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© Waters Corp. 2004©2004 Waters Corporation
Troubleshooting Common MS Problems
by Claude Mallet, Ph.D
claude_mallet@waters.com
presented by
Michael S. Young, Ph.D.
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Troubleshooting Common MS Problems
Overview of Troubleshooting Strategy
ESI sources parameters
Single and triple Quadrupoles
SIR vs MRM
Ion Suppression
Outline
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Try to simplify --assess impact on lab efficiency --
inspect the MS or /MS/MS --try to categorize
troubleshoot the easiest to fix items first
CHEMISTRY MECHANICAL IMPROPER SETTINGS
Adducts (Na+, K+)Multiple chargeIon stability (pH)Ion suppression
Ion beam instabilityProbe cloggingHeater/sensorN2 gas flow Loss of vacuumPower supply
ESI sources parametersQuadrupoles parametersAcquisition modes
MS Troubleshooting Strategy
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Sample Preparation Chromatography
MassSpectrometry
Polarity:Silica- C18, C8, C4, C2Hybrid- C18, C8, C4, C2Polymer- C18, C8, C4, C2Embedded polar groupCyano, PhenylParticle size:2.5, 3.5, 5 or 7 µmInternal diameter:4.6, 3.9, 2.1, 1.0, 0.32 mm and 75 µmLength:150, 100, 50, 30, 20 mm
Source:ESIAPcINano-ESIMass analyzers:magnetic sectorselectric sectorstime of flightquadrupoleion trapFT-ICR
Raw sample:- CaCO2, microsomes, P450,hepatocytes … etc- tissue, CSF, plasma, serumurine, tears … etc- water, sediment, food … etc
Extracted sampleFor LC/MS/MS
The Total Analysis
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BA MassSpectrometry
ESI source parameters
Part 1
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Quattro UltimaZQ
Quattro Premier
Mass Spectrometers
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First, let’s take a look at the first event in an ESI orthogonal source. The primary function of the probe is to transform a liquid (from LC column or other source) into a gas stream asshown in the red circle. Three parameters are used to optimize the probe, which are the 1- nebulizer gas, 2- the desolvation gas flow and 3-the desolvation temperature. The nebulizer gas is automatically set at maximum on the ZQ and manually on other mass spectrometers (i.e. Ultima, QToF, LCT … etc). The desolvation gas flow and desolvation temperature can be optimized to maximizesignal intensity. Highertemperatures are required when using mobile phasescontaining high percentage of water.
2,31
Mass Spectrometers
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Nebulizer gas flow off Nebulizer gas flow on
Notice the formation of a liquid drop. It can lead to source flooding if unattended for a long period of time. To avoid potential electrical hazard, the source is equipped with a drain valve.
Notice the formation of liquid droplets from condensation of the sprayer on the probe holder assembly.
ESI Probe Parameters
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Isolation valve
Cone shield and cone assembly
Baffle
Stainless steel capillary
Desolvation heater
Ion Block
ESI probe
ESI Source
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10 mm5 mm
Cone
Probe
ESI Probe Parameters
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Capillary Tip
Make sure capillary extends approx. 0.5 mm beyond probe tip.
Any corrosion, deposit constriction or other flow restriction will hinder proper nebulization.
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Probe too far from the cone?
Probe extends too far past cone?
ESI Probe Parameters
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Probe too close to the cone?
ESI Probe Parameters
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Response ofreserpine shows a good gaussian distribution with baselineresolution of the C13 isotopes
Temperature and gas flow are parameters that affect the desolvation efficiency of the probe. Improper settings can result in loss of signal. These values are optimized according to the column flow rate.
ESI Probe Parameters (tune page)
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In this case, a too lowdesolvation temperature resulted in a 50 % reduction in signal intensity.
This effect is compound dependent.
Desolvation Temperature
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Similar loss of signal intensity, in this case, it is due to a too lowsetting of the desolvation gas (113 vs 550 L/hr). The gas used for the desolvation is nitrogen
It must be of high purity (99.95%) and oil free. (traps can be used to increase the gas purity if needed).
Make sure delivery pressure is regulated to 100 psi.
Desolvation Gas Flow
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Column flow rate Desolvation temp Desolvation gas flowµL/min °C liters/hr
< 10 100 to 120 200 to 25010 to 20 120 to 250 250 to 40020 to 50 250 to 350 250 to 400>50 350 to 400 400 to 750
Higher desolvation temperatures give increased sensitivity. However, increasing the temperature above the range suggested reduces beam stability. Increasing the gas flow rate higher that the quoted values lead to unnecessary high nitrogen consumption. Avoid operating the desolvation heater for long periods of time without proper gas flow.To do so could damage the source.
Suggested Settings
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Dewar tanks Nitrogen generator
Both setups are widely used and the choice mostly depends on the consumption of nitrogen per day. Larger laboratories will have a tendency to choose the nitrogen generator for convenience and cost for long term operation.
