Taking Aim at Interference (Without Shooting Yourself in the Foot)

May 2020

3

IntroductionPopularity of LC-MS/MS-based methods for

clinical testing continues to increase

One of the major reasons:

superior analytical specificity

Despite that, these methods may still suffer from interference Affecting method accuracy and precision

Negatively impacting patient care

4

Aim of this presentation Sources of guidelines for interference testing and monitoring

What is analytical interference?

Where does it come from?

How do we define acceptable interference levels?

How do we test for interference in LC-MS/MS?

When do we test for interference?

Can we use internal standard to mitigate interference?

How do we monitor for interference in routine testing?

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Guidelines for interference testing

CLSI – Clinical and Laboratory Standards Institute EP7-A2: Interference testing in clinical chemistry EP14-A2: Evaluation of matrix effects C62-A: Liquid chromatography-mass spectrometry methods

FDA – Food and Drug Administration Bioanalytical Method Validation, Guidance for Industry

SWGTOX – Scientific Working Group for Forensic Toxicology SWGTOX Doc 003: Standard Practices for Method Validation in Forensic

Toxicology

WADA – World Anti-Doping Agency WADA Technical Document – TD2015IDCR: Minimum criteria for LC-MS

confirmation of the identity of analytes for doping control purposes

European Medicines Agency Guidelines on bioanalytical method validation

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What is analytical interference?

Interference =

the effect of a substance, identified or not, that causes the measured concentration of an analyte to differ from its true value.

Interferent or Interfering Substance =

the substance causing interference

Reference: CLSI document EP14-A2. Evaluation of matrix effects; Approved guideline – 2nd edition. Wayne (PA), 2005.

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What is analytical interference?

Interference May appear as partially or

completely co-eluting peaks in the analyte/internal standard mass chromatograms

May be invisible to the naked eye –a matrix effect Interferent alters the efficiency of

analyte/internal standard ions reaching the MS detector

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Time

Inte

nsity

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Where does interference come from?

Interfering substances May come from many different sources

May be introduced at any time before or during the testing workflow

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Compounds related to patient treatment medications parenteral nutrition plasma expanders

Metabolites produced in normal & pathological conditions

Substances ingested by patients alcohol drugs of abuse nutritional supplements food

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Examples of interfering substances

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Substances added during sample preparation anticoagulants – heparin & EDTA salts preservatives stabilizers

Contamination during sample handling hand lotion, soap serum separators collection tube stoppers leachables from plastic consumables

Interference arising from the sample matrix hemolysis – hemoglobin and RBC contents icterus – bilirubin lipemia – lipoproteins

Examples of interfering substances

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How do we define acceptable interference levels?

Reference: CLSI document EP7-A2. Interference testing in clinical chemistry; Approved guideline – 2nd edition. Wayne (PA), 2005.

Acceptability criteria must be decided prior to conducting evaluation to ensure objectivity

Key questions: 1. How large a discrepancy is considered clinically significant?

2. What are the allowable analytical error limits? Accuracy requirements:

Have been proposed for some analytes (total allowable error) Can be established based on physiological variability (inter- & intrapersonal) Can be derived from clinical experience (consensus of clinical experts) Can be based on analytical variability (long-term imprecision)

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Why do we test for interference?

Interference affects: Method accuracy Method precision Quality and validity of reported patient results

Assessing susceptibility to analytical interference– very important part of LC-MS/MS

method development and validation

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How do we test for interference in LC-MS/MS?

Interference testing falls into two categories:

1) Direct testing of the effect of specific substances on analyte concentration

2) Evaluation of unidentified interferents arising from sample matrix and anything added to it

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1. Testing for specific interference

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+

LC-MS/MS analysis

Patient specimens w/ analyte of interest Sample pool

Interferent 1Interferent 2

Interferent 3

Solvent

Analyze both test and control samples in the same manner as patient specimens with adequate replication within one analytical run

Test samples Control sample

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1. Testing for specific interference Evaluate interference as a relative bias of the target analyte

concentration in test vs control sample:

