The Role of Multiple MS pTechnologies in Bioanalytical Analysis
Daniel Pentek*February 3, 2011 -UCONNBioanalytical Chem 395
Introduction – Dan Pentek
Education:Wesleyan University: Bachelor’s and Master’s in ChemistryUCONN: Masters in Business AdministrationUCONN: Masters in Business Administration
Work Experience:Yale University Chemical Instrumentation Center (‘83-’93)
Mass SpectrometristPerkin-Elmer / Sciex Joint Venture (‘93-’99)
LCMS InstructorP d t S i li t (d li ti t )Product Specialist (demos, applications, etc.)Technology Sales ManagerApplication/ Demo/ Training Lab Manager
Bayer Healthcare – Pharmaceuticals (‘99-’04)Bayer Healthcare – Pharmaceuticals ( 99- 04)Sr ScientistPrinciple Scientist – Automation and New Technology
PerkinElmer, Inc (‘04-’present), ( p )MS Technology Manager, Product Owner, Product Manager, etc.
2
Topics to be covered:I t d ti Introduction Bioanalytical OverviewMS MS -
MS Market & TechnologiesBasic ionization methods
El t APCI APPIElectrospray, APCI, APPIInterfaces
Orifice, capillary
MS AnalyzersPerformance CriteriaStrengths & weaknesses for bioanalytical analyses
Quadrupole (single &triple quads)Ion traps (3-D and 2-D)Time of flight (linear, reflectron, MALDI)Hybrids (Quad TOF) and othersHybrids (Quad-TOF) and others…FTMS (ICRs and orbitraps)
Block Diagram - Bioanalytical Analysis
Sample PreparationSam
Sample Separation
mple
SPEED i th d i !!Sample Separation
Th
SPEED is the driver!!
Eliminate bottlenecks!
Sample Analysisroug
MS provided a paradigm shift in these areas
Data Analysis
ghpu
4
yt
Separation Based on Mass Has Been Around a Long Time…
Winnowing is a mass separation technique.
5 Slide from Dr. Athula Attygalle
Modern Grain Mill – Separation Based on Mass
A modern grain mill does this more efficiently. In fact, each process taking place inside a
i ill b dgrain mill can be compared to those taking place inside a
mass spectrometer
6 Slide from Dr. Athula Attygalle
What is a (Modern) Mass Spectrometer?
An instrument that separates molecules or atoms (e.g., ICP/MS) based on their mass/charge ratio (m/z).
m is the massΙzΙ is the absolute value of the number of charges on it.
Mass is reported in u mass units or the unified atomic mass unit which is Mass is reported in u, mass units, or the unified atomic mass unit which is defined as 1/12th the mass of carbon 12 (12C) or Da (Daltons) which is more prevalent in the biochemistry world.
Prior to 1993 mass was reported in atomic mass units (amu) which was defined Prior to 1993, mass was reported in atomic mass units (amu) which was defined as 1/16th the mass of oxygen 16 (16O). Masses are still frequently reported in amu, but this is incorrect!
A MS id 2 di i f i f tiAn MS provides 2 dimensions of information:A signal that “something is there” (a signal intensity)The mass of what is there, to a nominal or very precise level of accuracy
LC/MS Historical Perspective
Industry was desperate for a decent, rugged LC/MS interface.
GC/MS required derivatization etcGC/MS required derivatization, etc.Not applicable to most biomolecules (MW, etc.)
Atmospheric pressure ionization (API) ionization along withAtmospheric pressure ionization (API) ionization along withnew interface designs provided the solution.Now, all interfaces are differentially pumped.Now, all interfaces are differentially pumped.
Pumping of interfaces was criticalOrifice – skimmer (nozzle) designsH t d ( ld) ill d iHeated (or cold) capillary designs
LC/MS is a big business now!
