USP <1663> and <1664> in Practice: How GMP Labs Apply Them to Extractable & Leachable Studies

Introduction:

USP <1663> and <1664> E&L Testing is the analytical backbone that connects a packaging or delivery-system material to a documented, regulator-ready safety case for a drug product. For GMP laboratories, the objective isn’t simply to detect as many compounds as possible — it’s to generate scientifically defensible data that supports product quality, patient safety, packaging selection, regulatory submissions, and lifecycle management. Packaging components can contain polymers, elastomers, adhesives, coatings, lubricants, additives, pigments, and processing aids, and under appropriate conditions, chemical constituents may be released from these materials. Some substances identified during an aggressive laboratory extraction may never actually enter the drug product, while others can migrate into the formulation during normal storage — a distinction that sits at the center of how USP <1663> and <1664> E&L Testing programs are designed.

This article walks through how a GMP lab actually applies both chapters in day-to-day E&L work: what each one covers, how studies are sequenced, which analytical techniques carry the weight, and where programs commonly go wrong.

Summary:

• USP <1663> and <1664> E&L Testing gives manufacturers a science- and risk-based framework: USP <1663> covers extractables (what a packaging or delivery-system material could release), while USP <1664> covers leachables (what actually migrates into the drug product during storage and use).
• Neither chapter prescribes one universal extraction procedure, analytical method, or acceptance criterion — the study design must be scientifically justified for the specific product and packaging system.
• GMP labs apply both chapters as a connected workflow: risk assessment → material characterization → extraction design → orthogonal analytical testing → identification/quantification → toxicological assessment → documented reporting.
• Analytical strategies typically combine GC-MS/GC-HRMS, LC-MS/LC-HRMS, and ICP-MS depending on the expected chemical classes and the product’s route of administration.
• The Analytical Evaluation Threshold (AET) — not a generic detection limit — determines which detected substances require identification, quantification, and toxicological follow-up.
• FDA’s regulatory environment continues to move toward risk-based control of packaging-related chemicals: FDA issued a draft container closure systems guidance in August 2026, and the draft ICH Q3E E&L guideline (published November 2025) proposes a holistic, risk-based framework that is not yet finalized.

1: What Is the Difference Between USP <1663> and USP <1664>?

USP <1663> focuses on extractables generated under controlled laboratory extraction conditions, while USP <1664> focuses on leachables actually present in the drug product, evaluated under normal or accelerated storage/use conditions. The two assessments are connected stages of one program rather than interchangeable tests.

USP <1663>: The Extractables Assessment

USP <1663> provides a framework for designing and executing an extractables assessment, but it deliberately does not impose one fixed extraction condition or analytical procedure for every application — the manufacturer must justify conditions based on scientific and risk-management considerations. In practice, a GMP lab starts by defining the full packaging system under evaluation, which may include vials and bottles, elastomeric stoppers, syringe barrels and plungers, prefilled syringe and autoinjector components, plastic containers, caps and liners, tubing and connectors, administration sets, inhalation-device components, ophthalmic packaging, and single-use manufacturing components. The assessment should consider the complete relevant system rather than focusing on only the most obvious component.

A risk-based assessment then establishes the scope and severity of the extraction program, weighing factors such as route of administration, maximum daily dose, duration of treatment, drug formulation, contact surface area and time, temperature, material composition, sterilization conditions, manufacturing processes, patient population, and potential chemical toxicity. FDA’s container-closure guidance similarly emphasizes evaluating the specific packaging system, formulation, dosage form, route of administration, and dosing regimen when assessing packaging-related chemical safety.

Key elements a GMP lab builds into a <1663>-compliant extractables study:

• Representative material selection — production-grade components rather than generic substitutes.
• Multi-solvent extraction design — solvents of varying polarity chosen to model the drug product’s chemical environment plus worst-case surrogates, under justified temperature and duration.
• Orthogonal analytical techniques — GC-MS/GC-HRMS for volatiles and semi-volatiles, LC-MS/LC-HRMS for non-volatiles, ICP-MS for elemental extractables, and targeted LC-MS/MS for sensitive quantitative work.
• Compound identification and semi-quantitation — accurate mass, retention time, spectral library matching, and reference-standard comparison, distinguishing confirmed, probable, and tentative identifications.
• Reporting against a defined AET, so the extractables list is risk-ranked rather than an unfiltered chemical inventory.

