NVKC 2015 Niessen Final - The Matrix... · 2018. 1. 21. · 11/15/2015 5 Matrix effects/ionization...

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1 ©2015, hyphen MassSpecNVKC, PAOKC The Matrix, 17 November 2015, Arnhem, NL

Matrix effects/ionizationsuppression in LC‒MS

W.M.A. Niessenhyphen MassSpec

Warmond, The Netherlandswww.hyphenms.nl

Matrix effects/ionization suppression in LC‒MSIonization suppression/enhancement

2 ©2015, hyphen MassSpec

Electrospray ionization is a complex liquid-phaseionization process, influenced by droplet constituents: Mobile-phase additives:

• Buffer salts, acids, ion-pairing agents (TFA).

Co-eluting analyte or internal standards (IS).

Sample constituents (matrix effects):• (Non-volatile) salts.• Surface-active compounds, e.g., phospholipids.• (Non-volatile) biomacromolecules.• Compounds, e.g., with high proton affinity.

Matrix effects/ionization suppression in LC‒MSTargeted/untargeted analysis: the challenges


#targets Application of quantitative LC‒MS Aim of analysis1‒5 (Pre-)clinical studies in drug discovery/ drug

developmentReliable quantitative analysis for cohorts

1‒5 Clinical analysis of endogenous compounds Reliable quantitative analysis for individual patients

1‒5 Therapeutic drug monitoring

5‒20 Targeted clinical toxicology Screening and quantitative analysis

10‒300 Multiresidue analysis for environmental and food safety, sports doping

Screening and (semi-) quantitative analysis; targeted or untargeted≥1000 Systematic toxicological analysis

≥1000 Metabolomics and systems biology Comparison of states; targeted or untargeted

Matrix effects/ionization suppression in LC‒MSTargeted/untargeted analysis: the challenges


#targets Application of quantitative LC‒MS Sample matrices1‒5 (Pre-)clinical studies in drug discovery/ drug

developmentPlasma/serum

1‒5 Clinical analysis of endogenous compounds Plasma/serum/urine1‒5 Therapeutic drug monitoring Blood, plasma, serum,

urine, oral fluids, hair, …5‒20 Targeted clinical toxicology10‒300 Multiresidue analysis for environmental and

food safety, sports dopingMany different matrices: fruits, vegetables, food of animal origin, …

≥1000 Systematic toxicological analysis plasma, serum, urine, …≥1000 Metabolomics and systems biology Plasma, serum, urine/…

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Matrix effects/ionization suppression in LC‒MSIntroduction

5 ©2015, hyphen MassSpecW.M.A. Niessen et al., Mass Spectrom. Rev., 25 (2006) 881.

Matrix effects result in (unexpected) suppression or enhancement of the response due to co-elutingmatrix/sample constituents.Common matrix effects

Small retention time shifts, small interferences, poorer S/N.

Absolute matrix effects

Increase or decrease in response between solvent standard and spiked pretreated sample.

Relative matrix effects

Differences in response, accuracy and/or precision between different batches of the same matrix.

Matrix effects/ionization suppression in LC‒MSIntroduction


Matrix effects are dependent of:• Matrix• Analyte• Sample pretreatment and LC method• Ion-source geometryand independent of:• Type of mass analyzer

Matrix effects are ionization phenomena,occurring in the (electrospray) ion source.In ESI generally more matrix effects than in APCI.

Matrix effects/ionization suppression in LC‒MSDetection and monitoring of matrix effects


Different ways to detect and monitor matrix effects:• Compare response of post-extraction spiked

sample and solvent standard

• Post-column infusion method

• Post-extraction spike method

• Compare slopes of calibration curves• Multiple matrices and solvent standard• Multiple batches of a matrix

Matrix effects/ionization suppression in LC‒MSAbsolute matrix effect

8 ©2015, hyphen MassSpecA. Agüera et al., J. Chromatogr A, 1045 (2004) 125.

Increase or decrease in response between solventstandard and spiked pretreated sample.

Spiked pretreatedblank samples

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Matrix effects/ionization suppression in LC‒MSPost-column infusion method

10 ©2015, hyphen MassSpecR. King et al., J. Am. Soc. Mass Spectrom., 11 (2000) 942.