𝐁𝐁𝐁𝐁𝐁𝐁𝐁𝐁(%) = 𝐀𝐀𝐀𝐀𝐁𝐁𝐀𝐀𝐀𝐀𝐀𝐀𝐀𝐀 𝐁𝐁𝐀𝐀 𝐀𝐀𝐀𝐀𝐁𝐁𝐀𝐀 𝐁𝐁𝐁𝐁𝐬𝐬𝐬𝐬𝐀𝐀𝐀𝐀 −[𝐀𝐀𝐀𝐀𝐁𝐁𝐀𝐀𝐀𝐀𝐀𝐀𝐀𝐀 𝐁𝐁𝐀𝐀 𝐜𝐜𝐜𝐜𝐀𝐀𝐀𝐀𝐜𝐜𝐜𝐜𝐀𝐀 𝐁𝐁𝐁𝐁𝐬𝐬𝐬𝐬𝐀𝐀𝐀𝐀][𝐀𝐀𝐀𝐀𝐁𝐁𝐀𝐀𝐀𝐀𝐀𝐀𝐀𝐀 𝐁𝐁𝐀𝐀 𝐜𝐜𝐜𝐜𝐀𝐀𝐀𝐀𝐜𝐜𝐜𝐜𝐀𝐀 𝐁𝐁𝐁𝐁𝐬𝐬𝐬𝐬𝐀𝐀𝐀𝐀]

×100%

Analyte – Test at 2 (or more) concentrations

Potential interferent – Spike at the highest concentration expected in patient specimens When clinically significant interference present =>

evaluate further at lower concentrations

Reference:CLSI EP7-A2: Interference testing in clinical chemistry – guidelines for test concentrations for many analytes and potential interferents

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1. Testing for specific interferenceExample calculations table

Measured Concentration %Bias from Control Mean

Potential Interferent % Spike Volume

Intf. Conc. (µmol/L) Repl #1 Repl #2 Repl #3 Mean Repl #1 Repl #2 Repl #3 Mean

Control_Methanol Mean 5 52.1 52.5 52.2 52.3 -0.3% 0.5% -0.1%Caffeine 5 257 52.5 52.2 52.2 52.3 0.4% -0.1% -0.1% 0.1%Theobromine 5 278 52.1 51.9 52.1 52.0 -0.2% -0.7% -0.3% -0.4%Acetaminophen 5 331 52.2 51.6 48.3 50.7 0.0% -1.2% -7.7% -3.0%Aspirin (as salicylate) 5 362 52.3 51.6 52.1 52.0 0.0% -1.2% -0.4% -0.5%Diphenhydramine 5 196 52.0 52.0 51.8 51.9 -0.6% -0.6% -1.0% -0.7%Control_0.1 M NaOH 10 45.6 45.9 46.0 45.9 -0.5% 0.2% 0.4%Uric acid 10 1487 47.6 47.2 47.7 47.5 3.9% 3.0% 4.0% 3.6%Icterus - bilirubin 10 248 46.6 46.7 46.5 46.6 1.6% 1.7% 1.3% 1.6%Control_Normal Saline 10 47.4 47.1 47.4 47.3 0.3% -0.5% 0.2%Hemolysis - hemoglobin 10 166 41.6 41.4 41.5 41.5 -12.1% -12.4% -12.4% -12.3%Lipemia - triglycerides 10 N/A 48.6 48.3 48.0 48.3 2.7% 2.1% 1.5% 2.1%Ibuprofen 10 485 47.2 47.2 47.2 47.2 -0.3% -0.2% -0.3% -0.2%

Note: Think about the “mechanism” of the interference.Adding the interferent solution may not always give you a clear pictureof how the interferent acts in a biological system.

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1. Testing for specific interference

Advantages

Ability to define acceptable/unacceptable sample collection conditions

Ability to set limitations for sample abnormalities

Ability to provide guidelines for patient preparation(medications, supplements, and foods to avoid prior to sample collection)

To obtain a valid test resultTo reduce the need for repeat specimen collection and analysis

Disadvantages

Laboriousness of testing a large number of substances

No practical interference study can identify all potential interferents

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2. Testing for unidentified interference

LC-MS/MS allows testing for interference that cannot be anticipated or identified beforehand (matrix effects)

Interference arising from sample matrix can cause: Signal enhancementSignal suppression

Evaluation of matrix effects:A. Qualitative post-column infusion studyB. Quantitative matrix effects study

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A. Qualitative post-column infusion study Analyte solution infused into the LC column effluent

While analyzing blank sample matrix

When blank sample matrix not available/not representative of patient specimens IS may be infused while analyzing a patient specimen

