Endotoxin Testing by LAL Kinetic-Chromogenic Assay — Method Summary

A summary of the kinetic-chromogenic LAL endotoxin test as applied to peptide lot release. Method reference: USP <85> "Bacterial Endotoxins Test", and FDA Guidance for Industry on Pyrogen and Endotoxins Testing (June 2012).

Principle

The Limulus Amebocyte Lysate kinetic-chromogenic assay measures bacterial endotoxin by detecting the rate at which a chromogenic substrate (Boc-Leu-Gly-Arg-pNA) is cleaved by an enzymatic cascade activated by lipopolysaccharide (LPS). Onset time is inversely proportional to log endotoxin concentration over a standard range of 0.005–50 EU/mL.

Specification

Nexphoria lot-release specification: <0.25 EU/mg for parenteral-grade research peptides. Typical lot result: <0.05 EU/mg.

Procedure

  1. Reconstitute test sample in LAL Reagent Water (LRW) to 1 mg/mL.
  2. Prepare CSE (Control Standard Endotoxin) standard curve: 0.005, 0.05, 0.5, 5.0 EU/mL.
  3. Run positive product control (PPC) — sample + known endotoxin spike — to demonstrate sample does not inhibit the assay.
  4. Add LAL reagent to test wells; record onset time at 405 nm at 37 °C.
  5. Calculate concentration by linear regression of log onset time vs log EU/mL.

Validation Criteria

  • Standard curve r ≥ 0.980 absolute value
  • PPC recovery 50%–200%
  • Coefficient of variation <10% on replicates

Reporting

Every lot's EU/mg result is recorded on its Certificate of Analysis. See COA Library for the current lot release data.

RUO. US researchers only. Method summary for documentation purposes; not a clinical or human-use specification.