Qualitative assessment of suppression/enhancement

Continuous post-column infusion of analyte uponinjection of pretreated blank samples• Evaluate effect of sample pretreatment


11 ©2015, hyphen MassSpecM.D. Nelson and J.W. Dolan, LC-GC Eur., 15 (2002) 73.

Identifies regions in chromatogram where the analyte is susceptible to matrix effects.• Avoid early eluting analytes (k’ < 2‒3).

As matrix effects are compound-dependent, test must be performedfor each target analyte.


12 ©2015, hyphen MassSpecJ.L. Little et al., J. Chromatogr. B, 833 (2006) 219.

Monitoring ionization suppression by phospholipids

Eliminate phospholipids by sample pretreatment.

Glycerophosphocholinesabundantly present in plasma/serum.

Mixed hydrophobic/ioniccharacter.

Build-up of phospholipidson reversed-phase columns.

Ionization enhancement,not phospholipid related.

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Matrix effects/ionization suppression in LC‒MSPost-extraction spike method

14 ©2015, hyphen MassSpecM. Becker et al., Anal. Chim. Acta, 520 (2004) 19.

Quantitative assessment of suppression/enhancement

Matrix effects are compound and matrix dependent








Matrix effects/ionization suppression in LC‒MSCompare slopes of calibration curves

16 ©2015, hyphen MassSpecB.K. Matuszewski et al., Anal. Chem., 75 (2003) 3019.

1 Solvent standard Analyte and IS in solvent2 Post-extraction spike Add analyte and IS after extraction3 Pre-extraction spike Add analyte and IS before extraction

Matrix effect:%ME < 100: Suppression%ME > 100: Enhancement

Extraction recovery:

Overall processefficiency:

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Method development drug discovery in plasmaSample pretreatment:• Mix 1 ml plasma and 200 μl IS• Add 1 ml carbonate buffer (pH 9.8)• Extract with 7 ml MtBE• Separate layers, evaporate• Reconstitute in 300 μl mobile phase

Liquid chromatography:• Hypersil BDS C18 (50×4.6 mm; 3 μm)• Isocratic elution: 80% ACN; 0.1% FA

Mass spectrometry:• Positive-ion ESI or APCI• Sciex API3000• SRM m/z 394 > 326 (Analyte) and

m/z 358 > 290 (Analogue IS)



Evaluate the slopes of calibration curves for 5-6independent blank matrix samples.

Develop acceptance criteria based on slopes.


19 ©2015, hyphen MassSpecB.K. Matuszewski, J. Chromatogr. B, 830 (2006) 293.

Criteria to assess relative matrix effects

1 Range in %RSD of calibration samplesCompare solvent & post-extraction spike

Similar %RSD-range%RSD with ~12‒15%

2 %RSD of slopes of calibration plots of at least 5 sample batches

Slope %RSD < 3‒4%

3 Compare differences in slopes of calibration plots

Slope differences < 10%



Criteria to assess relative matrix effectsRe-evaluation of 52 validated bioanalytical methods accordingto three criteria:1. Similar %RSD-range;

%RSD with ~12‒15%2. Slope %RSD < 3‒4%3. Slope differences < 10%

45 out of 52 validated method comply with criteria 2 and 339 out of 45: APCI instead of ESI17 out of 45: Stable-isotope IS instead of Analogue IS

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Criteria to assess relative matrix effectsRe-evaluation of validated bioanalytical methods according to criteria

Compound %RSD with ~12‒15%

Slope %RSD< 3‒4%

Slope diff< 10%

50 (1 Batch) 4.2‒8.5 2.4 6.550 (5 Batches) 11.1‒27.8 13.2 34.3

7 1.2‒7.9 0.7 1.743 2.1‒7.5 3.2 7.348 1.5‒7.7 6.5 18.3

Matrix effects/ionization suppression in LC‒MSCompare different analytes and matrices

22 ©2015, hyphen MassSpecA.M. Botero-Coy et al., Anal. Bioanal. Chem., 407 (2015) 3667.

Matrix effects/ionization suppression in LC‒MSCompare slopes of calibration plots

23 ©2015, hyphen MassSpecA.M. Botero-Coy et al., Anal. Bioanal. Chem., 407 (2015) 3667.

Apply calibration plots to compare different matrices

Multi-residue LC-MS method for pesticide analysis in different tropical fruits.Areas within ±20% of solvent standards indicated as

Matrix effects/ionization suppression in LC‒MSAvoiding matrix effects


Multiple efforts needed to reduce matrix effects:• Apply matrix-matched calibration standards.• Improve sample pretreatment.