HPLC

Syringe Pump

Mass Spectrometer

LC column Tee

Sample

Analyte solution(IS solution)

Patient specimen

Void volume

Inte

nsity

(cps

)

Time (min)

Infusion trace

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A. Qualitative post-column infusion study

Signal suppression

Ion suppression (enhancement) evaluated as the presence of negative (positive) peaks in a steady signal trace of the infused analyte or IS

Advantage

Allows for visualization of the position and width of matrix effects regions

Useful in optimizing separation conditions

Calibrator

Void volume

Inte

nsity

(cps

)

No signal suppression

Time (min)

Infusion trace

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B. Quantitative matrix effects study

LC-MS/MS analysis

Extracted samples: Various blank matrices Patient specimens

with no/low analyte concentrations

Analyte Analyte Analyte Analyte Analyte

Analyze both test and control samples

in the same manner as patient specimens with adequate replication within one analytical run

#1 #2 #3 #n…Test samples

+Solvent

Control sample

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B. Quantitative matrix effects study

Analyte signal in test sample expressed as % of control:

𝐌𝐌𝐁𝐁𝐀𝐀𝐜𝐜𝐁𝐁𝐌𝐌 𝐀𝐀𝐞𝐞𝐞𝐞𝐀𝐀𝐜𝐜𝐀𝐀(%) = 𝐀𝐀𝐀𝐀𝐁𝐁𝐀𝐀𝐀𝐀𝐀𝐀𝐀𝐀 𝐁𝐁𝐀𝐀 𝐀𝐀𝐀𝐀𝐁𝐁𝐀𝐀 𝐁𝐁𝐁𝐁𝐬𝐬𝐬𝐬𝐀𝐀𝐀𝐀[𝐀𝐀𝐀𝐀𝐁𝐁𝐀𝐀𝐀𝐀𝐀𝐀𝐀𝐀 𝐁𝐁𝐀𝐀 𝐜𝐜𝐜𝐜𝐀𝐀𝐀𝐀𝐜𝐜𝐜𝐜𝐀𝐀 𝐁𝐁𝐁𝐁𝐬𝐬𝐬𝐬𝐀𝐀𝐀𝐀]

×100%

Matrix effect: <100% indicates signal suppression >100% indicates signal enhancement

Alternatively, as %Bias from control:

𝐌𝐌𝐁𝐁𝐀𝐀𝐜𝐜𝐁𝐁𝐌𝐌 𝐀𝐀𝐞𝐞𝐞𝐞𝐀𝐀𝐜𝐜𝐀𝐀(%) = 𝐀𝐀𝐀𝐀𝐁𝐁𝐀𝐀𝐀𝐀𝐀𝐀𝐀𝐀 𝐁𝐁𝐀𝐀 𝐀𝐀𝐀𝐀𝐁𝐁𝐀𝐀 𝐁𝐁𝐁𝐁𝐬𝐬𝐬𝐬𝐀𝐀𝐀𝐀 −[𝐀𝐀𝐀𝐀𝐁𝐁𝐀𝐀𝐀𝐀𝐀𝐀𝐀𝐀 𝐁𝐁𝐀𝐀 𝐜𝐜𝐜𝐜𝐀𝐀𝐀𝐀𝐜𝐜𝐜𝐜𝐀𝐀 𝐁𝐁𝐁𝐁𝐬𝐬𝐬𝐬𝐀𝐀𝐀𝐀][𝐀𝐀𝐀𝐀𝐁𝐁𝐀𝐀𝐀𝐀𝐀𝐀𝐀𝐀 𝐁𝐁𝐀𝐀 𝐜𝐜𝐜𝐜𝐀𝐀𝐀𝐀𝐜𝐜𝐜𝐜𝐀𝐀 𝐁𝐁𝐁𝐁𝐬𝐬𝐬𝐬𝐀𝐀𝐀𝐀]

×100%

Matrix effect: <0% indicates signal suppression >0% indicates signal enhancement

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B. Quantitative matrix effects study

The extent of a matrix effect can be calculated as: Non-normalized (using peak areas) or Normalized to internal standard (using concentrations)

Both matrix effect values provide valuable information: Non-normalized – actual magnitude of ion suppression/enhancement Normalized values – how well the IS compensates for the matrix effect