USP <1664>: The Leachables Study Design

USP <1664> addresses substances that migrate from packaging or delivery systems into the actual drug product under normal storage and use, or under appropriate accelerated conditions — which makes leachables testing fundamentally different from extractables testing because the drug product itself becomes part of the analytical challenge. A <1664>-aligned leachables program typically works through long-term and accelerated stability samples, multiple packaging configurations, multiple time points and storage conditions, and placebo, formulation-matched, or packaging-component controls, to determine whether compounds identified as potential extractables actually migrate into the product and at what concentration.

• Risk-based target list — extractables ranked by toxicological concern and analytical feasibility become the leachables target compound list.
• Product-matrix method development — methods validated in the actual drug product matrix, since real formulations (proteins, lipids, surfactants) can mask or enhance leachable detection through ion suppression or chromatographic interference.
• Stability-indicating leachables testing — samples pulled at defined stability timepoints across the intended shelf life.
• Toxicological risk assessment — leachable levels compared against Permitted Daily Exposure (PDE) or Safety Concern Threshold (SCT) values.

2: How Do GMP Labs Sequence a USP <1663>/<1664> Extractable & Leachable Testing Workflow?

A GMP lab sequences USP <1663>/<1664> E&L testing as a connected workflow — packaging risk assessment, extractables study, analytical characterization, compound identification, leachables study, quantification, toxicological assessment, risk evaluation, and regulatory documentation — rather than as a single isolated test. This staged approach avoids the two most common failure modes: running a leachables study without a proper extractables foundation, or over-testing every extractable regardless of toxicological relevance.

3: Which Analytical Techniques Are Used for USP <1663>/<1664> Extractable & Leachable Testing?

No single analytical technique can adequately characterize every potential extractable or leachable, so GMP laboratories generally combine complementary platforms covering volatile, semi-volatile, non-volatile, and elemental chemical classes.

The exact analytical package depends on the material, formulation, exposure scenario, and risk assessment — a polymeric component can generate a broad organic profile requiring both GC- and LC-based analysis, while elemental characterization typically requires ICP-MS. For elastomeric and rubber components specifically, rubber and elastomer leachables often warrant their own targeted extraction and identification strategy, since curing agents, vulcanization accelerators, and antioxidants behave differently than plastic or coating chemistries.

4: What Is the Role of AET in USP <1663>/<1664> Extractable & Leachable Testing?

The Analytical Evaluation Threshold (AET) is the concentration above which a detected extractable or leachable must be identified, quantified, and risk-assessed rather than treated as background noise — and it is not simply a generic laboratory detection limit. AET is tied to the toxicological assessment framework and the amount of product or packaging exposure represented by the study, and can depend on factors such as extract concentration, drug-product volume or mass, surface area, dose, extraction ratio, maximum daily exposure, route of administration, and overall study design. FDA’s 2026 draft container-closure guidance defines AET as the threshold above which an extractable or leachable should be identified, quantified, and further reported for potential toxicological assessment — so a laboratory should establish and document the AET calculation and rationale before interpreting the analytical dataset.

5: How Do GMP Labs Avoid False Positives in Extractable & Leachable Studies?

Contamination control matters because compounds can originate from solvents, laboratory plastics, tubing, instrument components, cleaning materials, or sample-handling procedures rather than from the packaging system itself. A robust program includes solvent blanks, method blanks, laboratory controls, packaging blanks where appropriate, matrix/placebo controls, procedural blanks, replicate samples, reference materials, and system suitability samples. If a compound appears in both the sample and the solvent blank at comparable levels, it should not automatically be attributed to the packaging component — this is where laboratory experience and scientific interpretation become particularly important, and where robust GMP-compliant extractables and leachables study design pays off downstream.

6: How Are Extractables and Leachables Evaluated Toxicologically?

Analytical detection alone does not establish patient risk — detected substances must be evaluated in the context of exposure and toxicological significance, considering estimated daily exposure, route of administration, duration of exposure, patient population, chemical identity, structural alerts, available toxicology data, mutagenicity considerations, and threshold concepts. FDA’s longstanding container-closure guidance emphasizes that safety assessment should account for the specific container-closure system, drug formulation, dosage form, route of administration, and dosing regimen — a consideration that becomes especially important for products with high patient exposure or sensitive routes of administration.

