Waters LCMSWaters LCMS Troubleshooting

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.
2004 Waters Corporation 2004 Waters Corporation
Troubleshooting Common MS Problems
Overview of Troubleshooting Strategy
ESI sources parameters
Single and triple Quadrupoles
SIR vs MRM
Ion Suppression
Outline
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 charge
Ion stability (pH)
Ion suppression
Ion beam instability
Probe clogging
Heater/sensor
N
2
gas flow
Loss of vacuum
Power supply
ESI sources parameters
Quadrupoles parameters
Acquisition modes
MS Troubleshooting Strategy
Sample
Preparation
Chromatography
Mass
Spectrometry
Polarity:
Silica- C
18
, C
8
, C
4
, C
2
Hybrid- C
18
, C
8
, C
4
, C
2
Polymer- C
18
, C
8
, C
4
, C
2
Embedded polar group
Cyano, Phenyl
Particle size:
2.5, 3.5, 5 or 7 m
Internal diameter:
4.6, 3.9, 2.1, 1.0,
0.32 mm and 75 m
Length:
150, 100, 50, 30, 20 mm
Source:
ESI
APcI
Nano-ESI
Mass analyzers:
magnetic sectors
electric sectors
time of flight
quadrupole
ion trap
FT-ICR
Raw sample:
- CaCO
2
, microsomes, P450,
hepatocytes etc
- tissue, CSF, plasma, serum
urine, tears etc
- water, sediment, food etc
Extracted sample
For LC/MS/MS
The Total Analysis
B A
Mass
Spectrometry
ESI source parameters
Part 1
Quattro Ultima
ZQ
Quattro Premier
Mass Spectrometers
First, lets 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 as
shown 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 maximize
signal intensity. Higher
temperatures are required
when using mobile phases
containing high percentage of
water.
2,3
1
Mass Spectrometers
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
Isolation valve
Cone shield and
cone assembly
Baffle
Stainless steel capillary
Desolvation heater
Ion Block
ESI probe
ESI Source
10 mm
5 mm
Cone
Probe
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.
Probe too
far from the
cone?
Probe
extends too
far past
cone?
Probe too
close to the
cone?
Response of
reserpine shows
a good gaussian
distribution with
baseline
resolution of the
C
13
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)
In this case, a too low
desolvation
temperature resulted
in a 50 % reduction in
signal intensity.
This effect is
compound dependent.
Desolvation Temperature
Similar loss of signal
intensity, in this case,
it is due to a too low
setting 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
Column flow rate Desolvation temp Desolvation gas flow
L/min C liters/hr
< 10 100 to 120 200 to 250
10 to 20 120 to 250 250 to 400
20 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
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
At this point lets 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
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
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.
With insufficient
capillary 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
The cone voltage is
applied 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 signal
intensity.
Cone Voltage
Poor response can
occur if cone voltage
is set too low. A
sufficient voltage is
required 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
Poor response can also
occur if cone voltage is set
too high. Too much energy
causes a phenomenon
known as In-source
fragmentation.
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 parent
ions.
In this case, the ion at m/z
609 shows a 90% reduction
in signal intensity.
Cone Voltage
The extractor voltage
is applied to a second
cone shaped metal
plate that separates
the 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
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 the
transition lens
hexapole assembly.
The RF lens value
should typically be set
to range from 0.1 to
0.5 volts.
RF Lens
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
B A
Mass
Spectrometry
ESI source parameters
Quadrupoles
Part 2
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
Source
Detector
Nonresonant
Ion
Resonant Ion
dc and rf voltages
+
U
dc
+ V cost
-
U
dc
V cost
Molybdenum Alloy
Quadrupole Schematic
Stable ion
Non-resonant
Trajectory
Pre-Filters
Resonant
Trajectory
Quadrupole
Resonant vs
Non-Resonant Trajectory
Quadrupole Unit Mass Resolution
572.8339
570 571 572 573 574 575 576 577
m/z 0
100
%
0
100
%
573.9185
574.8116
573.2997
574.3072
575.3155
Quadrupole
Resolution: 1000
Q-ToF
Resolution: 10 000
[M+H]
+
Isotopes
Bradykinin Frag 1-5:
Arg-Pro-Pro-Gly-Phe
Quadrupole
Resolution: 1000
285 286 287 288 289
m/z 0
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
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 HM
setting at the high
mass range of the
quadrupole.