Analytical Instruments Technology Segments…
SDI data on analytical instrument technology shows mass spectrometry is a $2.6B market in 2010, and predicts…
…that it will have fastest growth rate (8.6%) of any analytical instrument technology through 2014technology through 2014
*SDI Global 11th Ed. Sept. 2010
Distribution of Mass Spectrometry Techniques**
Avg. CGR >10% Growth Opportunities
$255MCGR 6.2%
$101MCGR 2.3%
$643M
CGR 9.5%$312M
CGR 9.4%$139M
CGR 17.0%$136M
CGR 9.7%$391M
CGR 4.9%
$241M$61M
*SDI Global 9th Ed. Sept. 2006
PKI has product offerings CGR 7.2%CGR 3.8%
LC/MS(/MS): A Marriage of Liquid Chromatography and Mass Spectrometry
Marriage (like any other close relationship) requires: COMPROMISE!COMPROMISE!
LC Person: “MS is just another detector”MS Person: “LC is just an inlet”
What’s good for LC may not be good for MS and vice versa.LC d l ti b f th fi d t h t i t f it LC was around a long time before they figured out how to interface it to an MS.
LC/MS Instrument Block Diagram
LC
Ion Source Interface Mass Analyzers Detectors
-ESI -Orifice – Sk -Quadrupole -CEM
Eluant
-APCI-APPI
-Capillary(Hot or cold)
p-Ion Trap (4 types)-Time of Flight
-Discrete dynode-CCDPhotomultiplier-Photomultiplier
MS System – under vacuumMS System under vacuum
Today’s LC/MS Ionization Methods: All Done at Atmospheric Pressure
On-line techniques:Electrospray (ESI) - Fenn @ Yale ~1984
Shared Nobel Prize in 2002 for this work with K. Tanaka (MALDI) and K. Wüthrich (NMR)Atmospheric Pressure Chemical Ionization (APCI)
Irabarne & Thomson ~1979At h i P Ph t I i ti (APPI)Atmospheric Pressure Photo Ionization (APPI)
Emerging, not as widely used yet.All of the above are done at atmospheric pressureSi ifi t h f t diti l i i ti th d hi h ll d ithiSignificant change from traditional ionization methods which were all done withinthe vacuum chamber.
Off-line techniques:Matrix Assisted Laser Desorption Ionization (MALDI)Desorption Electrospray (DESI)DART- Direct analysis in real timeand more…
PerkinElmer Acquires Analytica of Branford
In ~June of 2009, PKI acquired Analytica of Branford, Inc. (AoB)AoB was company started by John Fenn (share Nobel Prize 2002) and Craig Whitehouse (who built first electrospray source on an LCMS)( p y )
John Fenn Craig Whitehouse
(N b l P i L t 2002)
Company held exclusive IP on electrospray and multiple charging of moleculesIn 1987 AoB started designing ESI sources for many vendors
(Nobel Prize Lecture 2002)
Developed early ESI-TOF MSDeveloped over 75 patents in the field of MS
Ion source technologyIon guide technologyCoupling ESI to o-TOF MS
Significant Overlap in Ionization Techniquesht
1000 kDa
ar W
eigh
100 kDaProteins
Mol
ecul
a
10 kDaPeptides
Electrospray
M
1 kDa
APCIAPPI
Carbamates
APCISteroids
Phenyl ureas
PAHs100 Da
15 PolarityLow High
Electrospray- Spraying a Very (Charged) Fine Mist
High pressure nebulizer gas provides pneumatic assistance
16
High pressure nebulizer gas provides pneumatic assistance…
Electrospray: Ion Evaporation Process for LC/MSDesolvation Process and Ionization Mechanism
• Rayleigh limit is the maximum charge a droplet can hold while maintaining its volume
Evaporation Rayleigh Coulombic
maintaining its volume
Ions EjectedEvaporationLimit
ReachedExplosions
+ + ++ ++ ++ + ++
++ +--
Ions Ejected
+ ++
+++ +
+
+++
++
++ -
--
-
--
++
++ +
+ ++
+
+ +
---
--
++
+++- -- -
++
++
+
++
+---
-
- -
Droplet CapillaryHow well this works depends on the mobile phase composition and flow rate. Must have proper probe positioning, CCDG flow and CCDG temperature and
nebulizer pressure to insure proper ionization of analyte ion
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nebulizer pressure to insure proper ionization of analyte ion .
