Waters LCMSWaters LCMS Troubleshooting

8/12/2019 Waters LCMSWaters LCMS Troubleshooting

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Waters Corp. 20042004 Waters Corporation

Troubleshooting CommonMS Problems

by Claude Mallet, Ph.D

[email protected]

presented by

Michael S. Young, Ph.D.


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2004 Waters Corporation2004 Waters Corporation

Troubleshooting Common MS Problems

Overview of Troubleshooting Strategy

ESI sources parameters

Single and triple Quadrupoles

SIR vs MRM

Ion Suppression

Outline


3/82


Try to simplify --

assess impact on lab efficiency --

inspect the MS or /MS/MS --

try to categorizetroubleshoot the easiest to fix

items first

CHEMISTRY MECHANICAL IMPROPER SETTINGS

Adducts (Na+, K+)

Multiple chargeIon stability (pH)

Ion suppression

Ion beam instability

Probe clogging

Heater/sensor

N2 gas flow

Loss of vacuum

Power supply

ESI sources parameters

Quadrupoles parameters

Acquisition modes

MS Troubleshooting Strategy


4/82


Sample

Preparation ChromatographyMass

Spectrometry

Polarity:

Silica- C18, C8, C4, C2Hybrid- C18, C8, C4, C2Polymer- C

18

, C8

, C4

, C2Embedded 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

quadrupoleion trap

FT-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


6/822004 Waters Corporation2004 Waters Corporation

Quattro UltimaZQ

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 anddesolvation temperature can

be optimized to maximize

signal intensity. Higher

temperatures are required

when using mobile phases

containing high percentage of

water.

2,31

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 isequipped 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 toofar from the

cone?

Probe

extends toofar past

cone?



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Probe tooclose to the

cone?



14/82


Response ofreserpine shows

a good gaussian

distribution with

baseline

resolution of the

C13 isotopes

Temperature and

gas flow are

parameters thataffect the

desolvation

efficiency of the

probe. Improper

settings can result in

loss of signal. Thesevalues are optimized

according to the

column flow rate.

ESI Probe Parameters (tune page)


15/82


In this case, a too low

desolvation

temperature resulted

in a 50 % reduction in

signal intensity.

This effect is

compound dependent.

Desolvation Temperature


16/82


Similar loss of signal

intensity, in this case,

it is due to a too low

setting of thedesolvation 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


17/82


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 thetemperature 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


18/82


Dewar tanks Nitrogen generator

Both setups are widely used and the choice mostly depends on the consumption ofnitrogen 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


19/82


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 astainless 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 lensare used to guide the ion beam into

the mass analyzer

1 2

34

ESI Probe

ESI Source

ESI Source Parameters


20/82


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 notclean. 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



21/82


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 condensationproblems.

The source has a

maximum setting

of 150 C.



22/82


With insufficient

capillary voltage, the

signal shows an 80%

decrease in signalintensity.

Typical optimum

values for most small

molecules are

between 3.0 and 3.5kV.

Higher values usually

have little effect on

signal intensity.

Deviations from

experimentally

optimized value may

indicate problems in

the source.

Capillary Voltage


23/82


The cone voltage is

applied to a spherical

metal plate, the first gate

between the sprayer (atatmospheric 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 andincreased our signal

intensity.

Cone Voltage


24/82


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 conevolatage is optimized,

loss of sensitivity may

result from

contamination at the

cone.

Cone Voltage


25/82


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 highvelocities, 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


26/82


The extractor voltageis applied to a second

cone shaped metal

plate that separates

the ion block and the

mass analyzer. This

plate creates a second90 degree angle in the

ion beam, completing

the Z spray shape.

An incorrect voltagesetting of the extractor

resulted in a 70%

reduction in signal

intensity.

Typical extractorvoltage settings range

from 1 to 3 volts;

higher values will not

usually give better

sensitivity. Higher than

expected values mayindicate contamination

in the source block

Extractor Voltage


27/82


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


28/82


3.5

3.5

3. 3.

In the example shown,

we needed to increase

the RF lens to achieve

a symmetrical peakshape. This may

indicate that the

source is

contaminated.

RF Lens


29/82


BA MassSpectrometry

ESI source parameters

Quadrupoles

Part 2


30/82


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 ofRF/DC applied to the rods is proportional to a range or a specific mass to charge ratio.