The quadrupole can
only achieve mass
unit resolution,
which means that
multiple charged
peaks are not fully
resolved.
Low and
High Mass Resolution
.
U
dc
V
(DC voltage)
(R
f
voltage)
Correct V/U ratio
mass 1 & 2 are resolved
R: 1000
V/U slope
Stable trajectory
Unstable trajectory
Quadrupole Stability Diagram
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 in
signal 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
.
U
dc
V
(DC voltage)
(R
f
voltage)
Low V/U ratio
mass 1 & 2 merge together
R: 10
V/U slope
Stable trajectory
Unstable trajectory
In this case, the LH
and HM resolution
were set too high.
The ion beam falls in
the nonresonant
portion 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
.
U
dc
V
(DC voltage)
(R
f
voltage)
Incorrect V/U ratio
mass 1 & 2 are over resolved
V/U slope
Unstable trajectory
Stable trajectory
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 between
the peak and
isotopes.
Ion Energy
The multiplier is the
last step in the signal
production. The ions
produced by the ESI
source and filtered by
the quadrupole are
converted by the
multiplier into a
measurable 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
B A
Mass
Spectrometry
MS/MS
Part 3
Single Ion Recording
(SIR Mode)
Static
Full Scan
(MS mode)
Scanning
LOQ = 500 pg (quantity injected) LOQ = 5 pg (quantity injected)
Note: A quadrupole mass spectrometer is typically available with a
mass range of 2000 Daltons or 4000 Daltons
Single Q Mode of Acquisition
Full Scan Acquisition
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 4.00
Time 0
100
%
Scan ES+
TIC
2.06e9
2.76
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 4.00
Time 0
100
%
Scan ES+
TIC
3.12e9
2.76
2.56
3.18
2.98
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 4.00
Time 0
100
%
Scan ES+
TIC
5.60e9
3.18
2.96 2.54
2.74
3.27
[ ] = 50 ng/mL
[ ] = 5 ng/mL mixture of 5 basic compounds
[ ] = 500 ng/mL
Full Scan Acquisition
Single Ion Recording (SIR)
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 4.00
Time 0
100
%
SIR of 5 Channels ES+
TIC
3.77e7
2.76
2.55
0.85 2.26
3.18
2.97
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 4.00
Time 0
100
%
Scan ES+
TIC
2.06e9
[ ] = 5 ng/mL
[ ] = 5 ng/mL
Scan mode
SIR mode
Single Ion Recording (SIR)
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 MS
1
can be fragmented by collision, hence the term
collision induced dissociation. Depending if MS
1
and MS
2
are set in scan or park mode will determine the
desired type of acquisition mentioned earlier.
Tandem Mass Spectrometry
Collision Induced Dissociation (CID)
Parent Ion Scanning
MS1
MS2
Collision
Cell
Static
Scanning
Triple Q Modes of Acquisition
Daughter Ion Scanning
MS1
MS2
Collision
Cell
Static
Scanning
Constant Neutral Loss or Gain
MS1
MS2
Collision
Cell
Scanning
Scanning
Multiple Reaction Monitoring
MS1
MS2
Collision
Cell
Static
Static
MS
1
MS
2
Daughter
MS
1
CID
MS
2
A triple quadrupole mass
spectrometer offers lower
sensitivity 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 opened
windows for MS
1
, daughter
and MS
2
. Notice the mass
unit resolution of the
parent mass and isotopes
on both MS
1
and MS
2
.