7: Why Does Extractable & Leachable Testing Matter More for Injectable, Ophthalmic, Inhalation, and Single-Use Systems?

Products administered by sensitive routes, at high frequency, or manufactured using single-use systems require more rigorous E&L evaluation because packaging- or process-contact chemicals can have a direct impact on patient exposure or product quality. Parenteral products can carry stringent requirements because substances introduced into an injectable formulation may reach systemic circulation, and control of extractables from container-closure systems is particularly important for these products. Ophthalmic products present additional challenges from small dose volumes, direct exposure to ocular tissue, and multiple polymeric or elastomeric packaging components, where low-level contaminants can become clinically relevant. Inhalation products, including E&L compliance for a dry powder inhaler, involve very low dose levels, complex device components, propellants, elastomers, and large surface-area-to-dose relationships that make analytical sensitivity and identification strategy critical.

Biopharmaceutical manufacturing adds a further layer, since single-use bioprocessing systems (bags, tubing, filters, connectors) sit in direct contact with cell culture media, buffers, and drug substance for extended periods. A well-run single-use systems and GMP extractables and leachables testing program addresses E&L requirements for single-use bioprocessing components specifically, because these systems combine multiple polymer and elastomer contact surfaces across a single process train, and a leachable that is inconsequential in one unit operation can accumulate risk across the full manufacturing sequence.

8: How Does USP <1663>/<1664> Extractable & Leachable Testing Support Regulatory Submissions?

E&L data provides CMC evidence demonstrating that a selected container-closure or delivery system is appropriate for its intended use, and can support IND, NDA, ANDA, and BLA submissions, packaging qualification, stability programs, container-closure justification, post-approval packaging changes, and device-drug combination product filings. The regulatory landscape is actively evolving: FDA published a draft Container Closure Systems for Human Drugs and Biological Products guidance in August 2026 describing principles for evaluating container-closure systems, including those used in combination products, and separately published the draft ICH Q3E Guideline for Extractables and Leachables in November 2025, proposing a holistic, risk-based E&L framework. Because both remain draft guidance, they should not be represented as final enforceable requirements, but they signal the direction reviewers are already leaning toward.

Every result in the package — from extractables screening through leachables quantification — also needs to hold up as audit-ready extractables and leachables data, which means documentation, traceability, and interpretation rigor matter as much as the raw chromatography. That documentation trail is also inseparable from GMP data integrity for extractables and leachables testing — audit trails, controlled reporting, and chain of custody are what let a reviewer trust the conclusions, not just the numbers.

9: What Makes Extractable & Leachable Testing GMP-Compliant?

GMP-quality E&L testing depends on controlled laboratory practices, validated or appropriately qualified analytical procedures, traceability, data integrity, and scientifically justified conclusions — not merely on possessing sophisticated instruments. Key considerations include qualified instruments, controlled laboratory environments, approved analytical procedures, reference standards and qualified materials, system suitability, calibration and maintenance, proper sample storage, chain of custody, electronic data integrity, audit trails, deviation and OOS/OOT investigation procedures, technical review, and QA oversight. This distinction matters because an E&L report may eventually support CMC documentation, regulatory responses, packaging qualification, or lifecycle decisions.

10: USP <1663> vs USP <1664>: Quick Decision Guide

The strongest programs generally use both extractables and leachables assessments as complementary elements of a broader E&L strategy — treating either chapter in isolation tends to leave gaps that surface later, whether during scale-up, a container change, or an inspection.

11: Where Do USP <1663>/<1664> Extractable & Leachable Programs Commonly Fail?

USP <1663>/<1664> programs most often fail because of mismatched analytical scope, non-representative materials, or leachables studies that were never actually anchored to the extractables data. GMP labs see the same handful of root causes repeatedly across sponsor submissions:

• Treating USP <1663> as one fixed extraction recipe rather than a scientifically justified framework.
• Using only one analytical platform (GC-MS or LC-MS alone), leaving a chemical class undetected.
• Ignoring laboratory contamination, mistaking background peaks for packaging-derived compounds.
• Reporting every peak as a confirmed compound instead of applying identification-confidence tiers.
• Performing extractables testing without a leachables strategy to follow it.
• Ignoring the drug matrix during leachables method development, creating ion suppression or interference.
• Separating the analytical and toxicological assessments instead of translating findings into exposure and safety relevance.