Quadrupole Mass Analyzers


31/82


Source

DetectorNonresonant

Ion

Resonant Ion

dc and rf voltages

+Udc + V cost

-Udc V cost

Molybdenum Alloy

Quadrupole Schematic


32/82


Stable ion

Non-resonant

Trajectory

Pre-Filters

Resonant

Trajectory

Quadrupole

Resonant vsNon-Resonant Trajectory


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Quadrupole Unit Mass Resolution

1


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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-ToF

Resolution: 10 000

[M+H]+

Isotopes

Bradykinin Frag 1-5:Arg-Pro-Pro-Gly-Phe


35/82

L d


36/82


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 canonly achieve mass

unit resolution,

which means that

multiple charged

peaks are not fully

resolved.

Low andHigh Mass Resolution


37/82


.

Udc

V

(DC voltage)

(Rfvoltage)

Correct V/U ratio

mass 1 & 2 are resolved

R: 1000

V/U slope

Stable trajectory

Unstable trajectory

Quadrupole Stability Diagram


38/82


39/82


.

Udc

V

(DC voltage)

(Rfvoltage)

Low V/U ratio

mass 1 & 2 merge together

R: 10V/U slope

Stable trajectory

Unstable trajectory



40/82


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 massspectrometer and

produce a signal.

Low and High Mass Resolution


41/82


.

Udc

V

(DC voltage)

(Rfvoltage)Incorrect V/U ratio

mass 1 & 2 are over resolvedV/U slope

Unstable trajectory

Stable trajectory



42/82


The ion energy is

applied to a small

lens positioned

between the

quadrupole and

the multiplier. This

lens is used to

refocus the beamtoward 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


43/82


The multiplier is the

last step in the signal

production. The ions

produced by the ESI

source and filtered bythe quadrupole are

converted by the

multiplier into a

measurable current.

If the multiplier is settoo low, as shown

here, the signal

intensity will be

considerably reduced.

Too high a multipliersetting produces

saturation (flat-top

peaks) and poor

quantitation.

Multiplier


44/82


BA MassSpectrometry

MS/MS

Part 3


45/82


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 amass range of 2000 Daltons or 4000 Daltons

Single Q Mode of Acquisition


46/82


Full Scan Acquisition


47/82


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

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 4.00Time0

100

%

Scan ES+TIC

3.12e9

2.76

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


48/82


Single Ion Recording (SIR)


49/82


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

3.77e7

2.76

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)

Tandem Mass Spectrometry


50/82


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 termcollision 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 Spectrometry

Collision Induced Dissociation (CID)


51/82


Parent Ion Scanning

MS1 MS2

Collision

Cell

StaticScanning

Triple Q Modes of Acquisition


52/82


Daughter Ion Scanning

MS1 MS2

Collision

Cell

Static Scanning



53/82


Constant Neutral Loss or Gain

MS1 MS2

Collision

Cell

ScanningScanning



54/82


Multiple Reaction Monitoring

MS1 MS2

Collision

Cell

Static Static



55/82


MS1 MS2Daughter

MS1

CID

MS2

A triple quadrupole mass

spectrometer offers lower

sensitivity and

reproducable

fragmentation. With

Multiple ReactionMonitoring (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 MS1, daughter

and 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


56/82


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 inthe 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 theisotopes.

1

2

3


Optimizing an MRM


57/82


1

2

In this slide, LM/HM on MS1is slightly increased ( small

gain in resolution) just to the

point that the isotope is notseen. 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 foreach isotope of the parent

molecule.


Optimizing an MRM


58/82


12

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 aconsequence of lowering the

LM/HM values (2).


Optimizing an MRM


59/82


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 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 quitesimple; choosing a correct MRM

transition also requires us to park

MS1 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 MS1, thus leading to

a total loss of signal in MS2.

1

2

3

4


Optimizing an MRM


60/82


1

2

3

4

5

At this point, the quadrupoles

are 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 wereremoved so we can

concentrate on the daughter

ion scan (5). As we can see,

the tune page only shows the

parent ion without any daughterions. 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


61/82


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.

12

3

Optimizing an MRM

Optimizing an MRM


62/82


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), typicalvalues are between 2

to 3e-4 mbar.

p gTune Page with Gas Cell Pressure

Optimizing an MRM


63/82


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 isthe 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 enoughto see a small fraction of the

parent ion (3).


p gSetting Collison Cell Energy

Optimizing an MRM


64/82


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, thelevel 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 settingis 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.


p gSetting Collison Cell Energy


65/82


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

128

344.2

215

128

CID 0 volts

CID 10 volts

CID 20 volts

[M+H]+

NCH3

O

CH3

Cl215

128

Clemastine

(Different scale)

Daughter Ion Spectrum


66/82


Note: typical value of dwell times are between 0.2 and 0.05 seconds

Multiple MRM

M l i l MRM


67/82


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

2.91e5

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 4.00Time0

100

%

SIR of 5 Channels ES+TIC

2.29e6

2.272.75

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 4.00Time0

100

%

Scan ES+TIC

2.04e9

[ ] = 0.1 ng/mL

Scan mode

[ ] = 0.1 ng/mLSIR mode

[ ] = 0.1 ng/mLMRM mode

Multiple MRM

P t 4


68/82


BA MassSpectrometry

Ion suppression

Causes of Ion SuppressionTroubleshooting Ion Supression

Part 4


69/82


70/82


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 ?