Erratum: the optimized values of IE1 and IE2 are 0.4 and 0.8 respectively
Optimizing an MRM
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 MS
1
windows (2) will
broaden and show loss of
resolution. Conequently,
the ion beam passing from
MS
1
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 MS
2
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
2
3
Optimizing an MRM
1
2
In this slide, LM/HM on MS
1
is slightly increased ( small
gain in resolution) just to the
point that the isotope is not
seen. This step is crucial, if
LM/HM on MS
1
are too low,
additional ions will enter the
collision cell and will create
additional daughter ions for
each isotope of the parent
molecule.
Optimizing an MRM
1
2
3
Then, by decreasing the
LM/HM on MS
2
(1), the signal in
the daughter scan window has
increased (3). The resolution
on MS
2
also decreases as a
consequence of lowering the
LM/HM values (2).
Optimizing an MRM
Lets take a look at a common
problem when optimizing an MRM
transition. If we look at the LM/HM
values (1,2) on both MS
1
and MS
2
,
the quadrupoles are set at unit
mass resolution. This can be
verified in the MS
1
and MS
2
window 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
MS
1
on the top of the parent peak.
The parent peak has a molecular
weight 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 MS
1
, thus leading to
a total loss of signal in MS
2
.
1
2
3
4
Optimizing an MRM
1
2
3
4
5
At this point, the quadrupoles
are optimized to give maximum
signal intensity (1,2) and MS
1
is
correctly set at 344,2 Daltons
(4). In this tune page both MS
1
and MS
2
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
Optimizing an MRM
Prior to introduction of
collision cell gas (1). the
pressure on the collision
cell pirani guage
indicates 1.0 e-4 mbar
(2). Also, since there is
no argon gas in the
collision cell, the analyzer
penning guage should
show 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
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 MRM
Tune Page with Gas Cell Pressure
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).
Optimizing an MRM
Setting Collison Cell Energy
1
2
3
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.
Optimizing an MRM
Setting Collison Cell Energy
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
m/z 0
100
%
0
100
%
0
100
%
344.2
215
128
215
128
344.2
215
128
CID 0 volts
CID 10 volts
CID 20 volts
[M+H]
+
N
CH
3
O
CH
3
Cl 215
128
Clemastine
(Different scale)
Daughter Ion Spectrum
Note: typical value of dwell times are between 0.2 and 0.05 seconds
Multiple MRM
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 4.00
Time 0
100
%
MRM of 5 Channels ES+
TIC
2.91e5
2.95
2.54
2.76
3.18
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 4.00
Time 0
100
%
SIR of 5 Channels ES+
TIC
2.29e6
2.27
2.75
2.56
3.18
2.96
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 4.00
Time 0
100
%
Scan ES+
TIC
2.04e9
[ ] = 0.1 ng/mL
Scan mode
[ ] = 0.1 ng/mL
SIR mode
[ ] = 0.1 ng/mL
MRM mode
Multiple MRM
B A
Mass
Spectrometry
Ion suppression
Causes of Ion Suppression
Troubleshooting Ion Supression
Part 4
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
4
OH
Peak intensity: 5 143 003 136
Signal increase: + 41 %
100
0
%
472.63
50/50 Water/ACN
100
464 466 468 470 472 474 476 478 480 482 484
m/z 0
%
472.63
50/50 Water/ACN + 0.5 % TFA
Peak intensity: 893 059 072
Signal decrease: -75 %
Terfenadine
* All spectrums at same scale
suggest that ESI is
Scan ES+
Scan ES+
100
0
%
Scan ES+
472.63
50/50 Water/ACN + 0.5 % NH
4
OH
100
0
%
472.63
50/50 Water/ACN
100
464 466 468 470 472 474 476 478 480 482 484
m/z 0
%
472.63
Terfenadine
suggest that ESI is
Scan ES+
Scan ES+
100
0
%
Scan ES+
472.63
50/50 Water/ACN + 0.5 % NH
4
OH
100
0
%
Scan ES+
472.63
50/50 Water/ACN + 0.5 % NH
4
OH
100
0
%
472.63
50/50 Water/ACN
100
0
%
472.63
50/50 Water/ACN
100
464 466 468 470 472 474 476 478 480 482 484
m/z 0
%
472.63
Terfenadine
100
464 466 468 470 472 474 476 478 480 482 484
m/z 0
%
472.63
Terfenadine
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, lets 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 precipitation
Acetic acid Ammonium bicarbonate Oasis HLB 1-D
Formic Acid Ammonium biphosphate Oasis HLB 2-D
Oasis MCX
Basic additive Ion pairing additive
Ammonium hydroxide Tetraethylammonium hydroxide
Pyrrolidine Dimethylhexylamine
Detergents
Triton X100
SDS
What is ion suppression or enhancement ?
