Purpose: To establish a standard operating procedure (SOP) for performing the Limulus Amoebocyte Lysate (LAL) test to determine endotoxin levels in pharmaceutical products and medical devices.
Scope: This SOP applies to all personnel involved in the testing of endotoxin levels in pharmaceutical products and medical devices.
Responsibilities: All personnel involved in the LAL test are responsible for adhering to this SOP and for maintaining accurate records of their activities.
Materials Required: LAL Reagent
Endotoxin Standards
Sterile Pipettes and Pipette Tips
Microplates
Incubator
Spectrophotometer
Water Bath
Definitions:
Endotoxin: A lipopolysaccharide found on the outer membrane of Gram-negative bacteria.
LAL Reagent: An aqueous extract of horseshoe crab blood used to detect endotoxin levels.
Endotoxin Standards: Known concentrations of endotoxin used to validate the LAL test.
Microplates: Small plastic plates with wells used for holding the LAL reagent and test samples.
Spectrophotometer: An instrument used to measure the absorbance of light by the LAL reagent and test samples.
Description:
LAL Reagent: An aqueous extract of horseshoe crab blood used to detect endotoxin levels.
Endotoxin Standards: Known concentrations of endotoxin used to validate the LAL test.
Microplates: Small plastic plates with wells used for holding the LAL reagent and test samples.
Spectrophotometer: An instrument used to measure the absorbance of light by the LAL reagent and test samples.
Description:
The LAL test is used to determine the presence of endotoxins in pharmaceutical products and medical devices. The LAL reagent is mixed with the sample to be tested in a microplate and incubated at a specific temperature for a specific time. The resulting reaction is then measured using a spectrophotometer.
Preparation of Apparatus: Sterilize all pipettes and pipette tips.
Label all microplates and endotoxin standards.
Preheat the incubator to the required temperature.
Reconstitution of Reference Standard Endotoxin: Reconstitute the reference standard endotoxin according to the manufacturer's instructions.
Dilute the endotoxin standard to the required concentration with sterile water.
Reconstitution of Limulus Amoebocyte Lysate (LAL): Reconstitute the LAL reagent according to the manufacturer's instructions.
Dilute the LAL reagent to the required concentration with sterile water.
Performing the Test: Add the LAL reagent to the microplate wells.
Add the endotoxin standard to the appropriate wells.
Add the test sample to the remaining wells.
Incubate the microplate at the required temperature for the required time.
Measure the absorbance of the wells using a spectrophotometer.
Record the results in the appropriate logbook.
Reading and Recording the Results: Compare the absorbance of the test samples to the absorbance of the endotoxin standard.
Calculate the endotoxin concentration of the test samples using the standard curve.
Record the results in the appropriate logbook.
Specifications:
Preparation of Apparatus: Sterilize all pipettes and pipette tips.
Label all microplates and endotoxin standards.
Preheat the incubator to the required temperature.
Reconstitution of Reference Standard Endotoxin: Reconstitute the reference standard endotoxin according to the manufacturer's instructions.
Dilute the endotoxin standard to the required concentration with sterile water.
Reconstitution of Limulus Amoebocyte Lysate (LAL): Reconstitute the LAL reagent according to the manufacturer's instructions.
Dilute the LAL reagent to the required concentration with sterile water.
Performing the Test: Add the LAL reagent to the microplate wells.
Add the endotoxin standard to the appropriate wells.
Add the test sample to the remaining wells.
Incubate the microplate at the required temperature for the required time.
Measure the absorbance of the wells using a spectrophotometer.
Record the results in the appropriate logbook.
Reading and Recording the Results: Compare the absorbance of the test samples to the absorbance of the endotoxin standard.
Calculate the endotoxin concentration of the test samples using the standard curve.
Record the results in the appropriate logbook.
Specifications:
The LAL test must be performed using validated methods and materials.
The endotoxin levels of all pharmaceutical products and medical devices must be below the specified limits.
All personnel involved in the LAL test must be trained and qualified to perform the test.
Records: All LAL test results must be recorded in the appropriate logbook.
All materials used in the LAL test must be logged and tracked.
All personnel involved in the LAL test must sign the appropriate logbook.
References:
The endotoxin levels of all pharmaceutical products and medical devices must be below the specified limits.
All personnel involved in the LAL test must be trained and qualified to perform the test.
Records: All LAL test results must be recorded in the appropriate logbook.
All materials used in the LAL test must be logged and tracked.
All personnel involved in the LAL test must sign the appropriate logbook.
References:
United States Pharmacopeia Chapter 85: Bacterial Endotoxins Test.
ISO 10993-11: Biological evaluation of medical devices - Part 11: Tests for systemic toxicity.
ISO 10993-11: Biological evaluation of medical devices - Part 11: Tests for systemic toxicity.
Unites
States Pharmacopeia 44
Distribution: This SOP must be distributed to all personnel involved in the LAL test.
Distribution: This SOP must be distributed to all personnel involved in the LAL test.
END OF THE DOCUMENT
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