Presentation on theme: "Background and Theory of Electrospray and Tandem Mass Spectrometry"— Presentation transcript:
1 Background and Theory of Electrospray and Tandem Mass Spectrometry Why LC/MS/MS?Background and TheoryofElectrospray and Tandem Mass Spectrometry
2 LC/MS/MS: A Primer Advantages over GC/MS Importance of Chromatography Atmospheric Pressure IonizationFocus on ElectrosprayTandem Mass SpectrometryFocus on QqQModes of MS/MS analysisMRMProduct ion scan3200 Qtrap Parameters
3 Why LC/MS/MS? Why Liquid Chromatography? Why MS/MS? Analysis of labile analytesAnalysis of more polar compounds without derivatization.Analysis of significantly higher massesReduction of lengthy clean-upWhy MS/MS?Additional structural elucidationFurther reduction of clean-up (?)SpecificityUseful MS modes
4 System Configuration ESI Very important! Triple Quadrapoles APCI LiquidChromatographyIonizationMass AnalyzerDetector/DataCollectionESIAPCIAPPIVery important!Many columnsMany solvent systemsTriple QuadrapolesIon-TrapsHybrids
5 Atmospheric Pressure Ionization Revolutionized LC/MS opening it to a wide array of applications in the forensic laboratory.Desolvation and/or ionization of analytes occurs at atmospheric pressuresGas phase ions are sampled by the high vacuum mass spectrometer.
6 Types of IonizationSeveral common modes differing by method of ion formation:Electrospray (ESI)Atmospheric Pressure Chemical Ionization (APCI)Atmospheric Pressure Photo-Ionization (APPI)New dual sources (ESI/APCI) or (APCI/APPI)
7 Which is Best? It depends on the exact application. Increasing polarity and molecular weight and thermal instability favors electrospray.Most drugs of abuse are highly polar and are easily analyzed using electrospray.High molecular weight proteins also require electrosprayLower polarity and molecular weight favors APCI or APPI.Lower background, but compounds must be more thermally stable.
8 Focus on ElectrosprayElectrospray is a method of getting the solution phase ions into the gas phase so that they can be sampled by the mass spectrometer.
9 Electrospray: The Process Three Fundamental Processes:Production of charged droplets.Droplet size reduction, and fission.Gas phase ion formation.
11 ESI: Production of Charged Droplet A large voltage ( up to 6kV) is applied between the end of a capillary carrying the LC mobile phase and the mass spectrometer entrance.Ions (of the same polarity) are drawn out toward the counter electrode (curtain plate) pulling the mobile phase along.When the excess charge at the tip of the capillary overcomes surface tension, a droplet is formed.
12 ESI: Droplet size reduction & fission Droplet size reduction occurs by the continual repetition of two processes:Desolvation (evaporation of neutral solvent and volatile buffers)Droplet fission caused by electric repulsion between like charges.
14 ESI: Gas Phase Ion Formation Several models of bare ion formation; all seem to play some role.Charge Residue ModelAll the solvent evaporates, leaving a bare gas phase ion.Ion Evaporation ModelAs the droplet shrinks the charges (analyte) that reside on the surface get just enough energy to jump into the gas phase.Ion Emission ModelThe high voltage case some ion formation directly from the LC capillary.
15 ESI: Types of Ions Formed Electrospray can operate in either positive or negative mode.Positive mode:Best suited to basic drugs that form a stable HCl salt.[M+H]+ is the primary ion formed[M+nH]n+ and [M+Na+]+ can also be formed.Negative mode:Best suited to acidic drugs that form stable Na salts.[M-H]-, [M-nH]n- and [M+I-]-
16 ESI: Ionization Efficiency Enhanced by the production of smaller droplets.Lower mobile phase flow rate yield smaller droplets.Nebulizing gas promotes droplet formationUse of volatile mobile phases promotes desolvation and droplet fissionEnhanced by increasing the concentration of analyte ions at the end of the capillary tip.Matrix modifiers to promote solution ion formation.Chromatography to produce narrow highly concentrated bands of analyte.
17 ESI: Pros and ConsProsSoft ionization technique, resulting in little decomposition of labile analytes.Generally produces only molecular ions.Multi charged analytes easily produced, allowing proteins to be analyzed.Wide range of analytesHighly efficient ion production.
18 Ion Suppression ESI: Pros and Cons Cons Lower flow rates concentration dependentnL/min (nanospray)Analyte must form solution phase ion.HCl or Na salt good indicator of suitabilityIon Suppression
19 ESI: Ion Suppression The bogeyman of electrospray! Often results from inefficient droplet formation.Causes:Nonvolatile buffers or salts (phosphates)Nonvolatile materials in mobile phase Ion pairingReported that higher molecular weight analyte ions can suppress smaller analytes.Generally more prominent early in an RP-LC run, but can occur at anytime.Underscores the need for good chromatography!