2795
ESI-MS
Infusion pump
Used to add range of modifiers,
salts, ion pairs, pH additives,
Matrix extracts
50/50 ACN/ H
2
O
8 compounds
0.2mL/min
0.2mL/min
ES+
260.2 Propranolol
291.3 Trimethoprim
354.4 Pipenzolate*
411.4 Resperidone
472.6 Terfenadine
485.6 Methoxy-Verapamil
591.6 Benextramine
609.6 Reserpine
*quaternary amine drug
Compare 50/50 ACN/ H2O
to additive stream signal
(triplicates) blank, matrix, blank
Experimental design aimed to look for a better solution
removal of suppression
250 300 350 400 450 500 550 600 650 700 750 800 850 900 950
m/z 0
100
%
0
100
%
Scan ES+
6.41e9
354.4
291.3
669.78 609.6
581.71
472.6
411.4
537.60
485.6
713.74
757.83
801.86
845.88
889.91
933.93
977.95
Scan ES+
7.38e9 354.4
260.2
291.3
609.6
485.6
472.6
411.4
591.6
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 Propranolol
291.3 Trimethoprim
354.4 Pipenzolate *
411.4 Resperidone
472.6 Terfenadine
591.6 Benextramine
609.6 Reserpine
* Quaternary amine molecule
250 300 350 400 450 500 550 600 650 700 750 800 850 900 950
m/z 0
100
%
0
100
%
Scan ES+
6.41e9
354.4
291.3
669.78 609.6
581.71
472.6
411.4
537.60
485.6
713.74
757.83
801.86
845.88
889.91
933.93
977.95
Scan ES+
7.38e9 354.4
260.2
291.3
609.6
485.6
472.6
411.4
591.6
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 %
0.5 % Triton X 100
260.2 Propranolol
291.3 Trimethoprim
354.4 Pipenzolate *
411.4 Resperidone
472.6 Terfenadine
591.6 Benextramine
609.6 Reserpine
250 300 350 400 450 500 550 600 650 700 750 800 850 900 950
m/z 0
100
%
250 300 350 400 450 500 550 600 650 700 750 800 850 900 950
m/z 0
100
%
0
100
%
Scan ES+
6.41e9
354.4
291.3
669.78 609.6
581.71
472.6
411.4
537.60
485.6
713.74
757.83
801.86
845.88
889.91
933.93
977.95
0
100
%
Scan ES+
6.41e9
354.4
291.3
669.78 609.6
581.71
472.6
411.4
537.60
485.6
713.74
757.83
801.86
845.88
889.91
933.93
977.95
Scan ES+
7.38e9 354.4
260.2
291.3
609.6
485.6
472.6
411.4
591.6
354.4
260.2
291.3
609.6
485.6
472.6
411.4
591.6
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 %
0.5 % Triton X 100
260.2 Propranolol
291.3 Trimethoprim
354.4 Pipenzolate *
411.4 Resperidone
472.6 Terfenadine
591.6 Benextramine
609.6 Reserpine
Surfactant
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620
m/z 0
100
%
0
100
%
Scan ES+
609.6
354.4
260.3
291.3
485.6
472.6
411.5
591.7
Scan ES+
354.3
260.3
291.2
609.6
485.6
472.6
411.4
591.7
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 %
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620
m/z 0
100
%
0
100
%
Scan ES+
609.6
354.4
260.3
291.3
485.6
472.6
411.5
591.7
Scan ES+
354.3
260.3
291.2
609.6
485.6
472.6
411.4
591.7
260 280 300 320 340 360 380 400 420 440 460 480 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620
m/z 0
100
%
500 520 540 560 580 600 620
m/z 0
100
%
0
100
%
Scan ES+
609.6
354.4
260.3
291.3
485.6
472.6
411.5
591.7
Scan ES+
354.3
260.3
291.2
609.6
485.6
472.6
411.4
591.7
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
