Endotoxin and Sterility Testing Explained: LAL, USP <71> and How They Appear on a COA

Purity by HPLC answers one question about a sample. Endotoxin and sterility tests answer two others, using methods that have nothing in common with chromatography. This guide explains what each test is, what its result means, why neither can be inferred from a purity figure, and how to recognize the results on a certificate. The scope is analytical: what the tests report and how they are documented.

Why these tests are separate from purity

Reversed-phase HPLC with UV detection separates the species in a sample and records those that absorb light at a chosen wavelength, typically 210 to 220 nm. The purity percentage compares the main peak with the other peaks. Bacterial endotoxin is a lipopolysaccharide from the outer membrane of Gram-negative bacteria. Microorganisms are whole cells. Neither produces a peak in a peptide chromatogram at any practical concentration. A sample can therefore show a high HPLC purity and also contain endotoxin or viable organisms, and a certificate showing only HPLC has made no statement about either. The three measurements are independent, and a complete certificate lists them separately.

Bacterial endotoxin testing

The method

The standard method is the bacterial endotoxins test described in United States Pharmacopeia chapter <85>, with equivalents in the European and Japanese pharmacopeias. It uses Limulus amebocyte lysate (LAL), an extract from the blood cells of the horseshoe crab, which clots or changes color in the presence of endotoxin. Three formats are in use:

• Gel-clot. The sample is mixed with lysate and incubated. Formation of a firm gel indicates endotoxin at or above the lysate’s labeled sensitivity. The result is a limit test: pass or fail at a stated threshold, or a semi-quantitative value from a dilution series.
• Turbidimetric (kinetic or endpoint). The reaction produces turbidity that is read by an instrument. The rate or extent of turbidity change is compared against a standard curve to give a numeric result.
• Chromogenic (kinetic or endpoint). A synthetic substrate releases a colored product during the reaction. Absorbance is read and compared against a standard curve.

A recombinant Factor C (rFC) assay, which uses a recombinant protein instead of lysate, is accepted by several pharmacopeias as an alternative and is appearing on more certificates.

What the result looks like

Endotoxin is reported in endotoxin units (EU), referenced to an international standard. On a certificate the result is usually expressed per milligram of material (EU/mg) or per milliliter of the dissolved test solution (EU/mL), and most often as a less-than value: “< 0.5 EU/mg” or “< 5 EU/vial”. The less-than form means the result was below the method’s limit of detection or the lowest standard, not that endotoxin was measured at that exact value. A complete report states the method format, the lysate sensitivity or standard-curve range, the sample dilution and the acceptance limit it was tested against. It should also mention the inhibition and enhancement control (sometimes called the positive product control), which confirms the sample itself did not interfere with the assay.

Reading the number

Two certificates can report different numbers for the same material if one reports per milligram and the other per vial or per milliliter. Check the units before comparing. A report with no units, no limit and no method format is a number without context.

Sterility testing

The method

Sterility testing follows USP chapter <71>, with harmonized equivalents in the other major pharmacopeias. The sample is introduced into two culture media: fluid thioglycollate medium, incubated at 30 to 35 °C, which supports anaerobic and some aerobic organisms, and soybean-casein digest medium, incubated at 20 to 25 °C, which supports aerobic bacteria and fungi. Incubation lasts at least 14 days, with periodic inspection for turbidity or other visible growth.

Two sample-handling techniques are used. Membrane filtration passes the dissolved sample through a filter that retains microorganisms, and the filter is then placed in the media. Direct inoculation adds the sample straight into the media. Filtration is preferred when the material permits it because it removes substances that could inhibit growth. A method suitability test, run in advance, confirms that the sample does not suppress the growth of standard challenge organisms; without it a “no growth” result could be an artifact of inhibition.

What the result looks like

The result is qualitative. The certificate states “no growth observed” or “growth observed” for each medium, with the incubation period and temperatures, and a conclusion of complies or does not comply. There is no count and no identification unless growth occurred and the laboratory investigated further. A complete report also records the number of containers or the quantity tested, because <71> specifies minimums that depend on batch size.

The statistical limit

Sterility testing examines a sample, not the whole lot. A lot in which a small fraction of containers is contaminated can pass the test by chance. The chapter itself describes the test as a check on the sample tested rather than a guarantee for every container. This is why the result is tied to the lot and the quantity examined, and why it is reported alongside, not instead of, the process controls under which the material was filled.

How the two tests appear on certificates

Attribute	Endotoxin (USP <85>)	Sterility (USP <71>)
Type of result	Quantitative or limit value	Qualitative (growth / no growth)
Units	EU/mg, EU/mL or EU/vial	None; complies / does not comply
Typical duration	Hours	14 days or more
Typical presentation	Line item on the main certificate or a separate report	Almost always a separate report
Method detail to look for	Format (gel-clot, turbidimetric, chromogenic, rFC), sensitivity or curve range, dilution, limit, interference control	Technique (filtration or direct), media, temperatures, incubation period, quantity tested, method suitability
Laboratory	Often the same lab as HPLC	Often a different, microbiology-focused lab

Because sterility testing takes two weeks and needs a microbiology laboratory, a supplier that publishes it usually does so as a separate PDF, sometimes from a different laboratory than the one that ran the HPLC. Check that the lot number on the microbiology report matches the lot on the chemistry report. A mismatch means the two documents describe different samples.

How this site records them

Each supplier record carries two fields, testing.endotoxin and testing.sterility, each true, false or unknown. True means we found a published certificate showing the result; it does not mean every lot has one. In the testing factor each method shown earns a small number of points, so a supplier that publishes HPLC, mass spectrometry, endotoxin and sterility scores higher on methods than one that publishes HPLC alone. The point values are on the methodology page. As with every field, unknown earns nothing.

Across the suppliers in our dataset, HPLC results are common, mass spectrometry results are less common, and endotoxin and sterility results appear on a small minority of certificate libraries. Suppliers whose libraries are public are listed at public COAs; the method columns on each supplier page show which tests are present.

Summary

Endotoxin testing reports a quantity of bacterial lipopolysaccharide, in endotoxin units, against a stated limit. Sterility testing reports whether anything grew from the sample over 14 days in two media. Neither can be read from an HPLC chromatogram, and neither is implied by a purity percentage. On a certificate, look for the method, the units or the media, the lot number and the laboratory, and confirm that the microbiology results describe the same lot as the chemistry.