Dewar Tank vs Nitrogen Generator
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At this point let’s take a look at the second event. Once a spray is stable, ions are produced and directed toward the mass analyzer. Five parameters in the orthogonal source are used for this purpose. These parameters are: 1- capillary voltage, 2- cone voltage, 3-extraction voltage and 4- RF lens (transfer optics) 5- Source temperature. A high voltage, in the kV range, is applied to a stainless steel capillary tubing in the probe. This will produce charged droplets. With the assistance of the desolvation gas flow and desolvation temperature, those droplets will in turn produce ions in gas phase next to the cone. The cone voltage attracts positively charged ions from the spray into a reduced pressure chamber (ion block). The extractor and RF lens are used to guide the ion beam into the mass analyzer
1 2
3 4
ESI Probe
ESI Source
ESI Source Parameters
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Clean cone and cone shield
Notice the white residue on the cone shield, but the aperture of the cone is still clear. This is an indication that samples injected on this MS were not clean. In both pictures, the baffle shows brown spots, which indicates routine and normal usage. The white residue can result from long exposure to poorly prepared samples or from nonvolatile mobile phase additives.Over time, the aperture of the cone will become clogged, thus reducing signal intensity.
cone
baffle
Brown spot
ESI Source Parameters
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These are typical starting values to obtain a stable ion beam with flow rate ranging from 0.2 to 0.4 ml/min.
The ion block is heated to avoid any condensation problems.
The source has a maximum setting of 150 °C.
ESI Source Parameters
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With insufficientcapillary voltage, the signal shows an 80%decrease in signal intensity.
Typical optimum values for most small molecules are between 3.0 and 3.5 kV.
Higher values usually have little effect on signal intensity.
Deviations from experimentally optimized value may indicate problems in the source.
Capillary Voltage
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The cone voltage isapplied to a spherical metal plate, the first gate between the sprayer (at atmospheric pressure) and the inside of the mass analyzer (at 10-6
Torr of pressure). The cone creates the first bend of the ion beam in the orthogonal source.
This slide shows that we have optimized the cone voltage at 35 volts and increased our signalintensity.
Cone Voltage
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Poor response can occur if cone voltage is set too low. Asufficient voltage isrequired to atract a high population of ions into the ion block.
Once the cone volatage is optimized, loss of sensitivity may result from contamination at the cone.
Cone Voltage
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Poor response can also occur if cone voltage is set too high. Too much energy causes a phenomenon known as “In-sourcefragmentation”.
When ions are accelerated from the sprayer to the ion block with very high velocities, collisions among ions can create a high population of daughter ions at the expense of parentions.
In this case, the ion at m/z 609 shows a 90% reduction in signal intensity.
Cone Voltage
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The extractor voltage is applied to a second cone shaped metal plate that separatesthe ion block and the mass analyzer. This plate creates a second 90 degree angle in the ion beam, completing the Z spray shape.
An incorrect voltage setting of the extractor resulted in a 70% reduction in signal intensity.
Typical extractor voltage settings range from 1 to 3 volts;higher values will not usually give better sensitivity. Higher than expected values may indicate contamination in the source block
Extractor Voltage
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The RF lens focuses the ion beam as it passes into the mass analyzer. In the tandem mass-spectrometer, it focuses the beam to the center of thetransition lens hexapole assembly.
The RF lens valueshould typically be set to range from 0.1 to 0.5 volts.
RF Lens
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3.5
3.5
3. 3.
In the example shown, we needed to increase the RF lens to achieve a symmetrical peak shape. This may indicate that the source is contaminated.
RF Lens
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BA MassSpectrometry
ESI source parametersQuadrupoles
Part 2
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The quadrupole mass analyzer, like other type of mass analyzers (I.e. ToF, ion traps, sector … etc) separates ions according to their mass to charge ratio (m/z). The quadrupole is made of 4 highly polished metal rods positioned at precise angles from one another. These rods are connected to high voltage power supply (DC, positive/negative) and a radio frequency (RF) generator. The slope of RF/DC applied to the rods is proportional to a range or a specific mass to charge ratio.
Quadrupole Mass Analyzers
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Source
DetectorNonresonant Ion
Resonant Ion
dc and rf voltages
+Udc + V cosωt
-Udc – V cosωt
Molybdenum Alloy
Quadrupole Schematic
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Stable ion
Non-resonantTrajectory
Pre-Filters
ResonantTrajectory
Quadrupole
Resonant vsNon-Resonant Trajectory
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Quadrupole Unit Mass Resolution
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572.8339
570 571 572 573 574 575 576 577m/z0
100
%
0
100
%
573.9185
574.8116
573.2997
574.3072
575.3155
QuadrupoleResolution: 1000
Q-ToFResolution: 10 000
[M+H]+
Isotopes
Bradykinin Frag 1-5:Arg-Pro-Pro-Gly-Phe
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Quadrupole
Resolution: 1000
285 286 287 288 289m/z0
100
%
0
100
%
286.4118
287.1521
287.6505
288.1563
[M+H]+2
Q-ToF
Resolution: 10 000
Isotopes
Bradykinin Frag 1-5:Arg-Pro-Pro-Gly-Phe
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The low and high mass resolution are arbitrary values that are calculated from the RF/DC ratio.
The LM setting affects the resolution of ions at the low mass range of the quadrupole; the HMsetting at the high mass range of the quadrupole.