Electrospray: Ion Evaporation Process for LC/MSElectric field lines
-
-Electric field lines
(drawing (+) ions in
toward (-) charge) Heated drying gas
++
+
+
+ ++--
-
++
+++
+ -
-
-
-
+ ++ ++-
- -++
++
+--
-- -
+++
+
-- -
+++
+
---
-
+ ++++
--
-++++ + +- -
- -+ +++
+ +-- +++++
+
++ +
+ +
+
+--
- +
-
-
- 6000V at
MS entrance
(under vacuum)
1. Droplet from sprayer with (+) and (-) charges on it
2. Droplet shrinks with mobile phase evaporation
3. Coulombic repulsions rupture droplet ejecting ions (with solvent molecules)
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(under vacuum)4. Ions of desired polarity follow field lines to capillary tip while being strippedof residual solvent by heated drying gas and they are sucked into the MS entrance
Electrospray
ESI is most widely used LCMS ionization techniqueElectrospray is a concentration dependent technique.
Ease of use and LC flow rates drove source development.Started out as very low flow technique, which wasn’t very compatible with LC.
LC Person: “Use lower flows and narrower column”.MS Person: “Buy ESI probe that has higher gas flows and desolvates better”.
Nanospray takes ESI to much lower flow ratesPreserves precious sampleAllows multiple MS experiments to be run over time (which takes time)
Electrospray- Tips
ModifiersOrganic acids (e.g. formic, acetic) promote ionization of basic compounds (sp3
N t i i )N- containing)Neutral compounds containing nucleophilic lone pairs (sp2 N, sp3 O) can be desorbed by cationization with alkali metal or ammonium ions.Ammonium formate or acetate are recommended buffers ( 2-10 mM optimum, can see suppression effects over 20 mM, 50 mM max.)Salts can interfere with ionization and can cluster to complicate spectrum (but
l id i id tifi ti )also aid in identification)Strong bases or quaternary amines can interfere with positive mode analytesSulfonic acids interfere with negative mode analytesAVOID PHOSPHATE BUFFERS as much as possible
Contaminate ion pathSuppress ionizationpp
Atmospheric Pressure Chemical Ionization (APCI)
Field Free APCI Source Conventional APCI Source
Configuration
Nebulizer
Sample Inlet
Nebulization Gas Inlet
AuxiliaryG I l t
Nebulizer
Sample Inlet
Nebulization Gas Inlet
AuxiliaryG I l t
Nebulizer
Sample InletNebulization
Gas Inlet
AuxiliaryG I l t
Nebulizer
Sample InletNebulization
Gas Inlet
AuxiliaryG I l tGas Inlet
Gas Curtain
Gas Inlet
Gas Curtain
Heater
Gas Inlet
Heater
Gas Inlet
HeaterHot N2
To MSAnalyzer
ElectrodeHeaterHot N2
To MSAnalyzer
Electrode
CoronaNeedle
Hot N2
T MS
Gas CurtainElectrode
CoronaNeedle
Hot N2
T MS
Gas CurtainElectrode
CoronaNeedle
y
SamplingCapillaryCorona
Needle
y
SamplingCapillary
To MSAnalyzer
SamplingCapillary
To MSAnalyzer
SamplingCapillary
Increased analyte concentration around corona needle.
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Increased analyte concentration around corona needle. Decoupling of Ion production from Ion Transport Field.
APCI Reactions
N2 + e- N2+. + 2e-
N2+. + H2O H2O+. + N2
H2O+. + H2O H3O+ + OH.
H3O+ + M [M+H]+ + H2OH3O M [M H] H2O
APCI- Summary
HN is a high flow (0.5-2.0 mL/min.) inlet
Suitable for polar, thermally stable cmpds
Usually, MW < 1000 amu
Probe is heated to facilitate vaporization
Requires nebulizing and auxiliary gas
Requires corona discharge needle to produce ionization (APCI)
Atmospheric Pressure Photo Ionization (APPI)
Emerging technique (about 5 years old)Uses typical 10 eV UV lamp (similar to photo-ionization lamps for yp p ( p pGC). Similar to APCI, but applicable to broader range of compounds.
