Ion Suppression Esi Ms-ms Primer

Ion Suppression in Ion Suppression in

LCLC--ESIESI--MSMSMSMS

(a primer)(a primer)

Why ion suppression is an issue?Why ion suppression is an issue?

�� A cross disciplinary generated issueA cross disciplinary generated issue

…………………… it goes that more polar compounds are poorly retained on the it goes that more polar compounds are poorly retained on the

column in HPLC and will interfere with the detection and quantifcolumn in HPLC and will interfere with the detection and quantification ication

of the target compound. Sample cleanup is normally required.of the target compound. Sample cleanup is normally required.

In contrast, a mass detector can be configured to In contrast, a mass detector can be configured to

isolate and quantify specific ions; therefore, sample isolate and quantify specific ions; therefore, sample

cleanup and chromatographic separation can cleanup and chromatographic separation can

theoretically be reduced when analyte specific theoretically be reduced when analyte specific masses or product ions are monitored. masses or product ions are monitored.

misconceptions misconceptions

�� chromatographic separation can be minimized or chromatographic separation can be minimized or even eliminated, even eliminated,

�� the analytical column is needed only as a the analytical column is needed only as a mechanism to load sample on the system,mechanism to load sample on the system,

�� specimen cleanup can be minimized or eliminated,specimen cleanup can be minimized or eliminated,

�� the use of HPLCthe use of HPLC--MS or HPLCMS or HPLC--tandem MS provides tandem MS provides absolute accuracy and specificity. absolute accuracy and specificity.

ESI/MS/MS detection can fool you!!! ESI/MS/MS detection can fool you!!!

�� the presence of less volatile compounds that can the presence of less volatile compounds that can change the efficiency of droplet formation or droplet change the efficiency of droplet formation or droplet evaporation, competition for the ESI droplet surface evaporation, competition for the ESI droplet surface and hence ionization which in turn affects the and hence ionization which in turn affects the amount of charged ion in the gas phase. amount of charged ion in the gas phase. Mechanism? Mechanism? –––––––– still a debate!still a debate!

�� But it affects But it affects quantitationquantitation is a is a FACTFACT. .

MS/MS detection can fool you!!!MS/MS detection can fool you!!!

�� salts, ionsalts, ion--pairing agents, endogenous compounds, pairing agents, endogenous compounds,

drugs, metabolites, and proteins are responsible for drugs, metabolites, and proteins are responsible for Ion suppressionIon suppression

�� Also compound dependent, difficult to find a Also compound dependent, difficult to find a

common set of conditions that works for a large common set of conditions that works for a large number of compoundsnumber of compounds

Generally used approachesGenerally used approaches

�� Examining ion suppression: signal recovery studies Examining ion suppression: signal recovery studies using specimen extracts with added analyteusing specimen extracts with added analyte

�� Use Use postcolumnpostcolumn infusion of the analyte to evaluate infusion of the analyte to evaluate protracted ionization effects. protracted ionization effects.

�� Minimizing ion suppression by more rigorous Minimizing ion suppression by more rigorous cleanup, chromatographic changes, reagent cleanup, chromatographic changes, reagent modifications, and effective internal standardizationmodifications, and effective internal standardization

�� Use expected physiologic concentrations of the Use expected physiologic concentrations of the analyte under investigation. analyte under investigation.

�� The mass and charge of individual analytes are also The mass and charge of individual analytes are also

factors in making a compound a candidate for ion factors in making a compound a candidate for ion supsuppression or in making one compound a source pression or in making one compound a source

of ion suppression for another. of ion suppression for another.

�� Molecules with higher mass will suppress the signal Molecules with higher mass will suppress the signal

of smaller molecules and that more polar analytes of smaller molecules and that more polar analytes are more susceptible to suppression are more susceptible to suppression

The nature of matrix effectThe nature of matrix effect

““““““““How to?How to?”””””””” is to compare!is to compare!

�� the instrument response for calibrators (including the instrument response for calibrators (including any internal standards) injected directly in mobile any internal standards) injected directly in mobile phase, phase,

The data for the calibrator in mobile phase provide a The data for the calibrator in mobile phase provide a relative 100% response value.relative 100% response value.

