Endotoxin and sterility testing: what a purity number leaves out

Q: What do EU/mL and EU/mg mean?

EU is the Endotoxin Unit, a measure of endotoxin activity. EU/mL is the activity per millilitre of solution, and EU/mg is the activity per milligram of material.

By the PeptideLabCheck team · Last reviewed 14 November 2026 · 12 sources

Short answer

An HPLC purity percentage shows how much of the material seen by the detector is the target peptide. It does not measure bacterial endotoxins, and it does not show whether a sample is sterile. Those are two separate tests: a bacterial endotoxins test, reported in Endotoxin Units (EU) per mL or per mg, and a sterility test, which checks for microbial growth over at least 14 days. A lab report that shows only purity leaves both questions unanswered.

What does an HPLC purity number actually measure?

HPLC stands for high-performance liquid chromatography. It separates the components of a sample and records each one as a peak.

Peptides are generally detected by UV absorbance (measuring how much ultraviolet light is absorbed) at 210–220 nm ⁹. The purity figure is the area of the main peak as a percent of the total area of all reported peaks, a calculation called area percent or normalisation ¹².

So the purity figure is a ratio among the things that detector records. Our guide to HPLC purity covers what else it leaves out, such as water and counter-ions.

Regulatory guidance treats these as separate tests too. The ICH Q6A guideline, published by FDA on December 29, 2000, lists sterility and endotoxins as their own tests, each with its own procedure and acceptance criterion, for the sterile product category it calls parenteral products ¹⁰.

Test	Question it answers	How the result is written	Publicly cited standards
HPLC purity	What share of the detected peaks is the target peptide?	Percent of peak area	None cited in this guide
Bacterial endotoxins test	How much endotoxin activity is in the sample?	EU/mL or EU/mg, often "less than" a value	USP <85>, Ph. Eur. 2.6.14, JP 4.01 ⁴; recombinant methods: USP <86> ⁶, Ph. Eur. 2.6.32 ⁷
Sterility test	Did microorganisms grow from the units tested?	Growth or no growth after incubation	USP <71>, Ph. Eur. 2.6.1, JP 4.06 ⁵

USP is the United States Pharmacopeia. Ph. Eur. is the European Pharmacopoeia. JP is the Japanese Pharmacopoeia.

What are bacterial endotoxins?

Endotoxins are lipopolysaccharides (LPS). These are molecules found in the outer membrane of Gram-negative bacteria, a large group of bacteria. An FDA inspection guide says LPS is released after the bacterial cell dies and breaks apart ³.

This means endotoxin can remain in a material after the bacteria that made it are gone. The same FDA guide states that endotoxins are not removed by sterilizing filters. It lists the usual sources as water, raw materials, packaging components and equipment ³.

A sterile sample can therefore still contain endotoxin. The two properties need separate tests.

How is endotoxin tested and reported?

The classic test uses LAL, which stands for Limulus amebocyte lysate. This is an extract from the blood cells of the horseshoe crab. The harmonised pharmacopoeia text describes the bacterial endotoxins test as a test to detect or quantify endotoxins from Gram-negative bacteria using this lysate. It names three techniques: gel-clot, turbidimetric and chromogenic ¹.

Newer methods use recombinant reagents. These are made with gene technology instead of being taken from crabs. USP published the final text of chapter <86> on November 1, 2024. It covers recombinant cascade (rCR) and recombinant Factor C (rFC) reagents, and USP described these methods as alternatives to chapter <85> ⁶. The European Pharmacopoeia announced chapter 2.6.32 for the rFC method on July 1, 2020 ⁷.

Results are expressed in Endotoxin Units (EU). One EU equals one International Unit (IU) ¹. The EU is a unit of endotoxin activity ³. Limits and results are written per volume or per weight, such as EU/mL or EU/mg ¹.