LC/MS Instrument Block Diagram
LC
Ion Source Interface Mass Analyzers Detectors
-ESI -Orifice – Sk -Quadrupole -CEM
Eluant
-APCI-APPI
-Capillary(Hot or cold)
p-Ion Trap (4 types)-Time of Flight
-Discrete dynode-CCDPhotomultiplier-Photomultiplier
MS System – under vacuumMS System under vacuum
Example: Orifice-Skimmer Interface (AB-Sciex)
Nitrogen GasCurtain Interface
Orifice Plate
DifferentiallyPumped Interface
E V
Orifice Plate
QØSO
UR
CE V
AC
UU
M+
Skimmer
urt ai
nG a
sFlo
w
PI I
ON
S
M C
HA
M
CuA
MB
ER
Bioana
Flexar SQ 300 MS Detector Ion Optics and Vacuum System
Heated Countercurrent
Drying Gas (N2)
Dielectric Capillary
4 stage pumping system: 30/200/200 cartridge turbo with E2M28 rough pump
Patented ion guide technology traversing multiple vacuum stagesPatented ion guide technology traversing multiple vacuum stages
Vacuum Interface: “Protective” Gas & Differentially Pumped Interfaces
Regardless of interface used;UHP nitrogen counter current drying (CCDG) gas keeps non ionized species out of the analyzer regionnon-ionized species out of the analyzer regionCCDG aids in ion de-clustering (with CID potentials)Two stage transition from atmosphere to low pressure region Two stage transition from atmosphere to low pressure region of analyzer (1 x 10-5 torr) CCDG and ions are drawn in due to;;
Pressure differential (both ions & CG)Electric field gradients (ions only)
Interface exit into vacuum offers sample “fragmentation” opportunity
Poor man’s MS/MS ☺Poor man s MS/MS…☺
1st Vacuum Region- Supersonic Expansion Occurs (Mach Disk)
Within a Mach disk, all ions and gas molecules moving in same direction
It should project into the skimmer zone for ion transmission
CID induced by voltage difference between cap. exit and SK
It should project into the skimmer zone for ion transmission
Capillary ID, skimmer ID and pumping speed are all variables requiring optimization
LC/MS Instrument Block Diagram
LC
Ion Source Interface Mass Analyzers Detectors
-ESI -Orifice – Sk -Quadrupole -CEM
Eluant
-APCI-APPI
-Capillary(Hot or cold)
p-Time of Flight
(MALDI- TOF)
Ion Trap (4 types)
-Discrete dynode-CCDPhotomultiplier-Ion Trap (4 types) -Photomultiplier
MS System – under vacuumMS System under vacuum
Analyzer (and System) Criteria
Analyzer Considerations:Mass accuracyResolutionResolutionSensitivityScan speedDynamic range
System Considerations:“Sensitivity”Sample thru-put – Fast LC?Primary application: quantitation qualitative orPrimary application: quantitation, qualitative or…Software- application basedEase of use!!!P iPrice…
Single MS vs.MS/MS Systems:
Single MS systems (quadrupole or TOF)Have the added dimension of mass vs. UV or diode array detectors.However, chemical noise is the limiting factor for sensitivity (S/N) and dynamic range.
So sample preparation becomes a bigger factor!
“MS/MS Systems” Many variations now…
“Triple Quads” - 2 quadrupole MS’s separated by a collision cellTriple Quads - 2 quadrupole MS s, separated by a collision cell“QTOFs” - Quadrupole front end, collision cell, TOF back end“MSn” analyzers- ion traps (3D and 2D) and FTMS’sTOF-TOF separated by collision cellTOF-TOF, separated by collision cellIT – TOF (Shimadzu)
Purposes essentially the same (regardless of analyzer type), select one ion from all the others fragment it and study the fragment ionsfrom all the others, fragment it, and study the fragment ionsMS/MS systems ARE the paradigm shift in bioanalytical analysis
Mass Accuracy- Mass Analyzer Comparison (LC/MS)
Cost: 0 1 2 ($M)
m/z = 1000.00000Cost: ~ 0.1 2 ($M)
m/z 1000.00000 Quadrupole50-200 ppm50 200 ppm
Ion Trap10-100 ppm
FTMS-ICR0.05-1 ppm
Time of Flight1-20 ppm
FTMS –IT (Orbitrap)0.5-2 ppm
Resolution- What is it?