These experiments should be performed: At two analyte concentrations expected in the patient population With several native matrix sources, such as different patient specimens or

different vendor sources

Useful especially when: Qualitative post-column infusion experiment cannot be performed

(e.g. LC flow rate high => too high a backpressure for syringe pump) Testing matrices used for calibrator or QC preparation

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B. Quantitative matrix effects studyControl (solvent) spiked

with analyteTest sample (Plasma 1) spiked

with analyteTest sample (Plasma 1)

Baseline

Analyte Repl #1

Repl #2

Repl #3 Mean CV Repl

#1Repl #2

Repl#3 Mean CV Repl

#1Repl #2

Repl #3 Mean CV

Analyte 1 101 103 107 104 3% 113 114 112 113 1% 13 14 13 13 3%Analyte 2 98 99 96 98 2% 337 315 312 321 4% 213 225 233 224 5%Analyte 3 103 111 106 107 4% 103 102 104 103 1% 1 1 1 1 14%Analyte 4 103 107 104 104 2% 357 332 329 339 5% 256 254 254 255 0%Analyte 5 100 106 112 106 6% 98 102 101 101 2% 0 0 0 0 ####Analyte 6 94 93 99 95 3% 98 95 97 97 2% 8 8 9 8 6%Analyte 7 96 98 106 100 6% 212 214 187 204 7% 113 110 110 111 2%Analyte 8 105 103 104 104 1% 271 257 251 260 4% 139 146 146 144 3%Analyte 9 99 101 99 100 1% 280 287 276 281 2% 191 193 205 196 4%Analyte 10 102 100 104 102 2% 211 204 198 204 3% 110 115 115 113 2%Analyte 11 94 96 94 95 1% 185 182 176 181 3% 93 96 93 94 2%Analyte 12 97 100 100 99 2% 152 146 152 150 2% 53 55 56 55 3%Analyte 13 97 96 95 96 1% 190 191 183 188 2% 101 101 106 103 3%Analyte 14 97 102 101 100 3% 95 96 99 97 2% 7 7 7 7 0%Analyte 15 113 112 111 112 1% 506 483 489 493 2% 390 422 422 411 4%

Precision: %CV ≤15%10% ≤15% 16% >15%, ≤20%21% >20%

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B. Quantitative matrix effects study

AN Spike

Target Conc

Control (Solvent) Plasma 1 Plasma 2 Plasma 3 Plasma 4 Plasma 5 Plasma 6

Mean Conc

Mean %ME

Mean Conc Mean Rec

Mean %ME

Mean Conc Mean Rec

Mean %ME

Mean Conc Mean Rec

Mean %ME

Mean Conc Mean Rec

Mean %ME

Mean Conc Mean Rec

Mean %ME

Mean Conc Mean Rec

Mean %MEAnalyte Spkd Bsln Spkd Bsln Spkd Bsln Spkd Bsln Spkd Bsln Spkd Bsln

Analyte 1 100 104 4% 113 13 100 -4% 103 8 95 -9% 103 7 97 -7% 105 7 99 -5% 108 8 100 -4% 104 6 99 -5%Analyte 2 100 98 -2% 321 224 97 -1% 422 320 101 3% 413 300 113 15% 551 414 137 39% 344 227 117 19% 374 246 129 31%Analyte 3 100 107 7% 103 1 102 -4% 100 0 100 -6% 96 0 96 -9% 101 0 100 -6% 104 0 104 -2% 103 0 103 -4%Analyte 4 100 104 4% 339 255 85 -19% 415 340 76 -27% 302 222 79 -24% 636 513 123 18% 360 272 88 -16% 463 386 78 -26%Analyte 5 100 106 6% 101 0 101 -5% 95 0 95 -10% 96 0 96 -9% 97 0 97 -8% 101 0 101 -4% 103 0 103 -2%Analyte 6 100 95 -5% 97 8 88 -7% 96 11 85 -11% 92 10 82 -14% 99 10 89 -7% 110 19 91 -4% 108 16 92 -4%Analyte 7 100 100 0% 204 111 93 -7% 179 90 88 -12% 200 105 96 -4% 235 128 106 7% 198 96 102 2% 180 88 92 -8%Analyte 8 100 104 4% 260 144 116 12% 254 148 106 2% 226 110 116 12% 280 160 121 16% 310 201 109 5% 266 141 125 21%Analyte 9 100 100 0% 281 196 85 -15% 287 201 86 -13% 191 106 85 -14% 212 120 92 -8% 303 219 84 -16% 273 184 89 -11%Analyte 10 100 102 2% 204 113 91 -11% 243 154 88 -13% 194 108 87 -15% 152 56 96 -6% 211 114 97 -5% 207 113 94 -8%Analyte 11 100 95 -5% 181 94 87 -9% 168 87 81 -14% 125 43 82 -14% 171 78 93 -2% 157 63 94 -1% 147 56 91 -4%Analyte 12 100 99 -1% 150 55 95 -5% 106 16 91 -9% 101 8 93 -6% 127 30 98 -2% 118 20 98 -1% 110 13 97 -2%Analyte 13 100 96 -4% 188 103 85 -11% 188 111 77 -20% 140 49 91 -6% 146 56 90 -7% 212 119 93 -4% 188 98 90 -6%Analyte 14 100 100 0% 97 7 90 -10% 90 3 88 -12% 90 1 89 -11% 90 2 88 -12% 95 5 90 -10% 92 2 90 -10%Analyte 15 100 112 12% 493 411 81 -27% 697 620 77 -31% 701 625 76 -32% 702 586 117 4% 647 574 73 -34% 625 541 83 -26%