The quadrupole can only achieve mass unit resolution, which means that multiple chargedpeaks are not fully resolved.
Low andHigh Mass Resolution
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.
Udc
V
(DC voltage)
(Rf voltage)
Correct V/U ratiomass 1 & 2 are resolved
R: 1000
V/U slope
Stable trajectory
Unstable trajectory
Quadrupole Stability Diagram
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If LM and HM resolution are set too low, the quadrupole acts as a transmission cell (RF only). The isotope peaks merge with the main peak.
Notice the increase insignal intensity at the expense of a significant loss of resolving power.
On the other hand, if the values are too high, the quadrupole is over resolved with resulting poor sensititivity.
Low and High Mass Resolution
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.
Udc
V
(DC voltage)
(Rf voltage)
Low V/U ratiomass 1 & 2 merge together
R: 10V/U slope
Stable trajectory
Unstable trajectory
Quadrupole Stability Diagram
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In this case, the LH and HM resolution were set too high. The ion beam falls in the nonresonantportion of the stability diagram shown earlier.
Under these conditions, the ion beam will not reach the multiplier at the back of the mass spectrometer and produce a signal.
Low and High Mass Resolution
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.
Udc
V
(DC voltage)
(Rf voltage) Incorrect V/U ratiomass 1 & 2 are over resolved
V/U slope
Unstable trajectory
Stable trajectory
Quadrupole Stability Diagram
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The ion energy is applied to a small lens positioned between the quadrupole and the multiplier. This lens is used to refocus the beam toward the multiplier. Typical values range from 0.3 to 0.6.
As shown here, higher values will produce distortion and loss of resolution betweenthe peak and isotopes.
Ion Energy
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The multiplier is the last step in the signal production. The ionsproduced by the ESI source and filtered by the quadrupole are converted by the multiplier into ameasurable current.
If the multiplier is set too low, as shown here, the signal intensity will be considerably reduced. Too high a multiplier setting produces saturation (flat-top peaks) and poor quantitation.
Multiplier
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BA MassSpectrometry
MS/MS
Part 3
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Single Ion Recording(SIR Mode)
Static
Full Scan(MS mode)
ScanningLOQ = 500 pg (quantity injected) LOQ = 5 pg (quantity injected)
Note: A quadrupole mass spectrometer is typically available with amass range of 2000 Daltons or 4000 Daltons
Single Q Mode of Acquisition
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Full Scan Acquisition
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0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00Time0
100
%
Scan ES+TIC
2.06e92.76
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00Time0
100
%
Scan ES+TIC
3.12e92.76
2.56
3.182.98
3.29
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00Time0
100
%
Scan ES+TIC
5.60e93.18
2.962.54 2.743.27
[ ] = 50 ng/mL
[ ] = 5 ng/mL mixture of 5 basic compounds
[ ] = 500 ng/mL
Full Scan Acquisition
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Single Ion Recording (SIR)
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0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00Time0
100
%
SIR of 5 Channels ES+TIC
3.77e72.76
2.55
0.85 2.26
3.182.97
3.28
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00Time0
100
%
Scan ES+TIC
2.06e9
[ ] = 5 ng/mL
[ ] = 5 ng/mL
Scan mode
SIR mode
Single Ion Recording (SIR)
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A single quadrupole mass analyzer can be operated in two distinct modes, SCAN and SIR. A triple quadrupole mass spectrometer can offer 4 types of acquisition; 1- Daughter scan, 2- Multiple Reaction Monitoring (MRM), 3- Parent scan and 4- Constant neutral loss or gain scan. These types of scans rely on the middle quadrupole called the “collision cell”. The collision cell is in fact a hexapole (6 rods) that operates in RF mode only (no resolution capacity). The cell can be pressurize with argon gas. This provides a physical surface onto which ions filtered by MS1 can be fragmented by collision, hence the term “collision induced dissociation”. Depending if MS1 and MS2 are set in scan or park mode will determine the desired type of acquisition mentioned earlier.
Tandem Mass SpectrometryCollision Induced Dissociation (CID)
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Parent Ion Scanning
MS1 MS2Collision
Cell
StaticScanning
Triple Q Modes of Acquisition
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Daughter Ion Scanning
MS1 MS2Collision
Cell
Static Scanning
Triple Q Modes of Acquisition
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Constant Neutral Loss or Gain
MS1 MS2Collision
Cell
ScanningScanning
Triple Q Modes of Acquisition
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Multiple Reaction Monitoring
MS1 MS2Collision
Cell
Static Static
Triple Q Modes of Acquisition
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MS1 MS2Daughter
MS1
CID
MS2
A triple quadrupole mass spectrometer offers lowersensitivity and reproducable fragmentation. With Multiple Reaction Monitoring (MRM), up to 1000x in sensitivity can be achieved in comparison to scan mode. The next slides will describe some of the common problems associated with MRM and a guide on how to optimize MRM transitions.
We infused a basic drug (clemastine) and openedwindows for MS1, daughterand MS2. Notice the mass unit resolution of the parent mass and isotopes on both MS1 and MS2.