For sponsors weighing whether to build this capability in-house or hand it to a specialist partner, outsourcing extractables and leachables testing to a lab that already runs this workflow routinely is often what closes these gaps fastest, particularly for organizations running their first E&L program.

12: How Can ResolveMass Laboratories Support Extractable & Leachable Programs?

The value of an E&L testing partner isn’t limited to generating chromatograms — a capable lab connects study design, analytical chemistry, identification, quantification, data interpretation, and regulatory documentation into one coherent package. ResolveMass Laboratories Inc. supports pharmaceutical and biotechnology organizations across extractables assessment, leachables assessment, GC-MS/GC-HRMS and LC-MS/LC-HRMS analysis, targeted quantitative analysis, unknown compound identification, elemental analysis, method development and qualification, packaging-component investigations, risk-based study design, and regulatory-support documentation — with each program designed around the specific formulation, packaging configuration, route of administration, and anticipated patient exposure rather than a generic testing package. See a related example of a GMP-compliant extractables and leachables study for how this comes together on an actual project.

Conclusion:

USP <1663> and <1664> E&L Testing gives GMP laboratories and sponsors a practical, science-based path from raw material chemistry to a documented drug-product safety case — but only when the extractables and leachables stages are executed as a connected lifecycle, with orthogonal analytical coverage, a defensible AET, contamination controls, and product-matrix-specific leachables methods. Labs that treat <1663> and <1664> as a checklist rather than a scientific narrative are the ones that end up answering avoidable deficiency letters late in development. As the regulatory landscape continues to evolve — including the draft ICH Q3E framework and FDA’s 2026 draft container-closure guidance — a properly sequenced, GMP-executed USP <1663>/<1664> E&L testing program protects both the regulatory timeline and, ultimately, the patient.

Frequently Asked Questions:

Reference

• Ramamoorthy S, Chong NS, Hotha KK. Strengthening extractable & leachable study submissions: best practices to avoid regulatory deficiencies. American Journal of Analytical Chemistry. 2024;15(12):368-94.https://www.scirp.net/pdf/ajac20241512_32202357.pdf
• Parker W, DeCou D. Extractables and Leachables in Drug Products: An Overview. Handbook of Validation in Pharmaceutical Processes, Fourth Edition. 2021 Oct 28:943-51.https://www.academia.edu/download/124990488/HANDBOOK_OF_VALIDATION_IN_PHARMACEUTICAL_PROCESSES_2021_CRC_PRESS_.pdf#page=962
• Eissa M. An Exploratory Review of Elastomeric Closures in Pharmaceutical Packaging: From Material Science to Regulatory Compliance. Journal of Engineering Advancements. 2026 Jul 9;7(02):65-72.https://www.researchgate.net/profile/Mostafa-Eissa/publication/408625569_An_Exploratory_Review_of_Elastomeric_Closures_in_Pharmaceutical_Packaging_From_Material_Science_to_Regulatory_Compliance/links/6a4f4f165ec6581029902683/An-Exploratory-Review-of-Elastomeric-Closures-in-Pharmaceutical-Packaging-From-Material-Science-to-Regulatory-Compliance.pdf
• Otte T. Extractables and Leachables Testing for Inhaled Medicines. Pharmaceutical Technology. 2017 Jun 2;41(6).https://www.pharmtech.com/view/extractables-and-leachables-testing-inhaled-medicines
• Jahn M. Leachables and extractables: from regulatory expectations to laboratory assessment. InChallenges in protein product development 2018 Jun 21 (pp. 337-351). Cham: Springer International Publishing.https://link.springer.com/chapter/10.1007/978-3-319-90603-4_16
• Vas G, Fleck L, Comstock K, Cole J. Extractable and leachable testing for pharmaceutical packaging, finished pharmaceutical products, and medical devices: an analytical perspective. Curr Trend Mass Spectrom. 2020:5-14.https://www.chromatographyonline.com/view/extractable-and-leachable-testing-for-pharmaceutical-packaging-finished-pharmaceutical-products

About the Author

Priyal Darji