Ion Suppression in LC-ESI-MSMS (a primer)

Why ion suppression is an issue?

A cross disciplinary generated issue it goes that more polar compounds are poorly retained on the
column in HPLC and will interfere with the detection and quantification of the target compound. Sample cleanup is normally required.

In contrast, a mass detector can be configured to isolate and quantify specific ions; therefore, sample cleanup and chromatographic separation can theoretically be reduced when analyte specific masses or product ions are monitored.

misconceptions
chromatographic separation can be minimized or even eliminated, the analytical column is needed only as a mechanism to load sample on the system, specimen cleanup can be minimized or eliminated, the use of HPLC-MS or HPLC-tandem MS provides absolute accuracy and specificity.

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

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

MS/MS detection can fool you!!!

salts, ion-pairing agents, endogenous compounds, drugs, metabolites, and proteins are responsible for Ion suppression Also compound dependent, difficult to find a common set of conditions that works for a large number of compounds

Generally used approaches

Examining ion suppression: signal recovery studies using specimen extracts with added analyte Use postcolumn infusion of the analyte to evaluate protracted ionization effects. Minimizing ion suppression by more rigorous cleanup, chromatographic changes, reagent modifications, and effective internal standardization Use expected physiologic concentrations of the analyte under investigation.

The nature of matrix effect

The mass and charge of individual analytes are also factors in making a compound a candidate for ion suppression or in making one compound a source of ion suppression for another. Molecules with higher mass will suppress the signal of smaller molecules and that more polar analytes are more susceptible to suppression

How to? is to compare!

the instrument response for calibrators (including any internal standards) injected directly in mobile phase, The data for the calibrator in mobile phase provide a relative 100% response value. the same amount of compound added to preextracted samples The data for the same amount of compound added to preextracted samples show the effect of sample matrix on MS response (ion suppression)

How to? is to compare! (Contd)

the same amount of compound added to specimen matrix before extraction The response data for extracted samples containing the analyte can highlight whether any loss of signal is attributable to the extraction process or ion suppression

Ion suppression attributable to endogenous matrix components

100 A C E

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)

Response, %

B 120 240

D 360 480

F 600

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

How to? is to check!

interference checks for possible co-horde drugs or metabolites Just because a coeluting drug does not produce similar mass fragments does not mean that this compound is incapable of ion suppression.

How to? is to check! (contd)

postcolumn continuous infusion of compound into the MS detector

a syringe pump connected via a T to the column effluent, mobile phase or specimen extracts are injected into the HPLC system. The analyte being evaluated is continuously infused postcolumn and is mixed with the column effluent through a tee before entering the electrospray interface.
T HPLC Syringe Pump ESI/MS

How to? is to check! (contd)

8 4

Ion intensity x 103

8 4

Compound being tested is introduced into the mass detector at a constant rate, a constant ESI response should ideally be observed (A)

8 4

Minutes

How to? is to check! (contd)

8 4

Ion intensity x 103

8 4

8 4

serum liquid-liquid extract injection, ion suppression can be ~90% (a recovery time may exist, suppression is not limited to the solvent-front region)

Minutes

How to? is to check! (contd)

8 4

Ion intensity x 103

8 4

8 4

Minutes

serum protein precipitation extract injection, ion suppression can be ~90%

Mixed mode SPE vs others

Mix-mode SPE Protein Preciptn R-P SPE A: Propranolol B: Trimethoprime C: Pipenzolate D: Resperidone E: Terfenadine F: Methoxyverapamil G: Benextramine H: Reserpine

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

Non-conclusive Conclusion:
The degree of ion suppression and the recovery time to full response: 1. vary from compound to compound and from sample to sample 2. dependent on the sample preparation (cleanup) method 3. may not be evident initially but may still be present during subsequent runs

Non-conclusive Conclusion:
4. dependent on the analyte concentration, matrix/analyte ratio: a) decreasing the matrix/analyte ratio through more extensive cleanup or dilute the test solution and evaluate the dilution factor vs response b) performing ion suppression validations by using concentrations reflecting that encountered in real samples.

Non-conclusive Conclusion:
5. Issues of Ion-pairing agents: conventionally used TFA causes signal suppression, to tackle this: a) decrease the TFA concentration provided adequate separation b) use alternative weaker acids such as acetic, formic, propionic acid or hexafluorobutyric acid c) TFA fix: postcolumn addition of acids and solvent carriers to displace TFA, as reported, the use of 2-(2methoxyethoxy) ethanol as a signal enhancer to ~100-fold enhancement of the signal for the model drug ibuprofen.

(TFA issue)
In positive ion mode, Signal suppression is caused by strong ion pairing between the TFA anion and the protonated analyte cation of basic molecules, effectively making them neutral. In negative ion mode, complete suppression of analyte can occur by TFA competing for charge. Aqueous solutions of TFA have high conductivity and surface tension, which causes spray instability in ESI.

Non-conclusive Conclusion:
6. other approaches: a) Modify the chromatographic conditions so that the compound(s) of interest elute(s) in a region where ion suppression is not observed. (change run!!!) b) use an internal standard (stable isotope or structural analog), and to modify the chromatography so that the compound of interest and the IS coelute. However, if ion suppression deteriorate the signal of the analyte or IS, the S/N may be compromised to a point where accuracy or precision becomes @#$%.

Non-conclusive Conclusion:
7. Other odditites: SPE or sample well or sample vial may give off plasticizers over long standing of analyte solution check the components w/o stronger solvent sonication can locate the interference

QC 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