Extractables and leachables (E&L) studies can generate dozens—or even hundreds—of chromatographic peaks. But not every detected compound presents the same potential safety concern, and attempting to identify and quantify every analytical signal regardless of exposure would make many E&L programs unnecessarily complex. This is where the Analytical Evaluation Threshold (AET) becomes important. The AET provides a scientifically derived analytical threshold that helps determine which extractables or leachables detected during an E&L study should receive further analytical evaluation. In practical terms, it helps answer an essential question: At what analytical concentration must an extractable or leachable be reliably detected and considered for identification, quantitation, and subsequent safety assessment? The concept connects toxicological exposure thresholds with analytical method capability. A useful framework is: Patient safety threshold ↓ Product-specific exposure conditions ↓ Analytical Evaluation Threshold (AET) ↓ Analytical screening capability ↓ Detection of compounds at or above the AET ↓ Identification and quantitation ↓ Toxicological assessment Research on E&L analysis describes the AET as a practical mechanism for differentiating signals that require further evaluation from those below a toxicologically relevant analytical threshold. At topiox research, establishing an appropriate AET should therefore be viewed as part of the scientific design of an E&L program—not simply as a calculation performed after analytical testing.  

What Is an Analytical Evaluation Threshold?

The Analytical Evaluation Threshold is a concentration threshold derived from an appropriate safety-based exposure threshold and product- or study-specific parameters, used to establish the analytical level at which extractables or leachables should be evaluated further. The concept originated from safety-threshold approaches developed for pharmaceutical extractables and leachables, particularly through the Product Quality Research Institute (PQRI). The AET translates a patient-exposure threshold into a concentration that can be applied in the analytical laboratory. This distinction is critical: A safety threshold is exposure-based. An AET is the analytical concentration corresponding to that exposure threshold under the conditions of the study. The AET therefore connects toxicology and analytical chemistry.  

Why Is the AET Important in Extractables and Leachables Studies?

Modern analytical platforms are capable of detecting compounds at extremely low concentrations. Greater sensitivity is valuable, but sensitivity alone does not determine toxicological relevance. Without an evaluation threshold, analysts could spend considerable effort identifying extremely small signals that contribute negligible patient exposure while potentially losing focus on compounds that are more important. The AET creates a scientifically meaningful screening boundary. Compounds at or above the applicable AET generally warrant appropriate reporting and further evaluation according to the study framework. The developing ICH Q3E framework explicitly incorporates the AET into pharmaceutical E&L assessment and calls for extractables and leachables peaks above the AET to be included in relevant submission documentation. Because ICH Q3E remains draft guidance, however, its recommendations should currently be described as an emerging harmonized framework, not as finalized global requirements.  

AET vs SCT: What Is the Difference?

These terms are related but are not interchangeable.

Safety Concern Threshold

The Safety Concern Threshold (SCT) is a toxicologically based exposure concept. It represents a daily exposure level below which the likelihood of significant safety concern is considered sufficiently low within the framework for which that SCT was established. The historical PQRI recommendations for orally inhaled and nasal drug products introduced an SCT of 0.15 µg/day for that specific framework. That historical value should not automatically be applied to every dosage form or E&L program. Product type, route, duration of exposure, patient population, and the applicable regulatory or scientific framework must be considered.

Analytical Evaluation Threshold

The AET translates the relevant safety-based threshold into an analytical concentration appropriate for the particular study. Conceptually: SCT or other applicable safety threshold → patient exposure → study configuration → AET Therefore: SCT = toxicological exposure concept AET = analytical evaluation concentration Confusing these two concepts can lead to an incorrectly designed E&L study.  

How Is an AET Calculated?

The exact calculation depends on the applicable framework and study design. At a conceptual level: AET concentration ≈ Relevant safety-based daily exposure ÷ maximum daily exposure to the drug product Additional factors may be needed depending on how the analytical sample is prepared. These can include:

• number of doses per day;
• dose volume or mass;
• extraction volume;
• number or surface area of components represented;
• dilution;
• concentration;
• sample-preparation factors;
• extraction ratio;
• and analytical uncertainty.

This means the AET is product- and study-specific. It should not simply be copied from another E&L report.  

Example of the AET Concept

Consider two hypothetical products that use similar packaging materials.

Product A

The patient receives a relatively small quantity of drug product each day.