�� the same amount of compound added to the same amount of compound added to preextractedpreextracted samples samples

The data for the same amount of compound added to The data for the same amount of compound added to preextractedpreextracted samples show the effect of sample samples show the effect of sample matrix on MS response (ion suppression)matrix on MS response (ion suppression)

�� the same amount of compound added to specimen the same amount of compound added to specimen matrix before extractionmatrix before extraction

The response data for extracted samples containing The response data for extracted samples containing

the analyte can highlight whether any loss of signal the analyte can highlight whether any loss of signal

is attributable to the extraction process or ion is attributable to the extraction process or ion suppression suppression

““““““““How to?How to?”””””””” is to compare! (Contis to compare! (Cont’’’’’’’’d)d)

Ion suppression attributable to Ion suppression attributable to endogenous matrix components endogenous matrix components

0.1-mL aliquots Serum

A,C,E: 5-uL direct HPLC infusing

3 extraction techniques:

B: solid-phase extraction (Varian 60-mg Abselut column, water wash, methanol elution)

D: solvent extraction (0.8 mL of CH2CL2)

F: protein precipitation (0.5 mL acetonitrile)

Signal response comparisons (m/z 195) for caffeine added to serum extracts prepared by solid-phase extraction, solvent extraction, and protein precipitation.

100

Seconds

Respo

nse,

%

A

B

C

D

E

F

120 240 360 480 600

““““““““How to?How to?”””””””” is to check!is to check!

�� interference checks for possible cointerference checks for possible co--horde drugs or horde drugs or metabolitesmetabolites

Just because a Just because a coelutingcoeluting drug does not produce drug does not produce

similar mass fragments does not mean that this similar mass fragments does not mean that this compound is incapable of ion suppression. compound is incapable of ion suppression.

�� postcolumnpostcolumn continuous infusion of compound into continuous infusion of compound into the MS detector the MS detector

a syringe pump connected via a a syringe pump connected via a ““““““““TT”””””””” to the column effluent, to the column effluent,

mobile phase or specimen extracts are injected into the HPLC mobile phase or specimen extracts are injected into the HPLC

system. The analyte being evaluated is continuously infused system. The analyte being evaluated is continuously infused

postcolumnpostcolumn and is mixed with the column effluent through a and is mixed with the column effluent through a

tee before entering the electrospray interface.tee before entering the electrospray interface.

““““““““How to?How to?”””””””” is to check! (contis to check! (cont’’’’’’’’d)d)

HPLC ESI/MS

Syringe Pump

““TT””


�� Compound being tested is Compound being tested is

introduced into the mass introduced into the mass dedetector at a constant rate, tector at a constant rate,

a constant ESI response a constant ESI response

should ideally be observed should ideally be observed (A) (A)

4

8

1 3 5 7 9

4

8

1 3 5 7 9

4

8

1 3 5 7 9

Ion

in

ten

sity x

10

3

Minutes

�� serum liquidserum liquid--liquid extract liquid extract injection, ion suppresinjection, ion suppression sion

can be ~90% can be ~90%

(a recovery time may exist, (a recovery time may exist,

suppression is not limited suppression is not limited to the solventto the solvent--front region)front region)


4

8

1 3 5 7 9

4

8

1 3 5 7 9

4

8

1 3 5 7 9

Ion

in

ten

sity x

10

3

Minutes

�� serum protein precipitation serum protein precipitation

extract injection, ion extract injection, ion suppressuppression can be ~90% sion can be ~90%


4

8

1 3 5 7 9

4

8

1 3 5 7 9

4

8

1 3 5 7 9

Ion

in

ten

sity x

10

3

Minutes

Mixed mode SPE Mixed mode SPE vsvs othersothers

Level of ion suppression found for different sample preparation techniques. Comparedare protein precipitation, solid-phase extraction (SPE) with a simple reversed-phase protocol, andsolid-phase extraction with a mixed-mode reversed-phase ion-exchange protocol.