Erratum: the optimized values of IE1 and IE2 are 0.4 and 0.8 respectively
Optimizing an MRM
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Next, the LM/HM (1)values are lowered to the point that the first isotope and the parent ion are both passed into the collision cell. The peaks in the MS1 windows (2) will broaden and show loss of resolution. Conequently,the ion beam passing from MS1 to the collision cell is also increased (3). In the daughter scan window (middle window in the tune page), the parent peak is offscale and one isotope of the molecule is evident.Since MS2 is set with unit mass resolution setting (LM/HM = 15 ), good resolution is seen in the third window among the parent peak and the isotopes.
1
23
Erratum: the optimized values of IE1 and IE2 are 0.4 and 0.8 respectively
Optimizing an MRM
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1
2
In this slide, LM/HM on MS1is slightly increased ( small gain in resolution) just to the point that the isotope is not seen. This step is crucial, if LM/HM on MS1 are too low, additional ions will enter the collision cell and will create additional daughter ions for each isotope of the parent molecule.
Erratum: the optimized values of IE1 and IE2 are 0.4 and 0.8 respectively
Optimizing an MRM
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1 2
3
Then, by decreasing the LM/HM on MS2 (1), the signal in the daughter scan window has increased (3). The resolution on MS2 also decreases as a consequence of lowering the LM/HM values (2).
Erratum: the optimized values of IE1 and IE2 are 0.4 and 0.8 respectively
Optimizing an MRM
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Let’s take a look at a common problem when optimizing an MRM transition. If we look at the LM/HM values (1,2) on both MS1 and MS2, the quadrupoles are set at unit mass resolution. This can be verified in the MS1 and MS2window in the tune page. The peaks shows a gaussian distribution and resolution with the isotopes. However, the daughter window in the tune page shows no signal (4). The answer is quite simple; choosing a correct MRM transition also requires us to park MS1 on the top of the parent peak. The parent peak has a molecularweight of 344.2 Da (see previous slide). In this example, the setting was incorrect, 343.7 Da. The difference of 0.5 Da (3) was enough to miss the parent peak completely in MS1, thus leading to a total loss of signal in MS2.
1
2
3
4
Erratum: the optimized values of IE1 and IE2 are 0.4 and 0.8 respectively
Optimizing an MRM
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1
2
34
5
At this point, the quadrupolesare optimized to give maximum signal intensity (1,2) and MS1 is correctly set at 344,2 Daltons (4). In this tune page both MS1and MS2 windows were removed so we can concentrate on the daughter ion scan (5). As we can see, the tune page only shows the parent ion without any daughter ions. This is because the collision gas was not activated (6) and the collision cell was not optimized to produce daughters ions from collision with argon gas (3).
6
Erratum: the optimized values of IE1 and IE2 are 0.4 and 0.8 respectively
Optimizing an MRM
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Prior to introduction of collision cell gas (1). thepressure on the collision cell pirani guageindicates 1.0 e-4 mbar(2). Also, since there is no argon gas in the collision cell, the analyzer penning guage shouldshow a pressure in the vicinity of 1-2 e-5 mbar (3). This pressure indicates that the entire mass analyzer is under optimum vacuum.
1 2 3
Optimizing an MRM
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1
2
When the collision gas button is activated (1),argon gas will flow freely into the collision cell located inside the mass analyzer (between MS1 and MS2). Notice that the pressure on the collision cell gage will increase (2), typical values are between 2 to 3e-4 mbar.
Optimizing an MRMTune Page with Gas Cell Pressure
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1 2
3
Once the argon gas pressure is optimized in the collision cell, it requires some energy to produce fragments. In this case, the collision energy is set at 15 (1) (arbitrary units). The result is the production of two major fragments at 215 Da and 128 Da(2) of the parent ion of mass 344.20 Da. Notice that the energy level is still low enough to see a small fraction of the parent ion (3).
Erratum: the optimized values of IE1 and IE2 are 0.4 and 0.8 respectively
Optimizing an MRMSetting Collison Cell Energy
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1
23
In this scenario, the collision energy was purposely increased to higher values (1)that gives a 100 % conversion of the parent ion (3) into fragments ions. However, the level of energy is also high enough to produce further fragmentation of smaller daughter ions (2) and to reduce the intensity to the larger fragments. This type of setting is not favored for trace analysis. The optimum for sensitivity is to use conditions that will produce a 100 % conversion of the parent ion into one or two majors fragments. Hhowever, the production of more than two fragments may be desirable for verification of unknowns.
Erratum: the optimized values of IE1 and IE2 are 0.4 and 0.8 respectively
Optimizing an MRMSetting Collison Cell Energy
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100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400m/z0
100
%
0
100
%
0
100
%
344.2
215128
215
128344.2
215
128
CID 0 volts
CID 10 volts
CID 20 volts
[M+H]+
NCH3
O
CH3
Cl215
128
Clemastine
(Different scale)
Daughter Ion Spectrum
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Note: typical value of dwell times are between 0.2 and 0.05 seconds
Multiple MRM
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0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00Time0
100
%
MRM of 5 Channels ES+TIC
2.91e52.95
2.542.76
3.183.27
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00Time0
100
%
SIR of 5 Channels ES+TIC
2.29e6
2.27 2.75
2.56
3.182.96 3.27
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00Time0
100
%
Scan ES+TIC
2.04e9
[ ] = 0.1 ng/mLScan mode
[ ] = 0.1 ng/mLSIR mode
[ ] = 0.1 ng/mLMRM mode
Multiple MRM
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BA MassSpectrometry
Ion suppression
Causes of Ion SuppressionTroubleshooting Ion Supression
Part 4
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What is ion suppression or enhancement ?