Product B

The patient receives a much larger quantity each day. If the same concentration of a leachable is present in both products, the patient’s daily exposure could be substantially higher from Product B. Therefore, the analytically relevant threshold may need to be lower for the higher-exposure product. This illustrates the fundamental relationship: Higher daily product exposure → potentially lower analytical concentration corresponding to the same safety threshold The reverse can also apply. The calculation must therefore begin with patient exposure, not simply instrument sensitivity.  

Parameter 1: Applicable Safety-Based Threshold

The first parameter in establishing an AET is the toxicological threshold from which it is derived. The appropriate threshold depends on the regulatory and scientific framework applicable to the product. Historically, PQRI introduced SCT-based approaches for high-concern pharmaceutical products, including orally inhaled and nasal drug products, with later work extending threshold concepts to other dosage forms. The developing ICH Q3E framework represents an important movement toward broader international harmonization of E&L safety assessment. However: There is no scientifically defensible reason to assume that one historical SCT value automatically applies to every pharmaceutical product. The relevant framework should be established before calculating the AET.  

Parameter 2: Maximum Daily Patient Exposure

Daily exposure is one of the most important variables in an AET calculation. Relevant considerations may include:

• maximum number of doses per day;
• dose volume;
• dose mass;
• delivered volume;
• duration of treatment;
• and intended clinical use.

The calculation should represent a scientifically justified exposure scenario. For example, a multi-dose product may expose a patient to packaging-derived leachables repeatedly during a single day. A large-volume parenteral may involve substantially greater daily product volume than a small-volume injection. These differences can materially affect the resulting analytical threshold. Research on pharmaceutical leachables emphasizes that safety evaluation depends on both the concentration of a compound and the amount of product administered to the patient.  

Parameter 3: Extraction and Sample-Preparation Volume

For extractables studies, the analytical concentration observed in the extract depends partly on the experimental extraction configuration. Important variables may include:

• solvent volume;
• component surface area;
• number of components;
• extraction ratio;
• dilution;
• concentration steps;
• and final analytical sample volume.

The emerging ICH Q3E framework expects extraction conditions—including solvent, temperature, duration, and surface-area-to-volume relationships—to be scientifically justified. Therefore, the AET must be aligned with the actual analytical sample presented to the instrument. A mathematically correct threshold applied to the wrong sample basis can still produce an inappropriate analytical target.  

Parameter 4: Number of Components or Units Represented

An extract may represent:

• one component;
• multiple components;
• an entire container-closure system;
• a delivery-system assembly;
• or a defined surface area of material.

This affects how an analytical concentration translates back to potential patient exposure. For example, if one analytical extract contains material equivalent to several patient-use units, the relationship between measured concentration and exposure must account for that configuration. The calculation should maintain traceability between: Material extracted → analytical sample → measured concentration → product exposure → patient exposure This traceability is essential for defensible AET calculations.  

Parameter 5: Concentration and Dilution Factors

Sample preparation can substantially alter the effective analytical threshold. E&L samples may undergo:

• evaporation;
• solvent exchange;
• concentration;
• dilution;
• filtration;
• liquid-liquid extraction;
• solid-phase extraction;
• or other preparation steps.

If an extract is concentrated tenfold before injection, the instrument-level concentration corresponding to the original sample AET changes accordingly. Conversely, dilution may make a method incapable of detecting compounds at the required threshold. Every dilution or concentration step should therefore be incorporated correctly into the working AET.  

Parameter 6: Analytical Response-Factor Variation

One of the most important practical limitations of AET-based screening is that different compounds do not produce identical detector responses at identical concentrations. Suppose a surrogate standard produces a strong GC-MS or LC-MS signal at the AET. An unknown extractable present at the same concentration might produce a much weaker response. If analysts assume that every compound behaves exactly like the surrogate standard, a potentially relevant compound could appear to fall below the analytical threshold. Research has demonstrated substantial relative-response-factor variability in E&L analysis. This is why an AET calculation cannot be separated from method performance.  

Parameter 7: Uncertainty or Response-Factor Adjustment

Because detector response varies among compounds, an appropriate strategy may need to account for analytical uncertainty. Conceptually: Calculated AET ↓ Evaluate response-factor variability ↓ Apply scientifically justified adjustment where appropriate ↓ Establish working/final analytical threshold The purpose is to reduce the risk that a compound present at a potentially relevant concentration is missed simply because it responds poorly relative to the surrogate standard. However, uncertainty factors should not be applied mechanically. The magnitude should be scientifically supported by the method, detector response data, analytical framework, and applicable guidance. Research has shown that excessively large uncertainty adjustments can drive the working AET below practical analytical sensitivity and force aggressive sample concentration, which may itself create losses, degradation, or matrix effects. The objective is not simply to make the AET as low as possible. It is to establish a scientifically protective and analytically achievable threshold.  

