www.EurofinsLancasterLabs.com

An Alternative Approach to Quantitative Methods: Limit test for

Analysis of Deeming Analytes in Electronic Cigarettes

Carl Adams, Salem Chouchane

and Darius Grissom

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Questions

• 1000 or more samples of different electronic cigarettes are being analyzed

• Many constituents (deeming analytes), are reported as below detections, ND, below LOQ, BDL, BQL, NQ etc…

• When quantitated, levels are extremely low, in ppb, or even ppt• Aromatic amines, ammonia, BaP, etc…• Is it efficient, cost effective, and common sense to continue

quantitating, in E-liquid or aerosol, constituents that are consistently either not present in the matrices or at extremely low level?. Lower than what you can find in environment or food.

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Questions

• Can constituents in E-liquid or aerosol be considered impurities?

• Is Limit Test an acceptable analytical methodology to monitor such constituents in electronic cigarettes, or

ENDs products?

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Background

It is well known in industry that regulating agencies will require a product manufacturer to identify and control the level of impurities in a product, such that the integrity of the product is maintained with respect to identity, strength, quality, and purity for use by the consumer or patient.

As part of the purity analysis of a product during the development lifecycle, it is typical for product manufacturers to characterize impurities that may arise from the synthesis, manufacturing and packaging of those products and also the degradation products that occur over the shelf life of that product.

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Background

While it is common for many companies to quantitate and report specific levels of impurities in their developmental or commercial products, the specific quantitation of each impurity is not required provided that the overall total of impurity is below a limit of significance.

The significance of a limit can be based upon, but is not limited too:• Toxicological Assessment (i.e. Lowest Concentration Resulting in a

Toxic Effect or (TCLo), Acceptable Daily exposure (ADE) or Occupational Exposure Limit (OEL)

• Regulatory requirement (i.e. NNN in Smokeless Products)• Best Practice • Process Capability• Equipment Capability• Economic Factors (industry/region dependent)

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The LIMIT Test

Limit Test – A quantitative or semi-quantitative test designed and validated to allow an organization to assess the overall purity of their product with respect to a specific level of known or theoretical impurity.

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Regulatory, Compendia, and Guidelines

Are Limit Tests allowed according to Regulatory Agencies, compendia, and guidelines? Answer: Yes

Food and Drug Administration:• A review of the FDA website www.fda.gov allows the user to pull

up the guidance for industries whereby Q2A states: • Testing for impurities can be either a quantitative test or a

limit test for the impurity in a sample. Either test is intended to accurately reflect the purity characteristics of the sample. Different validation characteristics are required for a quantitative test than for a limit test.

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FDA Guidance for industry ANDAs: Impurities in Drug SubstancesInternational Conference on Harmonisation, ICH (2003) Q1A (R2) Stability testing of new drug substances and products, USA

International Conference on Harmonisation, ICH (1995) Q2A,Validation of analytical procedures(definitions and terminology), USA12. International Conference on Harmonisation, ICH (1996) Q2B,Validation of analytical procedures:methodology, USA

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Regulatory, Compendia, and Guidelines

United States Pharmacopeia:• Category II – Analytical procedures for the determination of impurities in bulk drug

substances or degradation compounds in finished pharmaceutical products. These procedures include quantitative assays and limit tests.

International Conference on Harmonization:• The discussion of the validation of analytical procedures is directed to the four

most common types of analytical procedures:• - Identification tests;• - Quantitative tests for impurities' content;• - Limit tests for the control of impurities;• - Quantitative tests of the active moiety in samples of drug substance or

drug• product or other selected component(s) in the drug product.

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What are the benefits of Limits test?

The benefits of limit tests come in the form of:• Laboratory Efficiency

• Faster development and validation• Fewer standard preparations• Reduced # of injections per sequence• Simplified data processing, reviewing and reporting• Higher throughput.

• Cost Savings• Reduced consumption of reference standards• Reduced consumption of mobile phase• Reduced labor hours for analysis and data processing and

review

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Example Efficiency Gain

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Typical Sequence Quantitative Limit

Solvent SolventCalibration Blank Calibration Blank

L1 L1L2 FMCS BackgroundL3 FMCS Level 1L4 Rep 1L5 Rep 2

Blank Rep 3ICV Rep 4

Rep 1 Rep 5Rep 2

40 Minute efficiency gainon instrument

Rep 3Rep 4Rep 5CCV

Efficiency Gain

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Regulatory, Compendia, and Guidelines

Are the ICH, USP, and FDA in agreement with respect to what validation parameters are required? Answer: YES

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Validation parameter required per ICH, USP, and FDA for different type of tests

Validation parameters Assaytesting for Impurities

IdentificationQuantitative Limit

Accuracy YES YES NO NO

Precision - Repeatability YES YES NO NO

Precision - Intermediate Precision YES (1) YES * NO NO

Specificity YES YES YES YES

Detection Limit NO NO YES NO

Quantitation Limit NO YES NO NO

Linearity YES YES NO NO

Range YES YES NO NO

Robustness YES YES NO NO

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Validation Strategies

We identified two ways in which limit test methods can be validated.

1. Validate as Limit Test; Implement at Limit Test• Confidence that analytes will not be present at significant

levels in Vapor products (i.e. PAAs, BaP, Ammonia, SVO).

