Endotoxin Test — EP 2.6.14 / USP <85> / ANSI AAMI ST72 · Pharmaceutical Analysis

What Is Endotoxin?

The endotoxin test measures the quantity of lipopolysaccharide (endotoxin) originating from the cell wall of Gram-negative bacteria. Endotoxin is heat-resistant: sterilisation kills the bacterium but does not destroy the endotoxin. A sterile product can therefore still be pyrogenic and must be tested separately.

Purpose of Endotoxin

The purpose is to show that the endotoxin level in devices that contact the body via the bloodstream, cerebrospinal fluid, or implantation is below the permitted limit.

Which Products Is Endotoxin Applied To?

Intravenous catheters and infusion sets
Implants
Dialysis sets
Surgical instruments and sets
Devices in contact with cerebrospinal fluid

In Which Sectors Is Endotoxin Used?

Medical DeviceHealthcarePharmaceuticals

Standards Related to Endotoxin

EP 2.6.14
USP <85>
ANSI/AAMI ST72
ISO 11737-3

Endotoxin Conditions

Method: Gel-clot, turbidimetric, or chromogenic LAL methods
Extraction: The product's body-contacting surfaces are extracted with endotoxin-free water
Interference control: It must be verified that the product does not inhibit or enhance the test

Equipment for Endotoxin

LAL test kits and reagents
Endotoxin-free glassware
Microplate reader (for kinetic methods)
Dry-heat oven (for depyrogenation)
Temperature-controlled incubator

Sample Requirements for Endotoxin

The final, sterilised product
The body-contacting surface area must be known
The number of specimens is determined according to the pharmacopoeia and batch information
The specimen must be prepared and transported in endotoxin-free containers

Duration of Endotoxin

The initial test, including the interference (inhibition/enhancement) study, takes several days; routine tests are shorter.

Endotoxin Procedure

1The endotoxin limit for the product is calculated.
2An interference study is performed, or reference is made to an existing study.
3The product is extracted with endotoxin-free water.
4The extract is mixed with the LAL reagent.
5It is incubated at the defined temperature.
6Clot formation, turbidity, or colour development is measured, depending on the method.
7The endotoxin quantity is calculated from the standard curve.

Endotoxin Acceptance Criteria

The acceptance criterion is the endotoxin limit calculated for the product. This limit is calculated using the formula defined in the pharmacopoeia, based on the device's route of contact and patient weight; the limit is markedly lower for devices in contact with cerebrospinal fluid.

Evaluation of Results

The result is compared with the calculated limit. If the interference study is not valid, the result cannot be interpreted; where the product suppresses the LAL reaction, a dilution factor must be determined.

Endotoxin Report

Applied pharmacopoeia and method
Specimen description, batch and surface area
Calculated endotoxin limit
Interference-study result and dilution factor
Measured endotoxin value (EU/device or EU/mL)
Comparison with the limit

Accreditation

EUROLAB holds internationally recognised accreditation obtained from accreditation bodies party to the IAF and ILAC multilateral recognition arrangements, under ISO/IEC 17025 for laboratory testing, ISO/IEC 17020 for inspection and surveillance, and ISO/IEC 17021-1 and ISO/IEC 17065 for system and product certification. This chain ensures the reports and certificates issued are accepted worldwide.

Whether this test can be offered within accredited scope depends on your product and the standard applied; since accreditation is method-based, the scope is confirmed in writing at the quotation stage.

Scope and authorisations

Related Regulations

MDR (EU) 2017/745
Pharmacopoeia requirements

Pharmaceutical Analysis — What Does This Testing Field Cover?

The section below describes the field that the Endotoxin test belongs to; the conditions and acceptance criteria of this particular test are given in the sections above. The scope specific to your product is confirmed in writing at the quotation stage.

Pharmaceutical analysis determines the identity, purity, quantity and behaviour of an active substance and of the finished product. What sets this family apart is that the methods are largely defined in PHARMACOPOEIAS: the European Pharmacopoeia (Ph. Eur.) and the United States Pharmacopeia (USP) give both the method and the acceptance framework together.