Parameter 8: Analytical Method Sensitivity

After establishing the AET, an essential question follows: Can the analytical methods reliably detect relevant compounds at that level? The analytical workflow may include:

• GC-MS for volatile and semivolatile compounds;
• LC-MS or LC-HRMS for nonvolatile and thermally labile compounds;
• headspace GC-MS for volatile species;
• ICP-MS for elemental extractables or leachables;
• and complementary detectors where appropriate.

Mass spectrometry plays a central role because E&L programs often require broad screening, tentative identification, and trace-level analysis. A method that cannot reliably reach the required AET is not suitable for the intended screening purpose simply because it produces a clean chromatogram.  

AET vs LOD vs LOQ: They Are Not the Same

These three concepts are frequently confused.

AET

A study- and exposure-driven evaluation threshold indicating the analytical level at which compounds should receive further evaluation.

Limit of Detection

The LOD describes the lowest amount or concentration that an analytical method can reliably distinguish from background under defined conditions.

Limit of Quantitation

The LOQ describes a concentration at which an analyte can be quantitatively measured with appropriate performance. The relationship should ideally support the intended study: Method capability → sufficient to address the AET The AET should not be selected simply because it matches the existing instrument’s LOD. Instead: Patient safety and study conditions define the analytical need; the analytical method should then demonstrate adequate capability to meet that need.  

Why Surrogate Standards Matter

E&L screening frequently encounters unknown compounds for which authentic reference standards are unavailable. Analysts therefore commonly use one or more surrogate standards to estimate concentrations. This is practical, but it introduces uncertainty because an unknown compound may not respond like the surrogate. The basic assumption can be represented as: Unknown response ≈ surrogate response at equivalent concentration In reality: Unknown response may be higher or lower than surrogate response This variation is a major source of uncertainty in semiquantitative E&L screening. Studies of internal standards and relative response factors have shown that standard selection can materially affect estimated concentrations. Therefore, surrogate-standard suitability should be part of method development.  

Why Orthogonal Analytical Methods Can Strengthen AET Coverage

No single detector responds equally well to every possible extractable or leachable. E&L compounds can span a broad range of:

• molecular weights;
• polarities;
• volatility;
• ionization behavior;
• functional groups;
• and chemical structures.

A compound that responds poorly using one detector may respond well using another. This is the rationale for orthogonal or multidetector approaches. Research combining LC-MS, charged aerosol detection, UV detection, GC-MS, and FID-related approaches demonstrated improved coverage across chemically diverse extractables and reduced some of the limitations associated with response-factor variability. This leads to an important principle: Broad E&L screening should focus on chemical coverage, not simply maximum sensitivity on one detector.  

What Happens When a Peak Is Above the AET?

Detection above the AET does not automatically mean the compound is unsafe. This is one of the most important distinctions in E&L interpretation. Instead, exceeding the AET generally means the signal has crossed the threshold for further evaluation within the applicable framework. That evaluation may include:

1. Confirming the analytical signal
2. Identifying or further characterizing the compound
3. Improving quantitation where necessary
4. Estimating patient exposure
5. Conducting toxicological assessment
6. Determining whether additional control is required

Therefore: Above AET ≠ unsafe Rather: Above AET → evaluate further The current draft ICH Q3E framework similarly calls for substances above the AET to undergo appropriate reporting and safety assessment.  

What Happens When a Peak Is Below the AET?

In a properly designed and applicable threshold framework, signals below the AET may generally receive reduced analytical attention because their estimated exposure is below the threshold used to derive the AET. However, this should not be interpreted as: “Everything below the AET is automatically safe under every circumstance.” Special considerations can arise for compounds associated with particular toxicological concerns or when the underlying threshold framework does not apply. The AET must therefore be used within a risk-based E&L strategy, not as an absolute universal rule.  

AET in Extractables Studies

Extractables studies use controlled conditions to identify compounds that could potentially migrate from materials or components. Conditions may intentionally be more aggressive than actual product use. The extractables profile can help:

• understand material composition;
• identify potential leachables;
• compare materials or suppliers;
• support component selection;
• identify compounds requiring targeted monitoring;
• and inform later leachables studies.