Ability to separate (resolve) adjacent ionsTypically defined as: M/∆MTypically defined as: M/∆M
M: Mass ∆M: Full Width at Half Max.
Quadrupoles: scan at constant peak widthQuadrupoles: scan at constant peak width30/1=30, 300/1=300, 3000/1=3000…
Resolution increases as you go to higher massy g g
TOFs: scan at constant resolution10k res: m/z 10.001, 100.01, 1000.1, 10,001, , , ,
Peak width increases as you go to higher mass
Analyzer Types: Quadrupoles (and hexapoles, octopoles, etc.)
Fundamental parts of virtually all LC/MS systemsServe one of two purposes:Serve one of two purposes:
Ion transmission devices (quad, hex, oct…)Capture and transmit ions from one place to another…
Ion filtering devices (quadrupoles only…)Act as a mass filter (analogous to a magnetic)
Quadrupole Theory
Quad. as a mass filterSeparates ions based on m/z ratioVoltages applied determine “width” of mass window
Quad. made of 4 rods“A pole” - vertical rods; “B pole” – y
L
horiz. rods (by convention)
DC, RF volt. imposed:ro r
++
x
U=(DC)A-(DC)B (FDC) V: RF volt. peak-peak (RFp-p)
2 2
z
V = 7.22 * M * r02 * f 2 ; i.e., V ~ M
‘Time-of-Flight’ (TOF) Analyzers
TOF AnalyzerLinear ModeLinear ModeReflectron Mode
Common Ionization Methods for TOF MSCommon Ionization Methods for TOF MSMALDI ESI
TOF Ion Optics (Linear)
t = time taken to reach the detector (seconds)
m = mass of the ion (kg)
z = number of charges on the ion. If singly charged z = 1charged z = 1
e = magnitude of the charge of an electron (coulombs)
V = Accelerating voltage (volts)
39
V Accelerating voltage (volts)
d = distance an ion travels (flight tube) (meters)Limitations: Kinetic energy distribution
Basic API TOF MS Ion Flight Path
Detector
Pulsing RegionPulsing Region
Horizontal TOF Drift TubeHexapole Ion Guide
Dielectric CapillaryIon Mirror
Grounded API Source
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Grounded API Source
CONFIDENTIAL AND PROPRIETARY INFORMATION OF PERKINELMER, INC © 2009 Perkin Elmer
Trap-Pulse Mode, Small Packet of Ions
High m/z0V
Bias +30V+10V
Ions filling ion guide, storing ions from last pulse,ion guide exit high Each ion packet smaller than usable pulsing region, mini TOF
0V
Bias +30V+10V
g
Low m/z0V
0V Over fill regionions lost
0V
0V
High m/z
Low m/z
0V
Bias -30V+10V
Initial ion release from ion guide, ion guide exit low
Pulser voltage applied, ions accelerated for TOF
Exclusion regionions lost
Low m/z
High m/z
Low m/z
0V
0V +400V
+200V
Bias +30V+10V
Short pulse of ions from ion guide, ion guide exit high, start storing for next pulse
High m/z
Low m/z
0V
0V
0VBias +30V+10V
start storing for next pulse
Usable pulsing region
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0V
Slide #: 3014
CONFIDENTIAL AND PROPRIETARY INFORMATION OF PERKINELMER, INC © 2009 Perkin Elmer
MS/MS Systems: Triple Quadrupole
ORIQ1
RNG
Q0 ST RO1
IQ2
ST3 RO3
DF
CEM
6 mTorr1 Torr
RO2 (LINAC)
DF
Varian 550S25B Pump
Leybold 361Backed by D10E
Triple Quad Scanning Modes: Multiple reaction Monitoring (MRM)
TQ’s remain the “Gold Standard” for quantitation because of TQ s remain the Gold Standard for quantitation because of MRMIf Q1 d Q3 idth 0 th MRMIf Q1 and Q3 width=0, then MRMMany (hundreds!) of precursor to product ion pairs can be
