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Why LC/MS/MS?
Background and Theory of Electrospray and Tandem Mass Spectrometry
LC/MS/MS: A Primer
Advantages over GC/MS Importance of Chromatography Atmospheric Pressure Ionization
Focus on Electrospray
Why LC/MS/MS?
Why Liquid Chromatography?
Analysis of labile analytes Analysis of more polar compounds without derivatization. Analysis of significantly higher masses Reduction of lengthy clean-up
Why MS/MS?
Additional structural elucidation Further reduction of clean-up (?) Specificity Useful MS modes
System Configuration
Liquid Chromatography
Ionization
Mass Analyzer
Types of Ionization
Several 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)
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 electrospray
Focus on Electrospray
Electrospray is a method of getting the solution phase ions into the gas phase so that they can be sampled by the mass spectrometer.
Electrospray: Overview
5kV
++ ++ + + + + ++ + + ++ + ++
Orifice Plate
Nebulizing Gas
+
+
+ +
Droplet Formation
+
+ +
To MS
+++++ + + + + + ++ ++ +
+++++ + + + + + ++ ++ +
+ + + + +
+++++ + + + + + ++ ++ +
Columbic Repulsion
=
Surface Tension
Negative mode:
Best suited to acidic drugs that form stable Na salts.
[M-H]-, [M-nH]n- and [M+I-]-
Enhanced 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.
Ion Suppression
Generally more prominent early in an RP-LC run, but can occur at anytime. Underscores the need for good chromatography!
~90%reduction
Questions?
Congratulations!
You have made an ion. Now what do you do with it?
RF only
Scanning RF/DC
Scanning RF/DC
Q1 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.
Triple Quads
In scanning mode 99% ions lost between the rods.
Poorer full scan sensitivity
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.
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.
Ion Traps
Ring Electrode
Ions fill the space between a ring electrode and a pair of end-cap electrodes. Mass analysis and fragmentation occur in the same space.
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.
Hybrid Instruments
Applied Biosystems 3200 Qtrap System
Q0
Q1
Q2
Q3
Typically 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.
LIT Scanning
Main RF Ramped
Radial Trapping
Axial Trapping
Q2
Auxiliary RF Ramped.
Radial Trapping
Modes of Operation
Triple Quads and Ion Traps
Full Scan (LC/MS) MRM (Multiple Reaction Monitoring) Product Ion Scan (PI)
H Y B R I D S
Q0
Q1
Q2
Q3
Fragmentation
N2 CAD Gas
Q1 Selects an [M+H]+ Q2 fragments the selected ion. Q3 monitors only one daughter ion
MRM
Steps MS2:
Ion accumulation
3 &4
Exit lens
Q0
Q1
Q2
Q3
Fragmentation
N2 CAD Gas
Only 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
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.
Q0
Q1
Q2
Q3
Fragmentation
N2 CAD Gas
Q1 selects a parent ion. Q2 fragments the selected ion Q3 traps then scans out all fragment ions.
Q0
Q1
Q2
Q3
Fragmentation
N2 CAD Gas
Selection of a single parent ion by Q1 allows separate product ion scans for coeluting compounds to be easily generated.
Provided they dont have the same mass
Oxycodone (316.2) is selected by Q1. Q2 fragments oxycodone Q3 operating as an LIT traps all the fragments, and the scans them out. Enhanced means using the LIT.
An ideal qualitative method would use MRM to look for drugs, and EPI to confirm them.
IS
ihe
++ ++ + + + + + + + + +++
+
++++ + + + + + ++
TEM &GS2
+
+
+
Orifice Plate
Source Parameters
IonSpray Voltage (IS) [5000]
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.
Source Parameters
Temperature (TEM) [400]
The temperature of the heater gas (the hairdryer). It promotes desolvation. The setting is optimized based on mobile phase flow rate and composition.
Higher liquid flow, and/or higher aqueous mobile phase composition, higher TEM and GS2 required. Needs to be optimized.
Source Parameters
Curtain Gas (CUR) [35]
High purity N2 that flows between the orifice and the curtain plate. It repulses large droplets and neutrals keeping the Q0 clean.
DP
EP
CEP CE
CXP CEM
Most potentials are relative to the entrance potential (EP). As ions move from the source to the detector they see increasingly negative volatage.
More Potentials
Declustering Potential (DP) [45]*
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.