Because controlled extraction can generate a large number of peaks, the AET helps prioritize which extractables require meaningful analytical evaluation. Research using AET-based screening has successfully characterized complex extractable profiles from pharmaceutical packaging materials.  

AET in Leachables Studies

Leachables studies address compounds that actually migrate into the drug product or relevant matrix under manufacturing, storage, or use conditions. These compounds are particularly important because they represent potential patient exposure. Leachables studies may involve:

• stability samples;
• multiple storage conditions;
• different time points;
• targeted analysis based on extractables;
• and non-targeted screening for unexpected compounds.

Broad analytical screening remains important because not every observed leachable can always be predicted from an extractables profile. A well-established AET provides a consistent analytical basis for deciding which signals warrant deeper characterization.  

Extractables, Leachables, SCT and AET: How They Fit Together

The complete relationship can be visualized as: Packaging / manufacturing / delivery materials ↓ Controlled extraction ↓ Extractables profile ↓ Potential leachables identified ↓ Drug product exposure during manufacture/storage/use ↓ Actual leachables ↓ Safety-based threshold ↓ Product-specific AET ↓ Analytical detection and evaluation ↓ Identification / quantitation ↓ Toxicological risk assessment ↓ Risk control where necessary This framework demonstrates why AET is not simply an analytical calculation. It is a bridge between patient safety and analytical science.  

AET and the Emerging ICH Q3E Framework

One of the most important developments in pharmaceutical E&L regulation is ICH Q3E: Guideline for Extractables and Leachables. As of October 2026, ICH Q3E remains in draft form and should not be presented as finalized harmonized guidance. The draft establishes a holistic framework for:

• E&L risk assessment;
• extractables studies;
• leachables studies;
• analytical evaluation;
• safety assessment;
• risk control;
• and regulatory documentation.

Importantly for AET programs, the draft expects E&L substances above the AET to be appropriately documented and evaluated. This development is significant because E&L expectations have historically relied on combinations of regional guidance, compendial standards, industry recommendations, and product-specific scientific practice. ICH Q3E represents an important movement toward greater international harmonization. Until finalized, organizations should distinguish carefully between: established regional requirements and standards and draft ICH Q3E recommendations.  

Common Mistakes When Applying the AET

Mistake 1: Copying the AET From Another Study

The AET depends on product exposure and study configuration. A threshold from another product may not be appropriate.

Mistake 2: Treating AET as a Toxicity Limit

AET is an analytical evaluation threshold, not a declaration that a compound above it is toxic.

Mistake 3: Confusing AET With LOD or LOQ

AET is exposure-driven; LOD and LOQ describe analytical capability.

Mistake 4: Ignoring Maximum Daily Dose

Daily patient exposure is central to translating a safety threshold into an analytical concentration.

Mistake 5: Ignoring Sample-Preparation Factors

Dilution, concentration, extraction volume, and component number can materially change the working AET.

Mistake 6: Assuming Every Compound Responds Like the Surrogate

Relative response factors can vary substantially between analytes.

Mistake 7: Setting an AET Below Realistic Method Capability Without a Strategy

An extremely low calculated threshold creates little value if the analytical workflow cannot reliably achieve adequate chemical coverage at that level.

Mistake 8: Treating Every Peak Above the AET as Unsafe

Crossing the AET triggers further evaluation; it does not automatically establish unacceptable toxicological risk.

Mistake 9: Applying Historical SCT Values Universally

Safety thresholds must be used within the product type and framework for which they are scientifically appropriate.  

Best Practices for Establishing an AET

Start With Patient Exposure

Determine the relevant safety-based threshold and maximum daily exposure before designing the analytical strategy.

Document Every Calculation Input

The AET should be reproducible and traceable. Document:

• safety threshold;
• daily dose;
• dose volume;
• extraction volume;
• number of units;
• concentration factors;
• dilution factors;
• and any uncertainty adjustment.

Establish the AET Before Finalizing Analytical Methods

Knowing the required analytical threshold helps determine whether GC-MS, LC-MS, ICP-MS, or complementary approaches provide sufficient sensitivity and coverage.

Evaluate Relative Response Factors

Understand how representative compounds respond relative to the selected surrogate standards.

Use Complementary Analytical Techniques

Broad chemical diversity generally requires more than one analytical platform.

Demonstrate Method Capability Around the AET

A screening method should show that relevant chemical classes can be detected at appropriate levels.

