6 Common Mistakes in Endotoxin Testing and How to Avoid Them
Endotoxin testing is a crucial step for keeping pharmaceuticals, medical devices, and biotech products safe. Besides product safety, endotoxin testing is also important for regulatory compliance and quality control. But even skilled professionals can make mistakes that lead to wrong results. This blog highlights the six most common mistakes in endotoxin testing and how to prevent them.
1. Ignoring Interference Factors
A common mistake is ignoring interference from the sample itself. Ingredients like proteins, surfactants, or chelating agents can affect the Limulus Amebocyte Lysate (LAL) test, causing false negative or false positive results.
How to avoid it:
Always perform an inhibition/enhancement (I/E) test as per pharmacopeial guidelines (USP <85>, EP 2.6.14) to validate the method for your specific product matrix [1].
2. Assuming Off-the-Shelf Kits Require No Validation
Many laboratories mistakenly believe that commercially available LAL or recombinant Factor C (rFC) kits come fully validated. While these kits are quality-controlled by the manufacturer, each laboratory must validate them for its own sample type and conditions.
How to avoid it:
Conduct a full method suitability test for your product, including recovery studies, to ensure the kit performs accurately in your matrix [2].
3. Inadequate Control of Environmental Conditions
Temperature and pH variations can significantly impact endotoxin detection. For example, the LAL assay is highly sensitive to pH outside 6.0–8.0, and poor temperature control can alter clot formation or fluorescence signals.
How to avoid it:
Maintain strict environmental controls during testing by using endotoxin-free glassware, disposables and reagents, and monitoring conditions according to the kit instructions and pharmacopeial standards [3]. Take particular care to comply with the manufactures recommended storage conditions. These will apply differently for the reagent ‘as delivered’ and when reconstituted.
4. Low Endotoxin Recovery
Low endotoxin recovery can result in a dangerous underestimation of the amount of endotoxin present in a sample. This issue has become increasingly important in biopharmaceutical products and complex biological raw materials and excipients. In general, low endotoxin recovery arises due to the endotoxin being ‘masked’ by a component(s) of a test mixture. Critically, it is time-dependent, with endotoxin masking developing over hours or days.
How to avoid it:
Endotoxin recovery studies must be carried out, over an extended period of time (hold-time), in order to assess the effect of time on the recovery of an endotoxin spike. This applies if your product contains either of the following [5,6]:
Biological or synthetic macromolecules
Highly charged excipients
Micro or nano particles
Carriers
5. Using Improper Dilutions
Incorrect sample dilution can mask endotoxins or exceed the maximum valid dilution (MVD), leading to invalid results.
Over-dilution may reduce sensitivity
Under-dilution can allow matrix interference to persist
How to avoid it:
Calculate the MVD correctly using the formula provided in USP <85>, and ensure dilutions are prepared with endotoxin-free water using validated pipettes and techniques [4].
6. Neglecting Routine Equipment Calibration and Maintenance
Plate readers, pipettes, and depyrogenation ovens require regular calibration. Even slight deviations in temperature or volume delivery can compromise test accuracy.
How to avoid it:
Implement a preventive maintenance and calibration schedule for all endotoxin testing equipment, and document each step as part of your quality system [5].
Conclusion
Avoiding these common mistakes in endotoxin testing is critical for regulatory compliance and patient safety. By validating your method, controlling interference, and following pharmacopeial guidelines, you can ensure accurate and reliable endotoxin detection.
References
United States Pharmacopeial Convention. (2024). United States Pharmacopeia - USP–NF 2024: Chapter <85> Bacterial Endotoxins Test. Rockville, MD: USP.
Council of Europe (2025). European Pharmacopoeia (12th ed.): General Chapter 2.6.14 Bacterial Endotoxins. Strasbourg, France: Council of Europe.
Williams, K. L. (2007). Endotoxins: Pyrogens, LAL Testing and Depyrogenation. CRC Press. Doi:10.3109/9781420020595
Petsch, D. & Anspach, F. B. (2000). Endotoxin removal from protein solutions. Journal of Biotechnology, 76(2-3): 97-119.
Center for Drug Evaluation and Research (2012). FDA Guidance for Industry: Pyrogen and Endotoxins Testing. Available at https://www.fda.gov/regulatory-information/search-fda-guidance-documents/guidance-industry-pyrogen-and-endotoxins-testing-questions-and-answers
Chen, D., von Wintzingerode, F., Barlasov-Brown, J., et al. (2019). PDA Technical Report No. 82 (TR 82). Low Endotoxin Recovery. Parenteral Drug Association, Inc. ISBN: 978-1-945584-07-7