20% Within ± 20% of mean analyte concentration in solvent - acceptable level of matrix effects (based on assay accuracy & precision)-25% < -20% of mean analyte concentration in solvent - signal suppression25% > 20% of mean analyte concentration in solvent - signal enhancement

Legend:

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When do we test for interference?

Interference testing often performed as part of method validation

Waiting until method validation to perform these experiments can result in unwanted surprises

To ensure a developed LC-MS/MS method is robust and provides high quality data, test for interferences: as part of the method development process by performing the experiments outlined above

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Post-column infusion study – very useful for: Designing LC conditions to maneuver analytes

out of suppression zones (especially in the case of dilute-and-shoot methods prone to matrix effects)

Assessing extract cleanliness when determining which sample preparation method or which conditions may best mitigate matrix effects

When do we test for interference?

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A couple of notes:1) Interference testing and the adjustment of

method parameters may need to be an iterative process

2) Use as many patient specimens as practical to ensure that they capture the biological variability of interference

When do we test for interference?

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Investigate interference EARLY on, and with a large variety of specimens!

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Don’t shoot yourself in the foot!

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Interference mitigation by internal standard

Many ways to reduce interference, but no method is completely immune to it

Use of stable isotope-labeled internal standards (SIL-IS) to mitigate interference (matrix effects) common

When analyte elutes in a suppression region, compensating with a SIL-IS often deemed adequate

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Interference mitigation by internal standard

Problems:1.Separation effect (kinetic isotope effect):

= IS and analyte not co-eluting => Differential suppression => Assay accuracy compromised

2.Severe suppression by matrix: = Coeluting IS not compensating for matrix effects

=> Analyte and IS S/N ratio drastically reduced => Assay performance compromised, especially near LLOQ

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Problem #1: Separation effect Deuterated analogs mostly affected esp. when deuteriums in positions

impacting chromatographic retention

Differences in physicochemical properties between H and D

=> differences in interactionwith mobile & stationary phases

=> differences in retention times

Degree of separation Molecule size Number of D labels Position of D labels LC conditions

IS Analyte

Time (min)

Analyte and IS suppressed by matrix to different degrees

Quantitation errors Over-quantitating:

if IS more suppressed Under-quantitating:

if analyte more suppressed

The larger the separation, the lower the IS ability to compensate for matrix effects

Separation effect results in:Differential suppression by sample matrix

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D&S

Infusion trace

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Example 1: Oxycodone/oxycodone-d6 matrix effect

Scenario: Opioid panel Dilute-and-shoot Differential

suppression between analyte and IS

Solution: Selective sample

cleanup

SPE

2.0 3.0 4.0

1.0e6

2.0e6

3.0e6

4.0e6

Inte

nsity

(cps

)

1.0

NOC

OC

M

OM

OC-d6

0.0 Time (min)

Infusion trace

0.0 1.0 2.0 3.0 4.0Time (min)