it d (A B A’ B’ A” B” t )monitored (A-B, A’-B’, A”-B”, etc.)MRM analysis is the best way to maximize signal intensity of product ions
MS/MS - Multiple Reaction Monitoring (MRM)
Precursor ion set
Product ion set
Fragmentation(CAD)set set(CAD)
MS/MS Systems: Quadrupole-TOF (QTOF)
Introduced commercially by Micromass around 1995.Brilliant innovation first commercial hybrid MS/MSBrilliant innovation, first commercial hybrid MS/MS.They charged “what the market would bear”
$500-600kNo competition!Great qualitative analyzer
TOF analyzer provided:TOF analyzer provided:Incredibly fast scan ratesAccurate mass capability (MW confirmation)Higher resolution (8k initially, now 10-12k w/ 1 reflectron)
QSTAR® XL System—Schematics
DC Quad250 L/s770 L/s
Acceleratorcolumn
4-anode detector
column
Sample
Ions
Conductingliner770 L/s
2.5 Torr
Q1
10-2 Torr
10 mTorr
C t iLINAC
Q2Q0
10-2 Torr
Field Free
Drift region
7x10-7 TorrEffective Flight
Curtain
Gas
• eliminate cross-talk
Ion Mirror
(reflector)
Path = 2.5 m• fast switching MS MS2
• broad dynamic range
• saturation correction
Triple Quads vs. Ion Traps Complementary MS/MS Approaches:
Tandem in Space: Triple QuadsTandem in Space: Triple QuadsPoor scanning sensitivity
Great for quant (MRM)Great for quant (MRM)
Very selective scans
T d i Ti I TTandem-in-Time: Ion TrapsVery sensitive scanning
O l d t iOnly product ion scans
Only scanning capability
I T 2D (2002)Ion Traps -2D (2002)“Ion bottles” for optical spectroscopyspectroscopy.
Minimize fringing fields to maximize performance.
Ion accumulation forIon accumulation for enhanced ms sensitivity.High quality mass
t tspectrometer:RCM, 2002, 16, 512-526.
3 D TLinear Traps
Feb. 1, 20073-D Traps
AB-Sciex Q TRAP™ System Ion Path
N CAD GDipolar Aux AC
N2 CAD GasSkimmer
Q0 Q1 Q2 Q3
ExitIQ3IQ2IQ1O ifi
linear ion trap
3 4x10-5 Torr
ExitIQ3IQ2
LINAC
IQ1Orifice
3-4x10-5 Torr
Trapping Forces in a Linear Ion Trap
Radial Trapping RF Voltage
AxialTrapping
AxialTrapping pp g
DCVoltage
Exit Lens
Radial Trapping RF VoltageResonance Excitation
Linear vs. 3-D Ion Traps:Li TLinear Trap
Trapping EfficiencyNo quadrupole field on center
3-D TrapTrapping Efficiency
Q d l fi ld i lit d No quadrupole field on center line.Longer flight path.
Quadrupole field gives amplitude and phase dependent injection eff’s.
1 cm to lose injection energy~1 cm to lose injection energy.
Extraction EfficiencyLinear trap is ~10X better
Extraction Efficiency18-20% (measured)
< 50% (Probably ~30%)
I C it3 D trap is 2X betterIon Capacity:
Ion Capacity:3-D trap is ~2X better
5-inch linear trap:5 inch linear trap:
45X greater capacity
Summary
Role and importance of MS in bioanalytical analysis continues to grow and evolve
MS is typically no longer the bottleneck (sample handling and data acquisition/processing slow thru-put)Power of new MS technology providing new dimension of information on biomoleculesgy p g
There are 3 fundamental MS analyzer technologies, each with it’s advantages and disadvantages.
‘H b id ’ t k d t f b t f 2 t h l i‘Hybrids’ can take advantage of best of 2 technologiesLC/MS continues to evolve at a rapid rate
Better, faster, cheaper…, , p