* All spectrums at same scale
Ion suppression or enhancement is a known phenomenon that occurs with a mass spectrometer equipped with an electrospray interface (ESI). Several papers in the literature explain in detail the formation of ions with this type of source, but several parameters, effects or observations are still unclear. For example, is ESI concentration or mass flow dependent? Some papers suggest that ESI is concentration/mass flow combination. Most applications use the ESI source in combination with LC (mostly reversed-phase column). The problem of suppression or enhancement occurs when additives in the mobile phase can either increase or decrease the intensity of the target analyte. Sample extracts also produce the same effect.
Scan ES+
Scan ES+
100
0
%
Scan ES+472.63 50/50 Water/ACN + 0.5 % NH 4OH
Peak intensity: 5 143 003 136Signal increase: + 41 %
100
0
%
472.63
50/50 Water/ACNPeak intensity: 3 636 985 856
100
464 466 468 470 472 474 476 478 480 482 484m/z0
%
472.63
50/50 Water/ACN + 0.5 % TFAPeak intensity: 893 059 072Signal decrease: -75 %
Terfenadine* All spectrums at same scale
Ion suppression or enhancement
occurs with a mass spectrometer equipped with an electrospray interface (ESI). Several papers in the literature explain in detail the formation of ions with this type of source, but several parameters, effects or observations are still unclear. For example, is ESI concentration or mass flow dependent? Some papers suggest that ESI is concentration/mass flow combination. Most applications use the ESI source in combination with LC (mostly reversed-phase column). The problem of suppression or enhancement occurs when additives in the mobile phase can either increase or decrease the intensity of the target analyte. Sample extracts also produce the same effect.
Scan ES+
Scan ES+
100
0
%
Scan ES+472.63 50/50 Water/ACN + 0.5 % NH 4OH
Peak intensity: 5 143 003 136Signal increase: + 41 %
100
0
%
472.63
50/50 Water/ACNPeak intensity: 3 636 985 856
100
464 466 468 470 472 474 476 478 480 482 484m/z0
%
472.63
50/50 Water/ACN + 0.5 % TFAPeak intensity: 893 059 072Signal decrease: -75 %
Terfenadine
Ion suppression or enhancement
occurs with a mass spectrometer equipped with an electrospray interface (ESI). Several papers in the literature explain in detail the formation of ions with this type of source, but several parameters, effects or observations are still unclear. For example, is ESI concentration or mass flow dependent? Some papers suggest that ESI is concentration/mass flow combination. Most applications use the ESI source in combination with LC (mostly reversed-phase column). The problem of suppression or enhancement occurs when additives in the mobile phase can either increase or decrease the intensity of the target analyte. Sample extracts also produce the same effect.
Scan ES+
Scan ES+
100
0
%
Scan ES+472.63 50/50 Water/ACN + 0.5 % NH 4OH
Peak intensity: 5 143 003 136Signal increase: + 41 %
100
0
%
Scan ES+472.63 50/50 Water/ACN + 0.5 % NH 4OH
Peak intensity: 5 143 003 136Signal increase: + 41 %
100
0
%
472.63
50/50 Water/ACNPeak intensity: 3 636 985 856
100
0
%
472.63
50/50 Water/ACNPeak intensity: 3 636 985 856
100
464 466 468 470 472 474 476 478 480 482 484m/z0
%
472.63
50/50 Water/ACN + 0.5 % TFAPeak intensity: 893 059 072Signal decrease: -75 %
Terfenadine
100
464 466 468 470 472 474 476 478 480 482 484m/z0
%
472.63
50/50 Water/ACN + 0.5 % TFAPeak intensity: 893 059 072Signal decrease: -75 %
Terfenadine
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Various type of additives can increase or decrease the signal of a target analyte. Furthermore, since ESI is compound dependent, it is expected to see variation in signal intensity as well as suppression or enhancement effect. At this point, let’s take a look at common additives used in LC and the response profile of various SPE extraction protocols.
Acidic additive Buffers SPE extracts
Trifluoroacetic acid Ammonium formate protein precipitationAcetic acid Ammonium bicarbonate Oasis HLB 1-DFormic Acid Ammonium biphosphate Oasis HLB 2-D
Oasis MCXBasic additive Ion pairing additive
Ammonium hydroxide Tetraethylammonium hydroxidePyrrolidine Dimethylhexylamine
Detergents
Triton X100SDS
What is ion suppression or enhancement ?
