Extractables and leachables (E&L) testing identifies and quantifies chemical substances that migrate from packaging, container closure systems, and drug delivery components into pharmaceutical products. Test objects include elastomeric stoppers, plastic containers, coatings, adhesives, filters, tubing, and single-use assemblies. An accredited laboratory performs the testing against compendial and ISO guidance and delivers documented evidence for regulatory submissions.
E&L Testing — Principle and Scope
E&L testing separates two related phenomena. Extractables are compounds released from a material under exaggerated laboratory conditions. These conditions use aggressive solvents and elevated temperatures to maximize migration. Leachables are the subset of those compounds, or their degradation products, that actually transfer into the drug product under normal storage and use. The workflow therefore proceeds from controlled extraction, through analytical profiling, to simulation and confirmatory leachables studies. The scope covers container closure systems for parenteral, oral, ophthalmic, and inhalation products. It also covers single-use bioprocess bags and filters, medical device materials, and packaging components such as seals, gaskets, and printing inks. Frameworks including USP General Chapters <1663> and <1664>, ICH quality guidance, and ISO 10993-12 for devices define study logic and reporting expectations. The outcome is a documented list of migrants with measured concentrations, carried forward to toxicological evaluation.
Sample Types and Extraction Study Design
Study design begins with a bill of materials listing polymers, elastomers, additives, colorants, and processing aids. Representative samples come from the same production lot as the marketed component. They are cut to a defined surface-area-to-volume ratio. Controlled extraction applies solvents of graded polarity: aqueous media, a polar organic mixture, a medium-polarity solvent such as isopropanol/water, and a nonpolar solvent such as hexane or dichloromethane. This range captures polar and lipophilic migrants. Extraction techniques include reflux, sealed-vessel heating at elevated temperature, sonication, and agitation across multiple time points. A simulation study with the placebo or a representative solvent bridges exaggerated extraction and actual product contact conditions. Headspace sampling of the extraction vessel documents volatile species that would otherwise be lost. Solvent volumes, sample masses, and time points are recorded so that every result remains traceable and repeatable.
Analytical
Methods — GC-MS, LC-MS, ICP-MS
Three platforms carry the analytical load. GC-MS resolves volatile and semi-volatile organic extractables. Headspace GC-MS screens residual volatiles. Capillary columns with a nonpolar 5%-phenyl-dimethylpolysiloxane phase or a polar polyethylene glycol (wax) phase separate compound classes. Quantitation is performed by GC-FID or SIM-mode acquisition. LC-MS addresses semi-volatile and nonvolatile migrants that GC cannot elute. UHPLC separation is coupled to high-resolution accurate-mass spectrometry (QTOF or Orbitrap-type). This enables formula assignment and library searching, with tandem MS confirming structures. ICP-MS quantifies elemental extractables such as sulfur, zinc, calcium, barium, and silicon. These elements originate from elastomer cure systems and glass contact. The three datasets are merged into a single extractables profile, and each peak receives an identification confidence level. Where reference standards are unavailable, quantitation applies relative response factors or semi-quantitative estimates, with the attached uncertainty stated in the report. Ion chromatography extends coverage to inorganic anions.
Analytical
Evaluation Threshold and Detection Limits
AET converts a health-based safety concern threshold (SCT) into an analytical reporting limit. Published guidance cites a common SCT for oral, parenteral, and ophthalmic routes on the order of 1.5 µg total daily intake. The AET divides the SCT by the number of daily doses. It then adjusts for dilution or concentration applied during sample preparation. Every reported extractable must be quantified at or below this level. For that reason, method LOD and LOQ are verified against the AET before sample analysis. Two adjustments follow. First, the uncertainty of semi-quantitative response is applied as an adjustment factor. This lowers the operational threshold so that under-response cannot hide an above-threshold compound. Second, compounds of toxicological concern are reported regardless of concentration. Detection limits are established from replicate blanks and low-level spike experiments. The achieved LOD and LOQ of each technique are documented to confirm threshold coverage.
Leachables
Monitoring in Stability Studies
Leachables monitoring runs within the formal stability program under ICH long-term and accelerated conditions. Drug product units are stored in the market container closure system and sampled at predefined time points. The leachables profile is measured by the same LC-MS and GC-MS methods validated during the extractables phase. Each leachable is tracked against time and storage condition. Its concentration is compared with the product-specific acceptance limit derived from the AET and toxicological qualification. New peaks appearing during storage trigger structural confirmation by accurate-mass MS and, where needed, isolation for further characterization. Trend evaluation addresses two questions: whether a leachable approaches its limit before shelf-life expiry, and whether degradation products accumulate. Placebo formulations confirm that matrix interference does not distort quantitation. The monitoring dataset informs specification setting. It shows, over the claimed shelf life, that patient exposure remains within qualified bounds.
Toxicological
Risk Assessment and Reporting
Risk assessment closes the chain from analytical data to a safety conclusion. Identified extractables and leachables are screened against lists of compounds of toxicological concern. This flags structures such as nitrosamines, mercaptobenzothiazole derivatives, phthalates, and epoxidized additives for priority review. For each compound, a toxicologist derives a permissible daily exposure (PDE or ADE) from published data. Where data are absent, the toxicologist uses read-across and structure-based assessment. Estimated patient daily exposure is calculated from measured concentration, dose volume, and dosing frequency. It is then compared with the PDE, and the resulting margin documents qualification status. The final report states the study scope, materials, extraction design, methods and their validated performance, the AET derivation, identified compounds with identification confidence, quantitation uncertainty, and the toxicological conclusion. Reports reference the applicable guidance frameworks and are formatted for regulatory submission. Reviewers can therefore trace every migrant from chromatographic peak to risk statement.
Frequently Asked Questions
What
happens if E&L testing results are disputed, and how is retesting of extractables and leachables data handled?
Disputed results are reviewed against the original study design, analytical methods (GC-MS, LC-MS, ICP-MS), and the analytical evaluation threshold applied. If discrepancies remain, targeted reanalysis or full retesting of the affected sample types can be performed. Findings are documented in a revised or supplemental report.
How long does Extractables and Leachables (E&L) testing take, and when is the final report delivered?
Turnaround depends on study scope: the number of sample types, whether leachables monitoring spans a stability study, and the extent of toxicological risk assessment. Reports are issued after methods, detection limits, AET, and risk conclusions are compiled. Laboratories typically confirm an estimated schedule before work begins.
What
samples are required for E&L testing, and how should they be prepared and submitted?
Sample requirements follow the study design: contact materials and final packaged products for extractables screening, and stability time-point samples for leachables monitoring. Quantities must support detection limits of GC-MS, LC-MS, or ICP-MS methods. Submit samples with material descriptions and storage conditions, ideally alongside blank or control articles.