The binding nature of the pharmacopoeial method shapes how the field works. In other sectors a laboratory can choose a suitable method, whereas here the method in the monograph governs; if an alternative method is to be used, its equivalence must be demonstrated. This is what makes results comparable worldwide.

Analyses are carried out in several groups. Identity and assay (HPLC, UV, titration) show what the product is and how much active substance it contains. The impurity profile and residual solvents look for unwanted compounds arising from the manufacturing process. Elemental impurities address metals originating from catalysts and equipment. Dissolution and disintegration behaviour is the most important in vitro indicator of how the drug will be released in the body.

The DISSOLUTION TEST is one of the most critical measurements in the field. It is not enough for a tablet to contain the right amount of active substance; that substance must dissolve at a suitable rate. The dissolution profile is used to assess the effect of formulation changes on bioavailability and holds a central place in generic drug licensing.

METHOD VALIDATION is a mandatory step in this family. If a pharmacopoeial method is used, it is verified that the method works properly under that laboratory's conditions. If an in-house method has been developed, it is validated against parameters such as linearity, precision, accuracy, specificity, limits of detection and quantification, and robustness. A result produced with an unvalidated method cannot be used in a licence dossier.

Stability studies determine shelf life and are the longest-running work in the field. The product is held under defined temperature and humidity conditions, analysed at set intervals, and the results become the basis of the shelf life and storage condition claims. Accelerated conditions give preliminary information but real-time data is what counts.

The microbiological side is a separate body of work: microbial enumeration, testing for specified microorganisms, sterility testing for sterile products and the bacterial endotoxin test. For sterile products, packaging integrity and sterilisation validation also come into play.

Data integrity has moved to the centre of inspections in recent years. It is not enough for the result to be correct; the raw data must be traceable, unalterable and auditable. Validation of electronic record systems has therefore become an inseparable part of analysis.

Method groups in this field

Identity and assay
HPLC, UPLC, UV-Vis spectrophotometry, titration, identity confirmation by FTIR.
Impurities and residues
Related impurities, degradation products, residual solvents (GC headspace), nitrosamine screening.
Elemental impurities
Determination of elemental impurities by ICP-MS and ICP-OES and the associated risk assessment.
Physicochemical
Dissolution and disintegration, hardness, friability, uniformity of dosage units, water content (Karl Fischer), particle size.
Microbiological
Microbial enumeration, specified microorganisms, sterility, bacterial endotoxin, preservative efficacy.
Stability
Real-time and accelerated stability studies; photostability and in-use stability.

Commonly confused in this field

The pharmacopoeial method is binding; an alternative method may be used only by demonstrating its equivalence.
A result produced without method validation cannot be used in a licence dossier.
Accelerated stability does not replace real-time data; shelf life is supported by real-time results.
The dissolution profile is sensitive to apparatus type, agitation rate and medium; the conditions must be complete in the report.
A shortcoming in data integrity produces a non-conformity in an inspection even when the result is correct.
A change of formulation or supplier requires analytical comparison and, where necessary, a new stability study.

Which decisions these results support

The licence dossier and batch release
Determining shelf life and storage conditions
Method transfer and method validation
Incoming raw material acceptance control from suppliers
Assessing the effect of a formulation change on bioequivalence

Related Tests

Frequently Asked Questions About Endotoxin

My product is sterile — why is an endotoxin test needed?

Endotoxin is heat-resistant; although sterilisation kills the bacterium, it does not break down endotoxin, which is a cell-wall residue. A sterile product can therefore still cause a pyrogenic reaction, and endotoxin must be measured separately.

How is the endotoxin limit determined?

It is calculated using the formula defined in the pharmacopoeia, based on the device's route of contact and the permitted dose. For devices in contact with cerebrospinal fluid, the limit is much lower; the calculation must be shown in the report.