Experimental design aimed to look for a better solution


71/82


2795

ESI-MS

Infusion pump

Used to add range of modifiers,

salts, ion pairs, pH additives,Matrix extracts

50/50 ACN/ H2O

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 Benextramine609.6 Reserpine

*quaternary amine drug

Compare 50/50 ACN/ H2O

to additive stream signal

(triplicates) blank, matrix, blank

p gremoval of suppression

S f t t


72/82


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

537.60

485.6

713.74

757.83

801.86

845.88

889.91933.93

977.95

Scan ES+7.38e9354.4

260.2

291.3

609.6

485.6472.6

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 Resperidone472.6 Terfenadine


591.6 Benextramine

609.6 Reserpine

* Quaternary amine molecule

250 300 350 400 450 500 550 600 650 700 750 800 850 900 950m/z0

100

%

0

100

%

Scan ES+

6.41e9

354.4

291.3

669.78609.6

581.71

472.6

411.4

537.60

485.6

713.74

757.83

801.86

845.88

889.91933.93

977.95

Scan ES+7.38e9354.4

260.2

291.3

609.6

485.6472.6

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 *



591.6 Benextramine

609.6 Reserpine


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

537.60

485.6

713.74

757.83

801.86

845.88

889.91933.93

977.95

0

100

%

Scan ES+

6.41e9

354.4

291.3

669.78609.6

581.71

472.6

411.4

537.60

485.6

713.74

757.83

801.86

845.88

889.91933.93

977.95

Scan ES+7.38e9354.4

260.2

291.3

609.6

485.6472.6

411.4

591.6

354.4

260.2

291.3

609.6

485.6472.6

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 *



591.6 Benextramine

609.6 Reserpine


Surfactant

A idi Additi


73/82


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

411.5 591.7

Scan ES+354.3

260.3

291.2

609.6

485.6472.6

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 620m/z0

100

%

0

100

%

Scan ES+

609.6354.4

260.3

291.3

485.6472.6

411.5 591.7

Scan ES+354.3

260.3

291.2

609.6

485.6472.6

411.4

591.7

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

411.5 591.7

Scan ES+354.3

260.3

291.2

609.6

485.6472.6

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

B i Additi


74/82


260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620

m/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

411.6

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 % NH4OH

50/50 Water/ACN

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620

m/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

411.6

591.7

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620

m/z0

100

%

500 520 540 560 580 600 620

m/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

411.6

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 % NH4OH

50/50 Water/ACN

Basic Additive

Ion Pairing Reagent


75/82


260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660

m/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 hydroxide

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/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

260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660

m/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 hydroxide

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

Salt Adducts


76/82


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

591.7

472.6411.5485.6 609.6

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

591.7

472.6411.5485.6 609.6

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

591.7

472.6411.5485.6 609.6

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

Rat Plasma


77/82


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

354.4 518.6

472.6 546.6

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

354.4 518.6

472.6 546.6

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

354.4 518.6

472.6 546.6

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

Human Plasma


78/82


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

411.5 591.6

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 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

411.5 591.6


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

411.5 591.6


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

Reversed Phase SPE


79/82


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

Reversed Phase SPE Rat Plasma


80/82


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

411.4

485.5

609.5

Scan ES+354.2

260.2

291.2

609.5

485.4472.5

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 620m/z0

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

485.4472.5

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 620m/z0

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

485.4472.5

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

Mixed Mode Cation Exchange SPE


81/82


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% NH4OH

Dilute with 0.5 ml water

Wash 1

1.0 mL Water + 2 % FALocks basic drug

on ion exchanger

Removes polar

interferences

* 30 mg Oasis MCX 96 well plate

Mixed Mode Cation-Exchange SPE

Mixed Mode SPE Rat Plasma


82/82

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 %

100

%

0

100

%

Scan ES+

354.4

291.3260.3

609.6

472.6

411.5

485.6

591.7

Scan ES+354.4

260.3

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 %

100

%

0

100

%

Scan ES+

354.4

291.3260.3

609.6

472.6

411.5

485.6

591.7

Scan ES+354.4

260.3

291.3

609.6

485.6

472.6

411.5 591.7


50/50 Water/ACN100

%

0

100

%

Scan ES+

354.4

291.3260.3

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

Waters LCMSWaters LCMS Troubleshooting

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