Extractables and leachables testing: what vials, stoppers and pens can add to a peptide

What extractables and leachables are

The FDA’s August 2026 draft guidance on container closure systems gives the definitions. Extractables are compounds released from a packaging part into an extraction solvent under laboratory conditions. Leachables are compounds present in the packaged product because they migrated from its packaging during normal storage and use, or during accelerated stability studies.

The ICH Q3E draft says leachables are generally a subset of the extractables, at lower levels. It also lists the exceptions: a leachable can degrade into a new compound, react with the drug, migrate from other packaging, or form as the material ages in light, heat or oxygen. The extractables study may also have missed it.

	Extractables study	Leachables study
What is tested	The part or system, treated as in use (sterilised, cleaned, siliconised)	The product in its final container
Conditions	Solvents of several pH values and polarities; worst-case heat and time	Real storage and in-use conditions, plus accelerated stability
Question	What could migrate?	What did migrate, and how much?
USP chapter	<1663>	<1664>

What vials, stoppers and pens can add to a peptide

The examples below come from regulators and published studies.

Part	Material	Compounds reported
Vial stopper or syringe plunger	Butyl rubber, plain or laminated with a film	Rubber oligomers, the antioxidant BHT, sulfur, vulcanisation accelerators such as 2-mercaptobenzothiazole, a peroxide polymerisation initiator
Glass vial or barrel	Glass	Particles from the glass surface when high heat, irradiation or sterilising gases make it delaminate
Plastic containers, bags and films	Polyethylene, polycarbonate, PVC	The antioxidant Irgafos 168 and its breakdown products, BPA from polycarbonate, phthalates such as DEHP from flexible PVC
Syringes, pens and other delivery devices	Plastic, glass, rubber and metal in one device	From prefilled syringes: silicone oil used as lubricant, acrylic acid from a syringe adhesive
Labels, inks, adhesives, cartons	Secondary packaging	Compounds that pass through semi-permeable plastic

The FDA’s 2026 draft lists what an incompatible container can do to a product: faster breakdown of the active ingredient, precipitation, particles, and leachables that react with a protein and change its structure. Published cases show the chemistry. Acrylic acid from a syringe adhesive reacted with proteins at lysine side chains, the N-terminal amine and histidine; many peptides carry the same groups. A peroxide initiator from a syringe plunger acted as an oxidant in a 2021 study, and oxidation is one route of peptide degradation. In agitation tests on two proteins, silicone-oil-lubricated glass syringes gave the largest protein loss. Plastic particles are a separate question, covered in microplastics in supplements.

Freeze-dried powder or solution: where the risk sits

The FDA’s 2026 draft rates the risk lower for a powder for injection than for a solution, because contact between powder and container is minimal. It gives two cases where the risk rises: a rubber stopper can interact with organic solvent vapour during freeze-drying, or with organic solvent in the reconstituted solution.

Published data point both ways:

• None found. A 2022 study followed five lyophilised protein products in rubber-stoppered glass vials and 19 liquid products in syringes and vials. No organic leachable was above the limit of detection at any time point.
• Found in the dry cake. In a 2017 study, a lyophilised product picked up rubber oligomers from its butyl stopper in measurable amounts, extracting like a 50/50 mix of isopropanol and water. The same product reconstituted in water showed no detectable leachables.

The FDA draft asks for a product-specific risk assessment; these two results show why.

How an extractable and leachable study works

1. Prior knowledge. The composition of each part, additives such as plasticisers and antioxidants, and supplier data.
2. Extractables study. The lab extracts the parts as used, with solvents of several pH values and polarities, at worst-case contact area, temperature and time. ICH Q3E asks for procedures that cover volatile, semi-volatile and non-volatile organic compounds. The routine tools are GC-MS, with headspace sampling for the most volatile compounds, and LC-MS.
3. Identification. Every peak above the analytical evaluation threshold (AET) is identified and quantified.
4. Leachables study. The product is tested in its container over its shelf life and in-use period. The FDA draft expects several batches (for example three), several time points, and accelerated and long-term conditions.
5. Safety assessment. The lab correlates leachables with extractables, and a toxicologist assesses every leachable above the threshold.

A 2021 study of 12 biotechnology products in glass vials, with stoppers from three rubber formulations, found each leachable profile was a subset of the extractable profile.