1.0e6

2.0e6

3.0e6

4.0e6 NOCOC-d6

M

OM

Dilute-and-shoot

OC

Inte

nsity

(cps

)Infusion trace

Example 2: Quantitation w/ HIAA-d5 vs HIAA-13C6Post-column infusion experiment: samples with < ±10% conc. deviation

35

[HIAA] = 8.97 mg/LDeviation = 2%HIAA

HIAA-13C6

Time (min)

HIAA-d5

Infusion trace

[HIAA] = 2.27 mg/LDeviation = 0%

HIAA

HIAA-13C6

Time (min)

HIAA-d5

Infusion trace

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[HIAA] = 1.66 mg/LDeviation = 27%

HIAA

HIAA-13C6

Time (min)

HIAA-d5[HIAA] = 4.82 mg/LDeviation = -11%

HIAAHIAA-13C6

Time (min)

HIAA-d5

HIAA over-quantitated using HIAA-d5

HIAA under-quantitated using HIAA-d5

Example 2: Quantitation w/ HIAA-d5 vs HIAA-13C6Post-column infusion experiment: samples with > ±10% deviation

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Example 2: Quantitation w/ HIAA-d5 vs HIAA-13C6

HIAA-d5 HIAA

ΔtR = 0.03 min

HIAA-13C6 HIAA

ΔtR = 0.00 min

Scenario: Urinary HIAA method with

HIAA-d5 IS Dilute-and-shoot Analyte and IS not coeluting Differential suppression

between analyte and IS Quadratic calibration curve fit

Solution:Replace HIAA-d5 with coeluting IS:

HIAA-13C6(Alternate isotope label)

Alternate isotope labels: 13C,15N,…

Advantages Do not suffer from label

instability

Closer co-elution with analyte

Better compensation for matrix effects

Drawbacks

More difficult to make

More expensive

Not always commercially available

38

39

Problem #2: Coeluting IS not compensating for matrix effects

Scenario: Urinary Vanillylmandelic acid (VMA)

and Homovanillic acid (HVA) Dilute-and-shoot

4 min LC method

Co-eluting IS (VMA-d3) Interferences:

~3% VMA samples

( vs. < 0.03% HVA samples) Suppression zone partially coeluting

with analyte in validated method

Inte

nsity

VMA

Time (min)

40

Problem #2: Coeluting IS not compensating for matrix effects

Normal scenario:No to very little signal suppression by matrix components

Time (min)

Inte

nsity

VMA

Time (min)In

tens

ity

VMA

Severe matrix effect:Signal suppression severely decreases S/N for analyte and IS=> Assay performance compromised, especially near LLOQ

41

Solution: Chromatographic separation

Primary LC method for regular testing

Alternate LC method for reinjection of samples with VMA interference

Injection-to-injection time remained 4 min

Inte

nsity

VMA

Time (min)

Primary method

Time (min)

Inte

nsity

VMAAlternate method

Problem #2: Coeluting IS not compensating for matrix effects

42

Interference mitigation by internal standard???

Only when…

Using IS exactly coeluting with analytei.e. one with stable isotope labels that do not

exhibit separation effect (13C, 15N)better compensate for matrix effects

AND

Signal suppression is NOT severe

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You validated a robust method, thoroughly tested for interference, and handed it off to the clinical lab.

Are you done with interference?

Even the best method development strategies rarely able to prevent interference completely

Monitor for interference in routine testing to avoid reporting compromised results

44

How do we monitor for interference?

Most relevant data quality metrics: Ion ratios

Interferents isobaric with analyte/IS(appear on analyte/IS transitions)

Absolute Internal Standard areas Interferents causing matrix effects

(signal suppression/enhancement)

Retention times Near-eluting isobaric interferent integrated instead of analyte/IS

Deviations can signal the presence of interference in either the analyte or internal standard mass chromatograms