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620
m/z 0
100
%
0
100
%
Scan ES+
354.5
260.3
291.4
471.6
411.5
609.6
485.6
591.7
Scan ES+
354.4
260.3
291.3
609.6
485.6
472.6
411.6
591.7
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 % NH
4
OH
50/50 Water/ACN
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620
m/z 0
100
%
0
100
%
Scan ES+
354.5
260.3
291.4
471.6
411.5
609.6
485.6
591.7
Scan ES+
354.4
260.3
291.3
609.6
485.6
472.6
411.6
591.7
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620
m/z 0
100
%
500 520 540 560 580 600 620
m/z 0
100
%
0
100
%
Scan ES+
354.5
260.3
291.4
471.6
411.5
609.6
485.6
591.7
Scan ES+
354.4
260.3
291.3
609.6
485.6
472.6
411.6
591.7
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 % NH
4
OH
50/50 Water/ACN
Basic Additive
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660
m/z 0
100
%
Scan ES+
1.95e8
354.44
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660
m/z 0
100
%
Scan ES+
7.38e9 354.44
260.28
291.31
609.55
485.62 472.57
411.49
591.66
591.66
50/50 Water/ACN Blank
50 mM Tetraethylammoniumhydroxide
260.2 - 100 %
291.3 - 100 %
354.4 - 88 %
411.4 - 100 %
472.5 - 100 %
485.5 - 100 %
591.6 - 94 %
609.5 - 100 %
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660
m/z 0
100
%
Scan ES+
1.95e8
354.44
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660
m/z 0
100
%
Scan ES+
7.38e9 354.44
260.28
291.31
609.55
485.62 472.57
411.49
591.66
591.66
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660
m/z 0
100
%
Scan ES+
1.95e8
Scan ES+
1.95e8
354.44
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660
m/z 0
100
%
Scan ES+
7.38e9 354.44
260.28
291.31
609.55
485.62 472.57
411.49
591.66
591.66
50/50 Water/ACN Blank
50 mM Tetraethylammoniumhydroxide
260.2 - 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
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620
m/z 0
100
%
0
100
%
Scan ES+
354.4
291.3
260.3
591.7
472.6
411.5
485.6
609.6
Scan ES+
354.4
260.3
291.3
609.6
485.6
472.6
411.5
591.7
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 %
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620
m/z 0
100
%
0
100
%
Scan ES+
354.4
291.3
260.3
591.7
472.6
411.5
485.6
609.6
Scan ES+
354.4
260.3
291.3
609.6
485.6
472.6
411.5
591.7
260 280 300 320 340 360 380 400 420 440 460 480 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620
m/z 0
100
%
500 520 540 560 580 600 620
m/z 0
100
%
0
100
%
Scan ES+
354.4
291.3
260.3
591.7
472.6
411.5
485.6
609.6
Scan ES+
354.4
260.3
291.3
609.6
485.6
472.6
411.5
591.7
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
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620
m/z 0
100
%
0
100
%
Scan ES+
591.7
354.4
518.6
472.6
546.6
609.6
Scan ES+
354.4
260.2
291.2
609.6
485.6 472.6
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
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620
m/z 0
100
%
0
100
%
Scan ES+
591.7
354.4
518.6
472.6
546.6
609.6
Scan ES+
354.4
260.2
291.2
609.6
485.6 472.6
411.4 591.6
.