Screening standards for GC and LC

ICH Q3E asks that semi-quantitative screens are qualified with several standard compounds typically seen as extractables. Suppliers sell certified mixtures for this. One pair, described in 2022, holds 21 compounds for LC-MS and 14 for GC-MS, each at 50 µg/mL, covering antioxidants, plasticisers, UV absorbers, monomers and curing agents. Detector response differs between compounds, instruments and ionisation modes, so for a semi-quantitative screen the AET is multiplied by an uncertainty factor of no more than 0.5.

Extractable and leachable calculation: the AET

The AET is the concentration above which a lab should identify, quantify and report a compound. It starts from a safety concern threshold (SCT) in µg per day; the FDA draft recommends 1.5 µg per day for most chronic-use drugs. A 2026 review works an example for a nasal spray with a maximum of 0.20 mL used per day. With an SCT of 1.5 µg per day and an uncertainty factor of 0.5, the AET is 3.75 µg/mL. Under the 2006 PQRI threshold of 0.15 µg per day, the same product needed 0.375 µg/mL, ten times lower. A lower AET needs a more sensitive method and can mean more peaks to identify.

Extractables and leachables guidelines: USP, FDA and ICH

Document	Status	What it covers
USP <1663> and <1664>	Official since 1 August 2015; informational	Study design and justification; no fixed tests or limits
USP <381>, <660>, <661.1>, <661.2>	Chapters cited in the FDA’s 2026 draft	Rubber components, glass containers, plastic materials and systems
FDA container closure guidance, May 1999	Final	Injections: highest concern, high likelihood of interaction. Sterile powders: highest concern, medium likelihood
FDA container closure draft, August 2026	Draft; replaces the 1999 guidance when final	Risk-based review, leachables over the shelf life, SCT of 1.5 µg per day
ICH Q3E	Step 2 draft, endorsed 1 August 2025	Study design, AET, thresholds and three classes of leachables

Q3E sorts leachables into three classes. Class 1 should be avoided where practical and has compound-specific limits. Besides mutagens of high concern, it names two compounds: benzo(a)pyrene, linked to carbon black, and BPA, linked to polycarbonate and epoxy resin. Class 3 covers common extractables of low potency, such as BHT, erucamide, a rubber oligomer and fatty acids. Our guide to BPA testing explains how BPA itself is measured.

Phthalates testing: method and report

Phthalates are plasticisers. A common source in medical products is flexible PVC, which can hold up to 50% phthalates by weight; DEHP is the one used most.

The US Consumer Product Safety Commission method CPSC-CH-C1001-09.4 (January 2018), written for children’s toys and child care articles, shows the steps. The lab cuts the plastic into pieces no larger than 2 mm, weighs at least 0.05 g and dissolves it in tetrahydrofuran. It precipitates the polymer with acetonitrile or hexane and analyses the solution by GC-MS in full-scan and selected-ion modes, for eight regulated phthalates.

The method warns that phthalates are a common laboratory contaminant, so the lab avoids plastic equipment and runs a reagent blank alongside the samples. A useful report names each phthalate, gives a result or “less than” the limit of quantification, states the unit (% of the material or µg per g) and shows the blank.

Extractables and leachables testing labs

E&L studies are run for the maker of a medicine or device, in its own lab or at a contract lab (CRO), because they support a marketing application. Among the peptide testing labs in our September 2026 research, none lists an extractables and leachables study. Two list targeted tests in the same field: Ozcanium Analytics offers a GC-MS phthalates screen next to its GC-MS odour profiles, and Krause Analytical offers BPA and BPS testing with confirmation by GC-MS and LC-MS.

Ask any E&L lab for the AET and its calculation, the solvents and conditions, the standards and uncertainty factor, the blanks, and every peak above the AET with its name, CAS number and level. Cost rises with the number of parts, solvents and techniques, a low AET, unknown peaks to identify, and a leachables study that runs over the shelf life on several batches.

What HK Analytical does

Extractables and leachables studies are not on our list of tests. BPA testing is the part of this field we are developing. HK Analytical is adding microplastic, PFAS and BPA testing. Write to contact@hkanalytical.com to hear when it starts. You can also use the contact form. How each of those tests works is explained in BPA testing, PFAS testing and how to test for microplastics.

Today we test the peptide in the vial: identity (LC-MS), purity (HPLC), content per vial and residual solvents left from synthesis, in any mix per sample. Solvents from manufacture are a different question from leachables; see residual solvents in peptides. Each report carries our photo of the vial as received, and every number comes with its raw data in a signed record you can check on our verify page. The example report shows the format. To price a panel, request a quote.