20 ESI: Ion Supression ~90%reduction Ion Suppression Study Oxycodone Infusion with solvent flow.Negative control injected at ~0.1min~90%reduction
21 ESI: Keys to RememberElectrospray is a soft ionization technique generally producing [M+H]+ ions in positive mode.Most drugs that form an HCl salt will be analyzable by positive mode electrospray.Volatile buffers and mobile phases will increase generally ionization efficiency.Good chromatography producing concentrated bands of analyte at the nebulizer tip will increase ionization efficiency.Poor clean-up can lead to significant ion suppression usually at the beginning of the LC run.
22 Now what do you do with it? Questions?Congratulations!You have made an ion.Now what do you do with it?
23 Tandem Mass Spectrometry Triple Quadrupole (QqQ)Two mass filtering quadrupoles bracket an Rf only collision cell.Ion Trap (IT)A single ion trap serves as mass analyzer and collision cell.Hybrids (e.g. LIT)Instrument is in the QqQ geometry, but one quadrupole can also trap and store ions.
24 Triple Quads v. Ion Traps Triple QuadrupoleAdvantagesVery sensitive. (SIM)Good for quantitationSome useful MS scanning modesLimitationsNo MSnExpensiveLimited to unit mass resolution.Less sensitive in full scan mode.AdvantagesHigher full scan sensitivityHigher mass resolutionMSnLimitationsNot as good for quantitations.Space Charge Effects1/3 cut-off rule.Cannot perform certain MS experiments.
25 Triple Quad Configuration ScanningRF/DCRF only Collision CellScanningRF/DCRF onlyQ1 and Q3 are standard mass filter quadrupoles.The can scan masses sequentially (e.g. 50 to 500 amu)The can be used to select a single mass.Q2 is an RF only quadrupole that is in a gas filled chamber.Q2 is the “collision cell” where mass fragmentation occurs.Q2 does not filter ions. It accepts all ion sent to it by Q1 and passes all ions formed by collision to Q3 to be sorted.
26 Triple Quads… In scanning mode 99% ions lost between the rods. Poorer full scan sensitivityIn SIM mode 100% of selected ion reaches detector.Makes them highly sensitive and great for quantitation!Mass resolution typically limited to “unit” (+/- 0.2 amu)Fragmentation is controlled by the energy ions have when they enter the collision cell.Higher energy >> greater fragmentation.
27 Collision Cell LINAC (linear accelerator) Collision Cell Filled with N2 gas at roughly 3x10-5 torr.Drives ions out, reducing “cross-talk”The analyte molecules undergo collision activated disassociation by energetic collision with the N2 molecules.The N2 also acts to “cool” fragments, facilitating transport to the detector.
28 Ion TrapsIons fill the space between a ring electrode and a pair of end-cap electrodes.Mass analysis and fragmentation occur in the same space.
29 Ion Traps…In full scan mode: Ions fill and are trapped in space then masses are scanned out of the trap sequentially.Ions are not lost, so full scan sensitivity is better, but filling/closing cycles make them poorer at quantitation.Mass resolution is controlled by the “speed” at which masses are scanned out of the trap.slower scanning = better mass resolution.In MS/MS mode: Ions trapped. Fragmentation occurs when the selected ion is excited by a so called “tickle” voltage and collides with bath gas (He). This process can occur recursively thus MS/MS/MS/MS….
30 Hybrid InstrumentsApplied Biosystems 3200 Qtrap SystemQ0Q1Q2Q3Typically in the same configuration as a triple quadrupole instrument.On the Qtrap Q3 is the hybrid quadrupole dubbed a “linear ion trap” or LIT.Q3 can function as a quadrupole OR an LIT.
32 Advantages of LIT vs. ITHas a larger “volume” so it can be filled with more ions before exhibiting space charge effects.Ions are formed outside the trap, so it is not limited by the 1/3 rule.Can perform MS/MS/MS experiments by selecting an ion and fragmenting it using the spillover collision gas. (1/3 rule applies here…)
33 Modes of Operation H Y B R I D S Triple Quads and Ion Traps Full Scan (LC/MS)MRM (Multiple Reaction Monitoring)Product Ion Scan (PI)Exclusively Triple QuadConstant Neutral LossPrecursor Ion ScanExclusively Ion TrapMSn
34 Multiple Reaction Monitoring (MRM) Steps MS2: &4Exit lensIon accumulationQ0Q1Q2Q3Precursor ionselectionFragmentationN2 CAD GasQ1 Selects an [M+H]+Q2 fragments the selected ion.Q3 monitors only one daughter ion
35 MRM Only the daughter ion reaches the detector. Steps MS2: &4Exit lensIon accumulationQ0Q1Q2Q3Precursor ionselectionFragmentationN2 CAD GasOnly the daughter ion reaches the detector.Sensitivity of MRM is a function of how much of the daughter ion is produced.The parent ion fragmentation to daughter ion is commonly referred to as a “transition”
36 Example MRM Data Many transitions can be stacked together in a method. Oxycodone: (316.2241)Parent : 316.2Daughter : 241Result of one MRM cycle of 130 drugs.Many transitions can be stacked together in a method.The instrument will monitor each pair for a short time.MRM is analogous to SIM on a GC/MS only more compound specific.