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2795 ESI-MS
Infusion pumpUsed to add range of modifiers, salts, ion pairs, pH additives,Matrix extracts
50/50 ACN/ H2O8 compounds
0.2mL/min
0.2mL/min
ES+260.2 Propranolol291.3 Trimethoprim354.4 Pipenzolate*411.4 Resperidone472.6 Terfenadine485.6 Methoxy-Verapamil591.6 Benextramine609.6 Reserpine
*quaternary amine drug
Compare 50/50 ACN/ H2Oto additive stream signal(triplicates) blank, matrix, blank
Experimental design aimed to look for a better solution –removal of suppression
©2004 Waters Corporation©2004 Waters Corporation
250 300 350 400 450 500 550 600 650 700 750 800 850 900 950m/z0
100
%
0
100
%
Scan ES+6.41e9
354.4
291.3
669.78609.6
581.71
472.6
411.4
537.60485.6
713.74757.83
801.86
845.88889.91
933.93 977.95
Scan ES+ 7.38e9354.4
260.2
291.3
609.6
485.6472.6
411.4591.6
260.2
260.2 - 80 %291.3 - 38 %354.4 - 13 %411.4 - 78 %472.6 - 59 %485.6 - 80 %591.6 - 71 %609.6 - 63 %
50/50 water/ACN Blank
0.5 % Triton X 100
260.2 Propranolol291.3 Trimethoprim354.4 Pipenzolate *411.4 Resperidone472.6 Terfenadine485.6 Methoxy-Verapamil591.6 Benextramine609.6 Reserpine* Quaternary amine molecule
250 300 350 400 450 500 550 600 650 700 750 800 850 900 950m/z0
100
%
0
100
%
Scan ES+6.41e9
354.4
291.3
669.78609.6
581.71
472.6
411.4
537.60485.6
713.74757.83
801.86
845.88889.91
933.93 977.95
Scan ES+ 7.38e9354.4
260.2
291.3
609.6
485.6472.6
411.4591.6
260.2
260.2 - 80 %291.3 - 38 %354.4 - 13 %411.4 - 78 %472.6 - 59 %485.6 - 80 %591.6 - 71 %609.6 - 63 %
50/50 water/ACN Blank
0.5 % Triton X 100
260.2 Propranolol291.3 Trimethoprim354.4 Pipenzolate *411.4 Resperidone472.6 Terfenadine485.6 Methoxy-Verapamil591.6 Benextramine609.6 Reserpine* Quaternary amine molecule
250 300 350 400 450 500 550 600 650 700 750 800 850 900 950m/z0
100
%
250 300 350 400 450 500 550 600 650 700 750 800 850 900 950m/z0
100
%
0
100
%
Scan ES+6.41e9
354.4
291.3
669.78609.6
581.71
472.6
411.4
537.60485.6
713.74757.83
801.86
845.88889.91
933.93 977.950
100
%
Scan ES+6.41e9
354.4
291.3
669.78609.6
581.71
472.6
411.4
537.60485.6
713.74757.83
801.86
845.88889.91
933.93 977.95
Scan ES+ 7.38e9354.4
260.2
291.3
609.6
485.6472.6
411.4591.6
354.4
260.2
291.3
609.6
485.6472.6
411.4591.6
260.2
260.2 - 80 %291.3 - 38 %354.4 - 13 %411.4 - 78 %472.6 - 59 %485.6 - 80 %591.6 - 71 %609.6 - 63 %
50/50 water/ACN Blank
0.5 % Triton X 100
260.2 Propranolol291.3 Trimethoprim354.4 Pipenzolate *411.4 Resperidone472.6 Terfenadine485.6 Methoxy-Verapamil591.6 Benextramine609.6 Reserpine* Quaternary amine molecule
Surfactant
©2004 Waters Corporation©2004 Waters Corporation
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0
100
%
0
100
%
Scan ES+
609.6354.4
260.3
291.3
485.6472.6
411.5591.7
Scan ES+ 354.3
260.3291.2
609.6
485.6472.6
411.4591.7
50/50 Water/ACN + 0.5 % FA
50/50 Water/ACN
260.3 + 5 %291.3 - 5 %354.4 - 5 %411.5 - 54 %472.6 - 7 %485.6 - 2 %591.7 - 52 %609.6 + 17 %
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0
100
%
0
100
%
Scan ES+
609.6354.4
260.3
291.3
485.6472.6
411.5591.7
Scan ES+ 354.3
260.3291.2
609.6
485.6472.6
411.4591.7
260 280 300 320 340 360 380 400 420 440 460 480260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0
100
%
500 520 540 560 580 600 620m/z0
100
%
0
100
%
Scan ES+
609.6354.4
260.3
291.3
485.6472.6
411.5591.7
Scan ES+ 354.3
260.3291.2
609.6
485.6472.6
411.4591.7
50/50 Water/ACN + 0.5 % FA
50/50 Water/ACN
260.3 + 5 %291.3 - 5 %354.4 - 5 %411.5 - 54 %472.6 - 7 %485.6 - 2 %591.7 - 52 %609.6 + 17 %
Acidic Additive
©2004 Waters Corporation©2004 Waters Corporation
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0
100
%
0
100
%
Scan ES+ 354.5
260.3291.4