45

𝑰𝑰𝑰𝑰𝑰𝑰 𝒓𝒓𝒓𝒓𝒓𝒓𝒓𝒓𝑰𝑰 =𝑸𝑸𝑸𝑸𝒓𝒓𝑰𝑰𝒓𝒓𝒓𝒓𝑸𝑸𝒓𝒓𝒊𝒊𝒓𝒓 𝑴𝑴𝒓𝒓𝑴𝑴𝑴𝑴 𝑻𝑻𝒓𝒓𝒓𝒓𝑰𝑰𝑴𝑴𝒓𝒓𝒓𝒓𝒓𝒓𝑰𝑰𝑰𝑰 𝑷𝑷𝒊𝒊𝒓𝒓𝑷𝑷 𝒓𝒓𝒓𝒓𝒊𝒊𝒓𝒓𝑸𝑸𝑸𝑸𝒓𝒓𝑸𝑸𝒓𝒓𝑸𝑸𝒓𝒓𝒊𝒊𝒓𝒓 𝑴𝑴𝒓𝒓𝑴𝑴𝑴𝑴 𝑻𝑻𝒓𝒓𝒓𝒓𝑰𝑰𝑴𝑴𝒓𝒓𝒓𝒓𝒓𝒓𝑰𝑰𝑰𝑰 𝑷𝑷𝒊𝒊𝒓𝒓𝑷𝑷 𝒓𝒓𝒓𝒓𝒊𝒊𝒓𝒓

Quantifier

Qualifier

Ion ratios –Monitoring for isobaric interferents in chromatograms

46

Ideally calculated for both analyte and internal standard

Individual specimen ion ratios compared to mean ion ratio

Mean ion ratio – calculated from ion ratios of calibration standards / quality controls

Acceptance limits set during method development

based on clinical requirements for the assay

±20 or 30% common

Ion ratios –Monitoring for isobaric interferents in chromatograms

47

No interference Interference

Quantifier

Qualifier

Quantifier

Qualifier

Quantifier peak area 1.02E+05Qualifier peak area 6.43E+04Ion ratio 1.58IR±20% range 1.26-1.89

Ion ratios –Monitoring for isobaric interferents in chromatograms

Quantifier peak area 1.18E+05Qualifier peak area 5.57E+04Ion ratio 2.11IR±20% range 1.26-1.89

48

Absolute Internal Standard areas –Monitoring for matrix effects (mainly signal suppression)

Mean or median value calculated For the entire run, or

For calibration standards set

Acceptance limits set during method development Based on clinical requirements for the assay, and

Overall method performance

Individual specimen peak area values compared to mean/median value

49

Absolute Internal Standard areas –Monitoring for signal suppression

0

50,000

100,000

150,000

200,000

250,000

300,000

0 50 100 150

IS P

eak

Area

(cou

nts)

Sample #

Internal Standard 1

Well-controlled assay

200% of peak area median

50% of peak area median

peak area median

50

Absolute IS areas –Monitoring for signal suppression

Not so well-controlled assay

0

50,000

100,000

150,000

200,000

0 50 100 150

Peak

Are

a (c

ount

s)

Sample #

Internal Standard 2

200% of peak area median

50% of peak area median

peak area median

Typical of dilute&shoot methods – large variations due to signal suppression

Can indicate samples extraction issues –variable recovery

51

Retention times (absolute or relative to IS) –Monitoring for near-eluting isobaric interferent integrated in place of analyte/IS

Analyte

IS

Interference

2.59 min

2.53 min

2.75 min Interference

IS2.53 min

2.75 min

52

Retention times (absolute or relative to IS) –Monitoring for near-eluting isobaric interferent integrated in place of analyte/IS

Set up acceptance limits in MS software for retention time (RT) differences between analytes and their internal standards

Absolute RT differences

Percentage of target RT for IS

Acceptance limits based on method performance evaluated during development and validation

Analyte RTs beyond the acceptance limits are flagged by the software

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Metanephrine interference

Original Qual

Original Quant

No interference

Interference on Quant

Interference on Qual

Scenario: Urinary metanephrines Deuterated (d3) IS SPE sample cleanup 5 min LC method

Interferences on both Quant and Qual MRM in original method

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Metanephrine interference

Original Qual

Original Quant

Alternate Qual

No interference Interference on Quant Interference on Qual

Use: Alternate Qual Original Qual as

new Quant MRM

Use: Alternate Qual Original Quant

Solution: Alternate quantitation method using a 3rd MRM transition

Use: Original Qual Original Quant

55

OC+IntfOC-d6

Specimen with Oxycodone and coeluting interference

316.15 241.2316.15 256.2

Coeluting Oxycodone interference

Scenario: Opioid panel

Unresolved Oxycodone interference in original opioid method

SPE sample prep

5 min run time

Added all viable MRMs

316.15 212.3316.15 181.3316.15 168.3316.15 128.2316.15 115.2

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316.15 168.3316.15 115.2316.15 241.2316.15 256.2316.15 181.3316.15 212.3316.15 128.2

Scenario: All MRMs give signal

for both analyte and interference

But at different intensities!!!