Four points help when reading a result:

"Less than" values. In the gel-clot quantitative test, when no reaction is seen, the harmonised text says to report the result as less than the sensitivity of the test ¹. A "less than" result means none was detected at that sensitivity.
Interference. The sample itself can weaken or strengthen the reaction. The harmonised text requires a test for interfering factors. In the photometric techniques, endotoxin added to the sample must be recovered within 50–200% ¹.
Handling. FDA states that storage and handling can affect the ability to detect endotoxins ².
No universal limit. The ICH evaluation notes that the test chapters contain no acceptance criteria. Limits are set in an individual product monograph or regulatory filing ⁴. A report should say which limit, if any, was applied.

What is a sterility test, and what are its limits?

A sterility test checks whether microorganisms grow from a sample. FDA calls USP <71> the principal source for sterility testing methods ⁸. ICH guidance published by FDA treats USP <71>, Ph. Eur. 2.6.1 and JP 4.06 as interchangeable, subject to conditions ⁵.

FDA's laboratory manual, which follows USP, describes the procedure. The sample is passed through a membrane filter or added directly to culture media. Two media are used: fluid thioglycollate medium and soybean-casein digest medium. They are incubated at 32.5 ± 2.5 °C and 22.5 ± 2.5 °C respectively, for not less than 14 days. A method suitability test checks that the product does not itself stop microorganisms from growing ¹¹.

The test has limits. FDA states that sterility tests are limited in their ability to detect contamination because of the small sample size. FDA gives an example: if a 10,000-unit lot with 0.1 percent contamination is tested using 20 units, there is a 98 percent chance the lot passes ⁸.

A passing result therefore means no growth was found in the units tested. It does not prove every unit in the batch is sterile. It also says nothing about endotoxin ³.

What should you look for on a lab report?

A separate endotoxin result. A purity percentage does not cover it.
The method named (for example gel-clot, chromogenic or rFC) and the standard cited.
The units (EU/mL or EU/mg), and for "less than" results, the sensitivity used ¹.
The limit applied and where it comes from ⁴.
For sterility: the method, the incubation time and how many units were tested ^{8, 11}.
The batch or lot number. A result describes the sample tested. FDA's inspection guide describes a case where a pooled sample met the endotoxin limit while some individual units exceeded it ³.

If a report does not include endotoxin or sterility results, those properties are unknown for that sample. A missing test is not a pass, and it is not a fail.

Key facts

The bacterial endotoxins test detects or quantifies endotoxins from Gram-negative bacteria using amoebocyte lysate from the horseshoe crab ¹.
The harmonised endotoxin test text names three techniques: gel-clot, turbidimetric and chromogenic ¹.
One International Unit (IU) of endotoxin equals one Endotoxin Unit (EU) ¹.
FDA's ICH Q4B Annex 14 guidance (October 2013) states that Ph. Eur. 2.6.14, JP 4.01 and USP <85> can be used as interchangeable, subject to conditions ⁴.
USP published the final text of chapter <86>, covering recombinant cascade (rCR) and recombinant Factor C (rFC) reagents, on November 1, 2024 ⁶.
FDA states that a 10,000-unit lot with 0.1 percent contamination has a 98 percent chance of passing a sterility test that uses 20 units ⁸.
FDA's Pharmaceutical Microbiology Manual gives an incubation period of not less than 14 days for sterility test media ¹¹.
Peptides are generally detected by UV absorbance at 210–220 nm, and HPLC purity by normalisation is the main peak's area as a percent of the total area of all reported peaks ^{9, 12}.

Common questions

Does 99% HPLC purity mean a peptide is free of endotoxin?

No. HPLC purity compares the target peptide peak with the other peaks the detector records ¹². Endotoxin is measured by a separate test and reported in Endotoxin Units ¹.

What do EU/mL and EU/mg mean?

EU is the Endotoxin Unit, a measure of endotoxin activity ^{1, 3}. EU/mL is the activity per millilitre of solution, and EU/mg is the activity per milligram of material.

Is LAL the only accepted endotoxin method?

No. USP chapter <86> and Ph. Eur. chapter 2.6.32 describe recombinant methods ^{6, 7}. FDA guidance says firms may use alternative methods if they are validated and shown to give equivalent or better results ².

If a sample passes a sterility test, is it also free of endotoxin?

Not necessarily. FDA's inspection guide says endotoxins are not removed by sterilizing filters, so lowering the number of microorganisms does not lower endotoxin to match ³.