471.6
411.5
609.6
485.6 591.7
Scan ES+ 354.4
260.3291.3
609.6
485.6472.6
411.6591.7
260.3 + 10 %294.4 + 4 %354.4 0 %411.5 + 16 %471.6 + 57 %485.6 + 46 %594.7 + 37 %609.6 - 6 %
50/50 Water/ACN + 0.5 % NH4OH
50/50 Water/ACN
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0
100
%
0
100
%
Scan ES+ 354.5
260.3291.4
471.6
411.5
609.6
485.6 591.7
Scan ES+ 354.4
260.3291.3
609.6
485.6472.6
411.6591.7
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0
100
%
500 520 540 560 580 600 620m/z0
100
%
0
100
%
Scan ES+ 354.5
260.3291.4
471.6
411.5
609.6
485.6 591.7
Scan ES+ 354.4
260.3291.3
609.6
485.6472.6
411.6591.7
260.3 + 10 %294.4 + 4 %354.4 0 %411.5 + 16 %471.6 + 57 %485.6 + 46 %594.7 + 37 %609.6 - 6 %
50/50 Water/ACN + 0.5 % NH4OH
50/50 Water/ACN
Basic Additive
©2004 Waters Corporation©2004 Waters Corporation
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660m/z0
100
%
Scan ES+ 1.95e8
354.44
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660m/z0
100
%
Scan ES+ 7.38e9354.44
260.28
291.31
609.55
485.62472.57
411.49591.66
591.66
50/50 Water/ACN Blank
50 mM Tetraethylammonium hydroxide260.2 - 100 %291.3 - 100 %354.4 - 88 %411.4 - 100 %472.5 - 100 %485.5 - 100 %591.6 - 94 %609.5 - 100 %
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660m/z0
100
%
Scan ES+ 1.95e8
354.44
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660m/z0
100
%
Scan ES+ 7.38e9354.44
260.28
291.31
609.55
485.62472.57
411.49591.66
591.66
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660m/z0
100
%
Scan ES+ 1.95e8
Scan ES+ 1.95e8
354.44
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660m/z0
100
%
Scan ES+ 7.38e9354.44
260.28
291.31
609.55
485.62472.57
411.49591.66
591.66
50/50 Water/ACN Blank
50 mM Tetraethylammonium hydroxide260.2 - 100 %291.3 - 100 %354.4 - 88 %411.4 - 100 %472.5 - 100 %485.5 - 100 %591.6 - 94 %609.5 - 100 %
Ion-Pairing Reagent
©2004 Waters Corporation©2004 Waters Corporation
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0
100
%
0
100
%
Scan ES+
354.4
291.3260.3
591.7472.6411.5
485.6 609.6
Scan ES+ 354.4
260.3
291.3
609.6
485.6472.6
411.5591.7
50/50 Water/ACN + 0.1M NaCl
50/50 Water/ACN
260.3 - 93 %291.3 - 95 %354.4 - 37 %411.5 - 62 %472.6 - 71 %485.6 - 84 %591.7 - 45 %609.6 - 95 %
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0
100
%
0
100
%
Scan ES+
354.4
291.3260.3
591.7472.6411.5
485.6 609.6
Scan ES+ 354.4
260.3
291.3
609.6
485.6472.6
411.5591.7
260 280 300 320 340 360 380 400 420 440 460 480260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0
100
%
500 520 540 560 580 600 620m/z0
100
%
0
100
%
Scan ES+
354.4
291.3260.3
591.7472.6411.5
485.6 609.6
Scan ES+ 354.4
260.3
291.3
609.6
485.6472.6
411.5591.7
50/50 Water/ACN + 0.1M NaCl
50/50 Water/ACN
260.3 - 93 %291.3 - 95 %354.4 - 37 %411.5 - 62 %472.6 - 71 %485.6 - 84 %591.7 - 45 %609.6 - 95 %
Salt Adducts
©2004 Waters Corporation©2004 Waters Corporation
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0
100
%
0
100
%
Scan ES+
591.7
354.4 518.6472.6 546.6 609.6
Scan ES+ 354.4
260.2
291.2
609.6
485.6472.6
411.4 591.6
.
50/50 Water/ACN
260.3 - 98 %291.3 - 98 %354.4 - 87 %411.4 - 94 %472.6 - 92 %485.6 - 95 %591.7 - 42 %609.6 - 94 %
50/50 Water/ACN + rat plasma supernatant
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0
100
%
0
100
%
Scan ES+
591.7
354.4 518.6472.6 546.6 609.6
Scan ES+ 354.4
260.2
291.2
609.6
485.6472.6
411.4 591.6
.
260 280 300 320 340 360 380 400 420 440 460 480260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0
100
%
500 520 540 560 580 600 620m/z0
100
%
0
100
%
Scan ES+
591.7
354.4 518.6472.6 546.6 609.6
Scan ES+ 354.4
260.2
291.2
609.6
485.6472.6
411.4 591.6
.
