CyA) (TRL) (SRL) (RAD)

LOD 1.3 0.1 0.1 0.1 [ng ml-1]

Linearity 0.9999 0.9997 0.9998 0.9998 [R2]

Recovery [%] concentration 1 88.3 114.4 116.9 118.8

concentration 2 103.7 101.9 114.5 102.8

RSD [%] Intra-day (n=100) 4.6 10.2 9.3 10.3

RSD [%] Inter-day (n=40) 12.3 13.5 12.3 10.9

Accuracy [%] 87.7 – 115.8 90.3 – 110.8 91.0 - 107.5 91.2 – 112.1 (n=40) (Mean: 99.2) (Mean: 96.9) (Mean: 98.9) (Mean: 103.1)

Koal et al.: J.Chromatogr. B 805 (2004) 215-222

Method performance parameters determined for 25 µl spiked blood sample

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MPA features

● MPA has a free acidic moiety which should make it

more sensitive in Negative Ion Mode.

● MPA is quite labile in the ionization process.

● Reported to be metabolized at a significant extent

(mainly to the Glucuronide conjugate).

● MPA prone to stick on surfaces.

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Fast 2D-LC MS/MS Method including MPA

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Conclusions

● LC/MS/MS technology and principles

● LC/MS/MS is superior to conventional methods with regard to specificity, accuracy, dynamic range and cost per assay

● LC/MS/MS is easy to use

● Wide range of potential uses for Therapeutic Drug Monitoring, Inborn Errors of Metabolism, Clinical Biomarkers – growing research into these areas focused on mass spectrometry.

Thank You For Your Attention!

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● Some of the publications cited in this presentation present off-label uses of Applied Biosystems products. Applied Biosystems’ citing of these publications does not constitute an endorsement of, nor warranty of, the data or conclusions contained in these publications.

● Applied Biosystems products have not been cleared or approved by the U.S. FDA or any other agency, or under the European IVD directive, for any purpose. Applied Biosystems does not promote or sell its instruments or other products for diagnostic or other clinical use.

● No claim or representation is made or intended by Applied Biosystems (i) as to any clinical use of any product (whether diagnostic, prognostic, therapeutic, blood banking or any other clinical use), or (ii) that any product has been cleared, approved, registered or otherwise qualified by Applied Biosystems with the united states Food and Drug Administration or any other regulatory agency for use in any diagnostic or other clinical procedure. Applied Biosystems’ products are labeled “For Research Use Only. Not For Use in Diagnostic Procedures,” or are General Laboratory Instruments.

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For Research Use Only. Not for use in diagnostic procedures.

Applera is a trademark and Applied Biosystems, POROS and AB (design) are registered trademarks of Applera Corporation or its subsidiaries in the U.S. and/or certain other countries.

API 2000, API 3200, API 4000, API 5000, Turbo V, Curtain Gas and Ionspray are trademarks. Photospray and LINAC are registered trademarks of Applied Biosystems/MDS SCIEX a joint venture between Applera Corporation, and MDS SCIEX Inc.

All other trademarks are the sole property of their respective owners

©2006 Applied Biosystems. All rights reserved.

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introduction to lc-ms to lc-ms dr. michael baynham senior specialist, mass spectrometry applications...

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