Scientifically reviewed by Dr Stephan Hansberg
Peptide endotoxin and sterility testing: LAL, USP <71>
What endotoxin (LAL and recombinant factor C), sterility and heavy metal tests measure, the pharmacopoeia methods behind them, and what research peptide certificates usually do and do not include.
Key takeaways
- •Endotoxins are lipopolysaccharides from the outer membrane of Gram-negative bacteria. They are measured with LAL reagents (USP <85>, Ph. Eur. 2.6.14) or recombinant factor C (Ph. Eur. 2.6.32), in endotoxin units (EU).
- •Sterility testing (USP <71>, Ph. Eur. 2.6.1) checks for living microorganisms by incubating samples in two culture media for at least 14 days.
- •Endotoxin and sterility are separate: a sterile sample can still contain endotoxin from bacteria that have been killed.
- •Elemental impurities (the old "heavy metals") are now assessed under USP <232>/<233> and ICH Q3D, usually by ICP-MS.
- •Most research peptide certificates report HPLC purity only. Contaminant tests are extra and should be read as separate results.
The short answer
Endotoxin testing measures bacterial cell-wall fragments, sterility testing checks for living microorganisms, and elemental impurity testing measures metals such as lead and arsenic. None of these is covered by an HPLC purity result. Most research peptide certificates report purity, sometimes identity, and only occasionally endotoxin, sterility or metals, so it is worth checking which tests a document actually contains.
What endotoxin is
Endotoxins are lipopolysaccharides (LPS) from the outer membrane of Gram-negative bacteria. They are heat-stable and are not removed by the filtration that removes bacteria, which is why a product can be sterile and still contain endotoxin. In laboratory research, endotoxin contamination is also an experimental variable: LPS is a potent activator of innate immune signalling in cell culture, so trace contamination can confound results.
How endotoxin is measured: LAL and recombinant factor C
The classic test uses Limulus amebocyte lysate (LAL), an extract of horseshoe crab blood cells that clots in the presence of endotoxin. USP <85> and Ph. Eur. 2.6.14 describe three LAL techniques: gel-clot, turbidimetric and chromogenic. Results are reported in endotoxin units (EU), commonly per ml or per mg, with one USP EU equal to one international unit.
Since 1 January 2021 the European Pharmacopoeia has included chapter 2.6.32, a test using recombinant factor C (rFC). It detects the same endotoxins with a synthetic reagent and fluorescence detection, without relying on horseshoe crab lysate. On a certificate, look for the method (LAL or rFC), the result with its unit, and the limit it was judged against.
Limits are product-specific. Pharmacopoeia limits for finished medicines are calculated individually for each product from a pharmacopoeia threshold and how the product is intended to be used, so there is no single universal number for a research reagent. For research material, a numeric result with its unit is more useful than a bare "pass".
Sterility testing
USP <71>, harmonised with Ph. Eur. 2.6.1 and the Japanese Pharmacopoeia, tests for viable bacteria and fungi. The sample is either passed through a membrane filter that is then placed in culture media (membrane filtration, preferred where possible) or added directly to the media (direct inoculation). Two media are used, fluid thioglycollate medium and soybean-casein digest medium, incubated at two temperatures for at least 14 days. The sample passes if no growth appears.
A sterility test result applies to the units tested, under the sampling plan used. It is a statistical check, not a guarantee for every container, which is why pharmaceutical manufacturing relies on validated aseptic processes rather than end testing alone.
Heavy metals and elemental impurities
The old USP <231> heavy metals test was a colorimetric method with poor specificity. It was replaced by USP <232> (limits) and <233> (procedures), aligned with the ICH Q3D guideline, and <231> was removed from the compendia from 1 January 2018. Arsenic, cadmium, lead and mercury form the core group; other elements, such as catalysts, are assessed if they could be present. Measurement is by ICP-OES or, more commonly at low levels, ICP-MS. Results are reported in ppm or as below the LOQ.
Why contaminant tests are reported separately
Purity, endotoxin, sterility and elemental impurities need different equipment, different sample preparation and often different laboratories. A chromatography lab may not run microbiology, and a microbiology lab may not run ICP-MS. Each test also consumes sample, and sterility testing in particular takes at least two weeks before a result can be issued. For these reasons, contaminant results normally appear as separate reports or as separate lines with their own method references, rather than inside an HPLC purity report.
When a supplier says a product is tested, the useful follow-up question is which tests, by which method, on which batch, with what result. A document that lists endotoxin as "tested" with no unit or limit gives much less information than one that reports, for example, a value in EU/ml against a stated specification and names the method used.
What research peptide COAs usually include
Independent testing services describe the same pattern. Finnrick's guide notes that HPLC purity covers only peptide-related impurities and does not measure salts, fillers, water, endotoxins or heavy metals, which are separate tests with separate reports.
| Test | Method | How often on research peptide COAs | Read the result as |
|---|---|---|---|
| Purity | HPLC-UV | Almost always | % main peak area |
| Identity | MS / LC-MS or HPLC vs reference standard | Often | Measured vs theoretical mass, or retention time match |
| Quantity | Quantitative HPLC vs reference standard | Sometimes | mg, mg/ml or % of label |
| Endotoxin | LAL (USP <85>) or rFC (Ph. Eur. 2.6.32) | Occasionally, as a separate test | EU/ml or EU/mg against a stated limit |
| Sterility | USP <71> / Ph. Eur. 2.6.1 | Rarely | No growth after 14 days |
| Elemental impurities | ICP-MS (USP <233>) | Rarely | ppm, or below LOQ |
Where Celyfe fits
Celyfe's published certificates are HPLC purity reports from Analiza Białek in Wrocław: 99% for NAD+ batch A26085 and 99% for WOLVERINE (BPC-157 15mg + TB-500 15mg) batch C26071, dated 2 October 2026. They are purity certificates, and this guide is written so readers can see exactly what that does and does not cover. GLOW and KLOW certificates are being re-issued and are listed in the COA library at /coa when published. Pens ship cold-chain and should be kept at 2 to 8 C and not frozen.
Sources
Sources used for this guide, checked October 2026:
- United States Pharmacopeia: General Chapter <85> Bacterial Endotoxins Test
- United States Pharmacopeia: General Chapter <71> Sterility Tests
- EDQM: Recombinant factor C, new Ph. Eur. chapter 2.6.32 available as of 1 July 2020 (edqm.eu)
- United States Pharmacopeia: General Chapters <232> Elemental Impurities, Limits and <233> Elemental Impurities, Procedures
- ICH Q3D(R2): Guideline for elemental impurities
- Finnrick: How to read a certificate of analysis (COA), 1 March 2026
Research use only
Celyfe supplies research peptides for laboratory and research use only. Nothing on this page is guidance on use in humans or animals.
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