How long does a sterility test take?

Under the USP-based procedure in FDA's laboratory manual, the media are incubated for not less than 14 days ¹¹.

Sources

"Bacterial Endotoxins Test" (PDG code Q-06, Rev. 2), harmonised text with sign-off cover sheet version 2. Pharmacopoeial Discussion Group, posted by Japan's Pharmaceuticals and Medical Devices Agency (PMDA). Cover sheet dated April 2026; Rev. 2 text signed June 2011. https://www.pmda.go.jp/files/000280573.pdf — Used for what the test is, the three techniques, units, "less than" reporting and interference checks.
"Guidance for Industry: Pyrogen and Endotoxins Testing: Questions and Answers (Edition 2)". FDA, March 2026. https://www.fda.gov/media/83477/download — Used for FDA's statements on sample storage and handling and on alternative methods.
"Bacterial Endotoxins/Pyrogens", Inspection Technical Guide No. 40. FDA, dated March 20, 1985; page content current as of November 17, 2014. https://www.fda.gov/inspections-compliance-enforcement-and-criminal-investigations/inspection-technical-guides/bacterial-endotoxinspyrogens — Used for what endotoxins are, their sources, filters, the meaning of EU and the pooled-sample example. Note that this is an older document.
"Q4B Evaluation and Recommendation of Pharmacopoeial Texts for Use in the ICH Regions, Annex 14: Bacterial Endotoxins Test General Chapter". FDA / ICH, October 2013. https://www.fda.gov/media/79183/download — Used for the interchangeable endotoxin chapters and the statement that the chapters contain no acceptance criteria.
"Q4B Evaluation and Recommendation of Pharmacopoeial Texts for Use in the ICH Regions, Annex 8(R1): Sterility Test General Chapter". FDA / ICH, September 2017. https://www.fda.gov/media/73134/download — Used for the interchangeable sterility chapters.
"Chapter for endotoxin testing using non-animal derived reagents published for early adoption". United States Pharmacopeia (USP), November 1, 2024. https://www.usp.org/news/chapter-for-endotoxin-testing-using-non-animal-derived-reagents-published-for-early-adoption — Used for USP chapter <86>, the reagents it covers and its status relative to <85>.
"Recombinant factor C: new Ph. Eur. chapter available as of 1 July 2020". European Directorate for the Quality of Medicines & HealthCare (EDQM), July 1, 2020. https://www.edqm.eu/en/-/recombinant-factor-c-new-ph.-eur.-chapter-available-as-of-1-july-2020 — Used for Ph. Eur. chapter 2.6.32 on the rFC method.
"Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing — Current Good Manufacturing Practice". FDA, September 2004. https://www.fda.gov/media/71026/download — Used for USP <71> as the principal source and for the limits of sterility testing.
Fields GB, Mant CT, Chen Y, et al. "HPLC Analysis and Purification of Peptides." Methods in Molecular Biology 386, 3–55 (2007); full text hosted by PubMed Central, U.S. National Library of Medicine. https://pmc.ncbi.nlm.nih.gov/articles/PMC7119934/ — Used for peptide UV detection at 210–220 nm.
"International Conference on Harmonisation; Guidance on Q6A Specifications: Test Procedures and Acceptance Criteria for New Drug Substances and New Drug Products: Chemical Substances". FDA, Federal Register, 65 FR 83041, December 29, 2000. https://www.govinfo.gov/content/pkg/FR-2000-12-29/pdf/00-33369.pdf — Used to show that sterility and endotoxins are listed as separate specification tests.
"Pharmaceutical Microbiology Manual" (ORA.007, Revision 02). FDA, Office of Regulatory Affairs, revised August 25, 2020. https://www.fda.gov/media/88801/download — Used for sterility test media, incubation temperatures and time, test methods and method suitability.
"How should the test requirements be applied in related substances tests?", EDQM FAQs, European Directorate for the Quality of Medicines & HealthCare (Council of Europe). https://faq.edqm.eu/x/_AGL — Used for the normalisation (area percent) calculation.