37 Product Ion Scanning Q1 selects a parent ion. Steps MS2: &4Exit lensIon accumulationQ0Q1Q2Q3Precursor ionselectionFragmentationlinear ion trap3x10-5 TorrN2 CAD GasQ1 selects a parent ion.Q2 fragments the selected ionQ3 traps then scans out all fragment ions.
38 Product Ion ScanSteps MS2: &4Exit lensIon accumulationQ0Q1Q2Q3Precursor ionselectionFragmentationlinear ion trap3x10-5 TorrN2 CAD GasSelection of a single parent ion by Q1 allows separate product ion scans for coeluting compounds to be easily generated.Provided they don’t have the same mass…
39 Example EPI Data Oxycodone (316.2) is selected by Q1. Q2 fragments oxycodoneQ3 operating as an LIT traps all the fragments, and the scans them out.“Enhanced” means using the LIT.
40 Linking MRM and EPI MRM is an excellent survey method. Allows for stacking of many transitionsRelatively fast* cycle timeEPI is an excellent for qualitative identificationParent ion linked fragmentation patternMany fragments that can be library matched.An ideal qualitative method would use MRM to look for drugs, and EPI to confirm them.
41 3200 Qtrap Overview: The Source ISiheGS1TEM &GS2Orifice PlateCurtain PlateVoltage, Temperature & Gas ParametersCUR
42 Source Parameters IonSpray Voltage (IS)  The voltage applied between the needle and orifice plate that “ionizes” and nebulizes the liquid flow. Polarity determines what type of ions will reach MS. In positive mode typically 4000 and 5500V; In negative mode –3000 to –4000V.Ion Source Gas 1 (GS1) The nebulizer gas pressure. Facilitates droplet formation. Higher flow, higher GS1.
43 Source Parameters Temperature (TEM)  Ion Source Gas 2 (GS2)  The temperature of the heater gas (“the hairdryer”). It promotes desolvation. The setting is optimized based on mobile phase flow rate and composition.Ion Source Gas 2 (GS2) The heater gas pressure. Aids in solvent evaporation, increasing ion efficiency. Heated gas stream intersects nebulized liquid stream at about 90o right in front of the curtain plate.Higher liquid flow, and/or higher aqueous mobile phase composition, higher TEM and GS2 required. Needs to be optimized.
44 Source Parameters Curtain Gas (CUR)  Interface Heater (ihe) [ON] High purity N2 that flows between the orifice and the curtain plate. It repulses large droplets and neutrals keeping the Q0 clean.Interface Heater (ihe) [ON]Orifice plate heater. I am sure it is important, but I cannot tell you why.
45 3200 Qtrap: PotentialsQ0Q1Q2Q3EPCEPCXPDPCEMCEMost potentials are relative to the entrance potential (EP).As ions move from the source to the detector they see increasingly negative volatage.
46 More Potentials Declustering Potential (DP) * The voltage applied to the orifice plate.Used to break up ion clusters e.g.( [M+H3O+]+) and reduce chemical noise (increase sensitivity).HOWEVER high DP values can induce fragmentation prior to mass analysis. Generally called “In source CID”. Great for LC/MS. Bad for LC/MS/MS.Entrance Potential (EP) *The voltage between the skimmer (ground) and the entrance to Q0. Typically set to -10V in positive mode.Collision Cell Entrance Potential (CEP) *The potential difference between Q0 and IQ2.Facilitates ion transmission to the collision cell.Most mass dependent parameter
47 Still More Potentials Collision Energy (CE) * The potential difference between the Q0 and Q2.Determines the degree of fragmentation in Q2.Greater CE is usually structurally elucidating unless so high it obliterates the parent molecule into small common mass fragments.(CE = EP – RO2; CE = -5V – (-25V) = 20V)Collision Energy Spread (CES) Since different analytes need different CE for optimized fragmentationCollision Cell Exit Potential (CXP) The potential difference between Q2 and IQ3.Always 4V.