Coeluting Oxycodone interference

CalibratorOC

OC-d6316.15 241.2316.15 256.2316.15 212.3316.15 181.3316.15 168.3316.15 128.2316.15 115.2

Specimen with OC interference only

OC intf

OC-d6

57

OC intf

OCOC-d6 OC-d6

OCOC intf

Coeluting Oxycodone interference

Solution: Chromatographic separation to resolve analyte from interferent Change in LC gradient profile

58

References:1. CLSI. Evaluation of matrix effects; Approved guideline – second edition. CLSI document EP14-A2.Wayne (PA): CLSI; 2005.2. CLSI. Interference testing in clinical chemistry; Approved guideline – second edition. CLSI document EP7-A2.Wayne (PA): CLSI;

2005.3. CLSI. Liquid chromatography-mass spectrometry methods; Approved guideline. CLSI document C62-A. Wayne (PA): CLSI; 2014.4. FDA. Guidance for Industry – Bioanalytical Method Validation, 2001. http://www.fda.gov/cder/guidance/index.htm.5. SWGTOX. Scientific Working Group for Forensic Toxicology Standard Practices for Method Validation in Forensic Toxicology,

Doc 003, 2013. https://academic.oup.com/jat/article/37/7/452/765476.6. World Anti-Doping Agency (WADA) Technical Document – TD2015IDCR. https://www.wada-

ama.org/sites/default/files/resources/files/td2015idcr_-_eng.pdf.7. European Medicines Agency. Guideline on bioanalytical method validation, 2012.

http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/08/WC500109686.pdf.8. Clark ZD, Balloch S, Calton L, Mason D. Interference Testing and Mitigation in LC-MS/MS Assays. Clinical Laboratory News

2017;43(8):22-5.9. Clark ZD, Cutler JM, Pavlov IY, et al. Simple dilute-and-shoot method for urinary vanillylmandelic acid and homovanillic acid by

liquid chromatography tandem mass spectrometry. Clin Chim Acta 2017;468:201–8.10. Clark ZD, Cutler JM, Frank EL. Practical LC-MS/MS method for 5-hydroxyindoleacetic acid in urine. J Appl Lab Med 2017;1:387–

99.11. Clark ZD, Strathmann FG, McMillin GA. Diluting and shooting yourself in the foot: Complications with sample-to-sample variations

in signal suppression. MSACL 2013 podium presentation.12. Clark ZD, Frank EF. Urinary metanephrines by liquid chromatography tandem mass spectrometry: Using multiple quantification

methods to minimize interferences in a high throughput method. J Chrom B 2011;879(31):3673-80.13. Matuszewski BK, Constanzer ML, Chavez-Eng CM. Strategies for the assessment of matrix effect in quantitative bioanalytical

methods based on HPLC-MS/MS. Anal Chem 2003;75:3019–30.14. Annesley TM. Ion suppression in mass spectrometry. Clin Chem 2003;49:1041–4.15. Bonfiglio R, King RC, Olah TV, et al. The effects of sample preparation methods on the variability of the electrospray ionization

response for model drug compounds. Rapid Commun Mass Spectrom 1999;13:1175–85.16. King R, Bonfiglio R, Fernandez-Metzler C, et al. Mechanistic investigation of ionization suppression in electrospray ionization.

J Am Soc Mass Spectrom 2000;11:942–50.17. Lynch KL. LC-MS/MS quality assurance in production: The real work begins after validation. Clinical Laboratory News

2017;43(5):28–9.18. Zabell APR, Stone J, Julian RK. Using big data for LC-MS/MS quality analysis. Clinical Laboratory News 2017;43(5):30–1.

AcknowledgementsMSACL organizers and committee members

Donald MasonLisa CaltonSteven Balloch

Dr. Marzia PasqualiDr. Frederick StrathmannDr. Elizabeth FrankDr. Gwen McMillin

Staff of ARUP’s Mass Spectrometry II, Clinical Toxicology, Analytic Biochemistry, and Biochemical Genetics Labs

ARUP Institute for Clinical and Experimental Pathology®

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