# Endotoxin Testing for Peptides: When You Need It and How LAL Works
Endotoxins are lipopolysaccharide fragments from the outer membrane of Gram-negative bacteria. They survive autoclaving. They survive most filtration. They are pyrogenic at nanogram-per-kilogram doses. If a peptide is going anywhere near an injectable route, endotoxins have to be measured.
When endotoxin testing is required
- Injectable use in humans — always. USP <85> is the reference standard.
- Injectable use in animal research — commonly required by institutional protocols.
- 503A and 503B compounding pharmacies — required for any injectable dosage form.
- Research peptides sold as not for human use — not required, but reputable suppliers test anyway.
- Topical or oral formulations — not typically required.
How LAL works
Limulus Amebocyte Lysate (LAL) is derived from horseshoe crab hemolymph. When LAL contacts endotoxin, a clotting cascade activates. Three assay formats read the cascade:
- Gel-clot — semi-quantitative, cheap, slow.
- Turbidimetric — measures cloudiness as clot forms.
- Kinetic chromogenic — measures color development from a synthetic peptide substrate. Fastest, quantitative, wide dynamic range. Standard for pharmaceutical-grade endotoxin testing today.
Reading a result
The unit is EU/mg (endotoxin units per milligram of peptide). A typical injectable peptide specification is under 5 EU/mg. The math:
- Endotoxin limit = K / M
- K = threshold pyrogenic dose (5 EU/kg body weight for most parenteral routes, 0.2 EU/kg for intrathecal)
- M = maximum peptide dose in mg/kg per hour
For a peptide dosed at 1 mg/kg subcutaneous, the limit is 5 EU/mg. At 5 mg/kg, the limit tightens to 1 EU/mg.