• Avoid “only protein precipitation” and “dilute & shoot”• Mixed-mode hydrophobic/ion-exchange against

phospholipids in plasma.• Improve LC separation.• Explore MS conditions: ESI vs APCI; positive vs negative.• Reduce injection volume and/or dilute extracts.• Apply stable-isotope-labeled internal standard (SIL-IS).

• Co-eluting analogue internal standard.• Standard addition method.

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Matrix effects/ionization suppression in LC‒MSAvoid matrix effects

25 ©2015, hyphen MassSpecM. Villagrasa et al., J. Chromatogr. A, 1157 (2007) 108.

Multiple efforts needed to reduce matrix effects:• Reduce injection volume and/or dilute extracts

5‒10× dilution oftensuccessful.

Requires sufficientlylow detection limits.

Therefore, limitedapplicability.


26 ©2015, hyphen MassSpecS. Wang et al., J. Pharm. Biomed. Anal., 43 (2007) 701.

Multiple efforts needed to reduce matrix effects:• Apply stable-isotope-labeled internal standard (SIL-IS)

Despite the use of SIL-IS,sample Lot 3 behaves differently.

SIL-IS does not correct for matrixeffects.

Results with 50×-dilution indicatethis is due to matrix effect.





SIL-IS does not correct for matrixeffects.

Post-column infusion shows criticalsuppression in Lot 3





Retention time shift between [D5]-IS and analyte results in different ionization suppression in Lot 3

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29 ©2015, hyphen MassSpecD. Remane et al., Rapid Commun. Mass Spectrom., 24 (2010) 859.

Effect of co-eluting SIL-IS on analyte response• Co-elution of analyte and SIL-IS leads to ionization suppression/

enhancement.• More pronounced effect in ESI (12 out of 14) than in APCI

(1 out of 14).

• Not necessarily affects reliable quantification of analyte, but may influence quantification limits.

• Does affect quantification if SIL-IS is applied as IS for several(related) analytes (no SIL-IS suppression for these analytes)

• Similar effects occur with co-eluting analytes.



Effect of co-eluting SIL-IS on analyte response



Effect of co-eluting SIL-IS on analyte response:• Co-elution of analyte and SIL-IS leads to ionization suppression/

enhancement.• More pronounced effect in ESI (12 out of 14) than in APCI

(1 out of 14).

• Not necessarily affects reliable quantification of analyte, but may influence quantification limits.

• Does affect quantification if SIL-IS is applied as IS for several(related) analytes (no SIL-IS suppression for these analytes)

• Similar effects occur with co-eluting analytes.

Matrix effects/ionization suppression in LC‒MSMatrix effects in systematic toxicological analysis

32 ©2015, hyphen MassSpecF.T. Peters and D. Remane, Anal. Bioanal. Chem., 403 (2012) 2155.

Review on matrix effects in clinical and forensic toxicology, incl. systematic toxicological analysis.

• Overview of experiments performed in multiresidue analysis in urine, serum, plasma, blood, oral fluid, hair, ...

• Impossible to develop multiresidue methods free frommatrix effects.

• Matrix effects can only be studied for limited number oftarget analytes, preferably 20‒30 model compounds, well-spaced over the chromatogram.

• Acceptance criteria less strict in STA?

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Matrix effects/ionization suppression in LC‒MSConclusion

33 ©2015, hyphen MassSpecF.T. Peters et al., Toxichem. Krimtech., 76 (2009) 185.B.K. Matuszewski, J. Chromatogr. B, 830 (2006) 293.

Matrix effects should be thoroughly investigatedto develop reliable and robust analytical methods.

Absolute matrix effects

Compare response between solvent standard and spiked pretreated sampleAcceptance criteria: <10‒15%

Relative matrix effects

Evaluate response in 5‒6 independent matrix batchesAcceptance criteria: • %RSD of %ME < 15‒25%• Slope %RSD < 3‒4%• Slope difference within 10%

DISCLOSUREArnhem – 17-11-2015

(Potentiële) Belangenverstrengeling Zie hieronder

Sponsoring GeenHonorarium/vergoeding GeenAandeelhouder GeenAnders, namelijk..• Eigenaar hyphen MassSpec 100%

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