A: A: PropranololPropranolol

B: B: TrimethoprimeTrimethoprime

C: C: PipenzolatePipenzolate

D: D: ResperidoneResperidone

E: E: TerfenadineTerfenadine

F: F: MethoxyMethoxy--

verapamilverapamil

G: G: BenextramineBenextramine

H: H: ReserpineReserpine

Mix-mode SPE

Protein Precipt’n

RR--P SPEP SPE

NonNon--conclusive Conclusion:conclusive Conclusion:

The degree of ion suppression and the recovery time The degree of ion suppression and the recovery time to full response:to full response:

1. vary from compound to compound and from 1. vary from compound to compound and from sample to samplesample to sample

2. dependent on the sample preparation (cleanup) 2. dependent on the sample preparation (cleanup) methodmethod

3. may not be evident initially but may still be present 3. may not be evident initially but may still be present during subsequent runsduring subsequent runs

4. dependent on the analyte concentration, 4. dependent on the analyte concentration, matrix/analyte ratio: matrix/analyte ratio:

aa) decreasing the matrix/analyte ratio through more ) decreasing the matrix/analyte ratio through more

extensive cleanup or dilute the test solution and extensive cleanup or dilute the test solution and evaluate the dilution factor evaluate the dilution factor vsvs responseresponse

bb) performing ion suppression validations by using ) performing ion suppression validations by using

concentrations reflecting that encountered in real concentrations reflecting that encountered in real samples.samples.


5. Issues of Ion5. Issues of Ion--pairing agents: conventionally used pairing agents: conventionally used TFA causes signal suppression, to tackle this:TFA causes signal suppression, to tackle this:

a) decrease the TFA concentration provided adequate a) decrease the TFA concentration provided adequate separationseparation

b) use alternative weaker acids such as acetic, formic, b) use alternative weaker acids such as acetic, formic, propionicpropionic acid or acid or hexafluorobutyrichexafluorobutyric acidacid

c) TFA fix: c) TFA fix: postcolumnpostcolumn addition of acids and solvent addition of acids and solvent carriers to displace TFA, as reported, the use of 2carriers to displace TFA, as reported, the use of 2--(2(2--methoxyethoxy) ethanol as a signal enhancer to methoxyethoxy) ethanol as a signal enhancer to ~100~100--fold enhancement of the signal for the model fold enhancement of the signal for the model drug ibuprofen. drug ibuprofen.


(TFA issue)(TFA issue)

�� In positive ion mode, Signal suppression is In positive ion mode, Signal suppression is

caused by strong ion pairing between the TFA caused by strong ion pairing between the TFA

anion and the protonated analyte anion and the protonated analyte cationcation of basic of basic molecules, effectively making them neutral. molecules, effectively making them neutral.

�� In negative ion mode, complete suppression of In negative ion mode, complete suppression of

analyte can occur by TFA competing for charge. analyte can occur by TFA competing for charge.

Aqueous solutions of TFA have high conductivity Aqueous solutions of TFA have high conductivity

and surface tension, which causes spray and surface tension, which causes spray instability in ESI. instability in ESI.


6. other approaches:6. other approaches:

a) Modify the chromatographic conditions so that the a) Modify the chromatographic conditions so that the

compound(scompound(s) of interest ) of interest elute(selute(s) in a region where ) in a region where ion suppression is not observed. (change run!!!)ion suppression is not observed. (change run!!!)

b) use an internal standard (stable isotope or b) use an internal standard (stable isotope or

structural analog), and to modify the structural analog), and to modify the

chromatography so that the compound of interest chromatography so that the compound of interest

and the IS and the IS coelutecoelute. However, if ion suppression . However, if ion suppression

deteriorate the signal of the analyte or IS, the S/N deteriorate the signal of the analyte or IS, the S/N

may be compromised to a point where accuracy or may be compromised to a point where accuracy or precision becomes @#$%.precision becomes @#$%.


�� 7. Other 7. Other oddititesodditites::

SPE or sample well or sample vial may give off SPE or sample well or sample vial may give off

plasticizers over long standing of analyte solution plasticizers over long standing of analyte solution

check the components w/o stronger solvent check the components w/o stronger solvent sonicationsonication can locate the interferencecan locate the interference

QC samples for specific purposesQC samples for specific purposes

� Accuracy Accuracy:

Calibration Control Standard (Calibration check)

Laboratory Control Standard: Standard reference materials (SRMs), Certified reference materials (CRMs) NIST, (US)EPA, (EU)BCR

� Precision:

Replicate samples, Replicate analyses

� Extraction efficiency:

Method Spikes, Matrix Spikes, Field Spikes, Surrogate Spikes

� Contamination:

Method Blank, Solvent Blank, Reagent Blank, Instrument Blank, Trip Blank

Ion Suppression Esi Ms-ms Primer

Documents

Transcript of Ion Suppression Esi Ms-ms Primer