Bacterial Endotoxin Testing (LAL) Procedure

Standard operating procedure for performing bacterial endotoxin testing using the Limulus Amebocyte Lysate (LAL) method on water, in-process samples, and finished injectables in accordance with USP <85>.

Steps

• Verify sample suitability and test request
  The analyst verifies that the sample identification, sample type, requested test, and applicable endotoxin limit match the approved specification or batch record. The analyst confirms that the sample is within hold time and that no visible condition suggests contamination, leakage, or improper storage. If the request is incomplete or the sample is unsuitable, the analyst stops and escalates to the supervisor.
• Prepare the controlled test area and materials
  The analyst gathers all approved reagents, controls, consumables, and instruments needed for the run. The analyst verifies reagent lot numbers, expiry dates, storage conditions, and method compatibility. The analyst confirms that the work surface is clean and that only endotoxin-free or depyrogenated materials will contact the test system.
• Review instrument status and environmental conditions
  The analyst verifies that the incubator, heating block, or reader is qualified and within calibration status. The analyst confirms the setpoint, timer, and any required environmental controls match the validated method. If equipment status is out of date or the setpoint is incorrect, the analyst stops and escalates before proceeding.
• Prepare standards, controls, and sample dilutions
  The analyst prepares the standard curve or control series required by the validated method. The analyst prepares sample dilutions using endotoxin-free water and the approved dilution scheme. The analyst labels all tubes or wells clearly and prevents cross-contamination between samples, controls, and standards.
• Run the LAL assay
  The analyst adds reagents and samples in the sequence defined by the validated method. The analyst starts the timer immediately after the final reagent addition and places the test system into the incubator or reader without delay. The analyst avoids vibration, disturbance, or temperature excursions during incubation.
• Verify control performance
  The analyst reviews the negative control, positive control, and product control results against the validated acceptance criteria. The analyst confirms that the run meets system suitability requirements and that no control indicates contamination, inhibition, or enhancement. If any control fails, the analyst documents the deviation and escalates the run for investigation.
• Interpret sample results against the endotoxin limit
  The analyst compares each valid sample result to the approved endotoxin limit and the method-specific acceptance criteria. The analyst records whether the sample passes, fails, or requires retest or investigation based on the validated rules.
• Document and release acceptable results
  The analyst records the final results, reagent lots, instrument status, control outcomes, and any required calculations in the approved data system or laboratory record. The analyst submits the completed run for review or release according to site procedure.
• Escalate non-conforming or invalid results
  The analyst documents the non-conformance, including failed controls, invalid assay conditions, suspected contamination, or out-of-specification results. The analyst notifies the supervisor or quality unit and follows the site deviation, retest, or investigation procedure before any further testing or reporting.