Investigate Peaks Above the AET

Identification confidence, quantitation, and toxicological evaluation should increase as required by the study framework.

Recalculate When Important Parameters Change

AET calculations may need revision when:

• dose changes;
• formulation changes;
• packaging changes;
• extraction design changes;
• sample preparation changes;
• or the applicable safety threshold changes.

 

AET and Analytical Method Development

One of the most useful ways to apply the AET is before analytical screening begins. Consider the following development sequence: Define product exposure ↓ Determine applicable safety threshold ↓ Calculate preliminary AET ↓ Select analytical technologies ↓ Evaluate surrogate responses ↓ Assess response-factor variability ↓ Confirm method sensitivity and coverage ↓ Establish working AET ↓ Conduct E&L screening This is more scientifically defensible than: Run available method → determine sensitivity → call that sensitivity the AET The AET should help define analytical requirements. The analytical instrument should not arbitrarily define the patient-safety threshold.  

Why Identification Confidence Matters Above the AET

Once a compound exceeds the relevant AET, its identity becomes increasingly important because toxicological assessment depends on knowing what the patient may be exposed to. Identification may progress through levels such as:

• unknown;
• tentative identification;
• probable identification;
• and confirmed identification using an authentic reference standard.

Useful evidence can include:

• accurate mass;
• isotope pattern;
• fragmentation;
• retention behavior;
• spectral-library matching;
• and comparison with authentic standards.

LC-HRMS and GC-MS are particularly valuable because they provide structural information in addition to chromatographic separation. The stronger the identification, the stronger the subsequent toxicological evaluation can be.  

AET as a Link Between Chemistry and Toxicology

E&L programs work best when analytical chemistry and toxicology are connected from the beginning. The chemist asks: What is present, and at what estimated concentration? The toxicologist asks: What is the resulting patient exposure, and is it acceptable? The AET connects these questions. That collaboration can prevent two common problems: Over-analysis: spending excessive resources identifying toxicologically insignificant trace peaks. Under-analysis: failing to identify a weakly responding compound that may exceed a relevant exposure threshold. The AET is therefore most valuable when treated as a risk-management tool, not merely a reporting threshold.  

Analytical Evaluation Thresholds at topiox research

Establishing and applying an AET requires integration of:

• extractables study design;
• leachables study design;
• patient exposure calculations;
• safety-threshold selection;
• analytical method development;
• GC-MS;
• LC-MS/LC-HRMS;
• ICP-MS where applicable;
• relative-response-factor assessment;
• compound identification;
• semiquantitation;
• and toxicological risk assessment.

At topiox research, AET strategy can be integrated into broader extractables and leachables programs so that analytical methods are designed around the relevant scientific and safety questions. The objective is not simply to detect as many chromatographic peaks as possible. It is to determine: Which compounds require further evaluation, whether the analytical methods can detect them reliably, and how the resulting data support a scientifically defensible patient-safety assessment.

Conclusion

The Analytical Evaluation Threshold is one of the most important links between analytical chemistry and patient safety in extractables and leachables studies. A scientifically sound AET strategy should:

1. Identify the applicable safety-based threshold.
2. Determine maximum patient exposure.
3. Account for study and extraction configuration.
4. Include dilution and concentration factors.
5. Consider the number of components or units represented.
6. Evaluate surrogate-standard response.
7. Address relative-response-factor uncertainty appropriately.
8. Confirm that analytical methods can achieve the required sensitivity and chemical coverage.
9. Further evaluate relevant compounds at or above the AET.
10. Connect analytical results to toxicological risk assessment.

The central principle is: The AET should translate patient-safety considerations into a scientifically meaningful analytical target. It should not be treated as an arbitrary instrument limit, a universal concentration, or a toxicity threshold. Research shows that relative response factors, surrogate-standard selection, analytical coverage, and detector selection can materially influence whether compounds near the AET are detected and accurately estimated. With the emergence of the draft ICH Q3E, the AET is also becoming increasingly important within the evolving international framework for pharmaceutical extractables and leachables assessment. At topiox research, integrating AET calculations with E&L study design, analytical method capability, compound identification, and toxicological interpretation can help generate data that are both analytically meaningful and relevant to patient safety. Planning an extractables or leachables study? Connect with topiox research for AET strategy, extractables and leachables study design, GC-MS and LC-MS screening, compound identification, trace-level analytical support, and integrated E&L risk assessment.