2. Validate as Quantitative Test; Implement as Limit Test with option to Quantitate.• Confidence that most formulations will result in trace level

impurities, but new product formulations may vary (i.e. Diacetyl; Acetyl Propionyl).

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Preparing Your Limit Standard

How to prepare the limit standard:• External Standard diluted in solvent (clean).• External Standard spiked/extracted/diluted in matrix (dirty,

builds in comparative recovery).• Deuterated Internal Standard for direct comparison.• Surrogate Internal Standard with defined relative response

factor• Blank Corrected Response

We used external Standard in Matrix, blank corrected where applicable.

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Some examples

Deeming Method Validations in Electronic Cigarette Aerosol and Liquid:

• Benzo-a-pyrene (Limit)• Select Volatile Organics (Limit)• Ammonia (Limit with Option to Quant)• Primary Aromatic Amine (Limit with Option to Quant)• Diacetyl; Acetyl Propionyl (Limit with Option to Quant)

Leachables Method Validations in Electronic Cigarette Aerosol:• Organic Leachables by LCMSMS – Quantitative with level 1 standard

at or below Analytical Evaluation Threshold (AET).• Organic Leachables by GCMS – Quantitative with level 1 standard at

or below AET.

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Some examples

Vapor Analysis: External Limit Standards in Matrix

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50 ng/mL Ammonia Standard vs. Aerosol (IC) 0.04-0.05ng/mL PAA Standards vs. Aerosol (GCMS)

0.1 ng/mL BaP Standard vs. Aerosol(UPLC/Fluorescence) 1.1-3.7 ng/mL SVO Standards vs. Aerosol (GCMS)

Note: Specifics about each method will be available during the Poster session.

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Example Results

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Example Results for PAAs in Electronic Cigarette Aerosol

Sample Reps 1-Amino 2-Amino 4-Aminobi

2242397 1-5 NMT 0.004ng/puff NMT 0.005ng/puff NMT 0.004ng/puff2242393 1-5 NMT 0.004ng/puff NMT 0.005ng/puff NMT 0.004ng/puff2242376 1-5 NMT 0.004ng/puff NMT 0.005ng/puff NMT 0.004ng/puff2242395 1-5 NMT 0.004ng/puff NMT 0.005ng/puff NMT 0.004ng/puff

• Sequence #1

2245465 1-5 NMT 0.004ng/puff NMT 0.005ng/puff NMT 0.004g/puff2245461 1-5 NMT 0.004ng/puff NMT 0.005ng/puff NMT 0.004g/puff2245469 1-5 NMT 0.004ng/puff NMT 0.005ng/puff NMT 0.004g/puff2245466 1-5 NMT 0.004ng/puff NMT 0.005ng/puff NMT 0.004g/puff

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Example Results

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Example Results for Ammonia in Electronic Cigarette Aerosol

Samples (µg NH3/E-Cig)2294113_1 NMT 1.022294113_2 NMT 1.022294113_3 NMT 1.022294113_4 NMT 1.022294113_5 NMT 1.022294117_1 NMT 1.022294117_2 NMT 1.022294117_3 NMT 1.022294117_4 NMT 1.022294117_5 NMT 1.022294151_1 NMT 1.022294151_2 NMT 1.022294151_3 NMT 1.022294151_4 NMT 1.022294151_5 NMT 1.02

Sample ID CalculatedConcentration

(µg NH3/E-Cig)2295548_1 NMT 1.022295548_2 NMT 1.022295548_3 NMT 1.022295548_4 NMT 1.022295548_5 NMT 1.02

Limit 1.02 µg/E-cig

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Example Results

Final concentration pg/puffSample ID NNN NNK2459342-3 <9.6 <9.42459344-1 <9.6 <9.42459344-2 <9.6 <9.42459344-3 <9.6 <9.42459350-1 <9.6 <9.42459350-2 <9.6 <9.42459350-3 <9.6 <9.42459352-1 <9.6 <9.42459352-2 <9.6 <9.42459352-3 <9.6 <9.42459345-1 <9.6 <9.42459345-2 <9.6 <9.42459345-3 <9.6 <9.42459347-1 <9.6 <9.42459347-2 <9.6 <9.42459347-3 <9.6 <9.42459346-1 <9.6 <9.42459346-2 <9.6 <9.42459346-3 <9.6 <9.42459348-1 <9.6 <9.42459348-2 <9.6 <9.42459348-3 <9.6 <9.4

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Final concentration pg/puffSample ID NNN NNK2459339-1 <9.6 <9.42459339-2 <9.6 <9.42459339-3 <9.6 <9.42459341-1 <9.6 <9.42459341-2 <9.6 <9.42459341-3 <9.6 <9.42459343-1 <9.6 <9.42459343-2 <9.6 <9.42459343-3 <9.6 <9.42459349-1 <9.6 <9.42459349-2 <9.6 <9.42459349-3 <9.6 <9.42459351-1 <9.6 <9.42459351-2 <9.6 <9.42459351-3 <9.6 <9.42459340-1 <9.6 <9.42459340-2 <9.6 <9.42459340-3 <9.6 <9.42459342-1 <9.6 <9.42459342-2 <9.6 <9.4

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Take Aways

• Limits Tests are acceptable for use in the industry• Economically beneficial• Improve laboratory efficiency/throughput• Simplified laboratory operations• For selected constituents only

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Questions

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