260 280 300 320 340 360 380 400 420 440 460 480 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620
m/z 0
100
%
500 520 540 560 580 600 620
m/z 0
100
%
0
100
%
Scan ES+
591.7
354.4
518.6
472.6
546.6
609.6
Scan ES+
354.4
260.2
291.2
609.6
485.6 472.6
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
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620
m/z 0
100
%
0
100
%
Scan ES+
591.7
354.4
518.5
472.6 485.5 609.6
Scan ES+
354.4
260.2
291.3
609.6
485.6 472.6
411.5
591.6
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 %
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620
m/z 0
100
%
0
100
%
Scan ES+
591.7
354.4
518.5
472.6 485.5 609.6
Scan ES+
354.4
260.2
291.3
609.6
485.6 472.6
411.5
591.6
50/50 Water/ACN
260 280 300 320 340 360 380 400 420 440 460 480 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620
m/z 0
100
%
500 520 540 560 580 600 620
m/z 0
100
%
0
100
%
Scan ES+
591.7
354.4
518.5
472.6 485.5 609.6
Scan ES+
354.4
260.2
291.3
609.6
485.6 472.6
411.5
591.6
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
Condition/Equilibrate
1.0 mL methanol / 1.0 mL water
Load
1.0 mL plasma
Wash
1.0 mL 5% methanol in water
Elute
0.5 mL MeOH
Dilute with 0.5 ml water
Plasma Sample
* 30 mg HLB 96 plate
Reversed Phase SPE
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620
m/z 0
100
%
0
100
%
Scan ES+
354.4
260.1 291.2
591.6
472.5
411.4
485.5
609.5
Scan ES+
354.2
260.2
291.2
609.5
485.4
472.5
411.4
591.6
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 %
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620
m/z 0
100
%
0
100
%
Scan ES+
354.4
260.1 291.2
591.6
472.5
411.4
485.5
609.5
Scan ES+
354.2
260.2
291.2
609.5
485.4
472.5
411.4
591.6
50/50 Water/ACN
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620
m/z 0
100
%
0
100
%
Scan ES+
354.4
260.1 291.2
591.6
472.5
411.4
485.5
609.5
Scan ES+
354.2
260.2
291.2
609.5
485.4
472.5
411.4
591.6
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
Condition/Equilibrate
1.0 mL methanol / 1.0 mL water
Load
1.0 mL plasma
Prepare Sample Solution
Wash 2
1.0 mL MeOH
Elute
0.5 mL MeOH + 2% NH
4
OH
Dilute with 0.5 ml water
Wash 1
1.0 mL Water + 2 % FA
Locks basic drug
on ion exchanger
Removes polar
interferences
* 30 mg Oasis MCX 96 well plate
Mixed Mode Cation-Exchange SPE
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 %
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640
m/z 0
100
%
0
100
%
Scan ES+
354.4
291.3
260.3
609.6
472.6
411.5
485.6
591.7
Scan ES+
354.4
260.3
291.3
609.6
485.6
472.6
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 %
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640
m/z 0
100
%
0
100
%
Scan ES+
354.4
291.3
260.3
609.6
472.6
411.5
485.6
591.7
Scan ES+
354.4
260.3
291.3
609.6
485.6
472.6
411.5 591.7
50/50 Water/ACN
260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640
m/z 0
100
%
0
100
%
Scan ES+
354.4
291.3
260.3
609.6
472.6
411.5
485.6
591.7
Scan ES+
354.4
260.3
291.3
609.6
485.6
472.6
411.5 591.7
50/50 Water/ACN
Mixed Mode SPE Rat Plasma

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