50/50 Water/ACN
260.3 - 98 %291.3 - 98 %354.4 - 87 %411.4 - 94 %472.6 - 92 %485.6 - 95 %591.7 - 42 %609.6 - 94 %
50/50 Water/ACN + rat plasma supernatant
Rat Plasma
©2004 Waters Corporation©2004 Waters Corporation
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0
100
%
0
100
%
Scan ES+
591.7
354.4 518.5472.6 485.5 609.6
Scan ES+ 354.4
260.2
291.3
609.6
485.6472.6
411.5591.6
50/50 Water/ACN + human plasma supernatant
50/50 Water/ACN
260.2 - 97 %291.2 - 96 %354.4 - 86 %411.4 - 93 %472.6 - 93 %485.6 - 95 %591.6 - 89 %609.5 - 93 %
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0
100
%
0
100
%
Scan ES+
591.7
354.4 518.5472.6 485.5 609.6
Scan ES+ 354.4
260.2
291.3
609.6
485.6472.6
411.5591.6
50/50 Water/ACN + human plasma supernatant
50/50 Water/ACN
260 280 300 320 340 360 380 400 420 440 460 480260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0
100
%
500 520 540 560 580 600 620m/z0
100
%
0
100
%
Scan ES+
591.7
354.4 518.5472.6 485.5 609.6
Scan ES+ 354.4
260.2
291.3
609.6
485.6472.6
411.5591.6
50/50 Water/ACN + human plasma supernatant
50/50 Water/ACN
260.2 - 97 %291.2 - 96 %354.4 - 86 %411.4 - 93 %472.6 - 93 %485.6 - 95 %591.6 - 89 %609.5 - 93 %
Human Plasma
©2004 Waters Corporation©2004 Waters Corporation
Condition/Equilibrate1.0 mL methanol / 1.0 mL water
Load1.0 mL plasma
Wash1.0 mL 5% methanol in water
Elute0.5 mL MeOH
Dilute with 0.5 ml water
Plasma Sample
* 30 mg HLB 96 plate
Reversed Phase SPE
©2004 Waters Corporation©2004 Waters Corporation
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0
100
%
0
100
%
Scan ES+
354.4
260.1 291.2
591.6
472.5
411.4485.5
609.5
Scan ES+ 354.2
260.2
291.2
609.5
485.4472.5
411.4591.6
50/50 Water/ACN + rat plasma HLB 1D extract
50/50 Water/ACN
260.2 - 41 %291.2 - 26 %354.4 - 9 %411.4 - 32 %472.6 - 23 %485.6 - 38 %591.6 + 26 %609.5 - 49 %
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0
100
%
0
100
%
Scan ES+
354.4
260.1 291.2
591.6
472.5
411.4485.5
609.5
Scan ES+ 354.2
260.2
291.2
609.5
485.4472.5
411.4591.6
50/50 Water/ACN + rat plasma HLB 1D extract
50/50 Water/ACN
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620m/z0
100
%
0
100
%
Scan ES+
354.4
260.1 291.2
591.6
472.5
411.4485.5
609.5
Scan ES+ 354.2
260.2
291.2
609.5
485.4472.5
411.4591.6
50/50 Water/ACN + rat plasma HLB 1D extract
50/50 Water/ACN
260.2 - 41 %291.2 - 26 %354.4 - 9 %411.4 - 32 %472.6 - 23 %485.6 - 38 %591.6 + 26 %609.5 - 49 %
Reversed Phase SPE - Rat Plasma
©2004 Waters Corporation©2004 Waters Corporation
Condition/Equilibrate1.0 mL methanol / 1.0 mL water
Load1.0 mL plasma
Prepare Sample Solution
Wash 21.0 mL MeOH
Elute0.5 mL MeOH + 2% NH4OH
Dilute with 0.5 ml water
Wash 11.0 mL Water + 2 % FA
Locks basic drugon ion exchanger
Removes polar interferences
* 30 mg Oasis MCX 96 well plate
Mixed Mode Cation-Exchange SPE
©2004 Waters Corporation©2004 Waters Corporation
260.2 - 9 %291.2 - 11%354.4 - 0.5 %411.4 - 13 %472.6 - 9 %485.6 - 2 %591.6 - 8 %609.5 - 8 %
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640m/z0
100
%
0
100
%
Scan ES+
354.4
291.3260.3
609.6
472.6
411.5
485.6
591.7
Scan ES+ 354.4
260.3291.3
609.6
485.6472.6
411.5 591.7
50/50 Water/ACN + rat plasma MCX extract
50/50 Water/ACN
260.2 - 9 %291.2 - 11%354.4 - 0.5 %411.4 - 13 %472.6 - 9 %485.6 - 2 %591.6 - 8 %609.5 - 8 %
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640m/z0
100
%
0
100
%
Scan ES+
354.4
291.3260.3
609.6
472.6
411.5
485.6
591.7
Scan ES+ 354.4
260.3291.3
609.6
485.6472.6
411.5 591.7
50/50 Water/ACN + rat plasma MCX extract
50/50 Water/ACN
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640m/z0
100
%
0
100
%
Scan ES+
354.4
291.3260.3
609.6
472.6
411.5
485.6
591.7
Scan ES+ 354.4
260.3291.3
609.6
485.6472.6
411.5 591.7
50/50 Water/ACN + rat plasma MCX extract
50/50 Water/ACN
Mixed Mode SPE – Rat Plasma