Photometric testing should be performed using a suitable instrument, generally at a temperature of 37 °C ± 1 °C.
To ensure the validity of turbidimetric and chromogenic tests, the reliability of the standard curve and the interference of the test sample solution should be evaluated in advance. If any change occurs in the testing environment that could affect the test results, the validity of the test should be reassessed.
Prepare a standard endotoxin solution and make dilutions at no fewer than three concentrations. The dilution factor at each step should not exceed 10, and the lowest concentration should not be lower than the labeled detection limit of the LAL reagent being used.
The mixing time at each dilution step should be the same as that used for the gel-clot method. At least three replicate tubes should be prepared for each concentration. In addition, two negative controls should be included. If the negative controls show no reaction within the specified reaction time, all data should be subjected to linear regression analysis.
Select an endotoxin concentration at the midpoint of the standard curve or one close to the midpoint. Prepare Solutions A, B, C, and D according to Table 4.
The volumes of test sample solution and LAL reagent, the ratio of test sample solution to LAL reagent, incubation time, and other parameters should be performed according to the relevant instructions for the instrument and reagents being used. At least two replicate tubes should be prepared for each solution.
| Reagent | Endotoxin Content (per 0.1 ml) EU/ml | Negative Control Tube (add β-glucan 0.1 ml) | ||||
|---|---|---|---|---|---|---|
| 0.5 | 0.25 | 0.125 | 0.06 | 0.03 | ||
| LAL Reagent (Complete Composition) | + | + | + | − | − | + |
| LAL Reagent (Specific Formulation) | + | + | + | − | − | − |
A: Test sample solution diluted by a factor not exceeding the Maximum Valid Dilution (MVD).
B: Test sample solution containing a known endotoxin concentration at the midpoint of the standard curve or a concentration close to the midpoint, with the same dilution factor as Solution A.
C: Standard endotoxin solution used to prepare the standard curve, as described under “Standard Curve Reliability Test.”
D: Negative control.
Using the linear regression equation obtained from the standard curve, calculate the endotoxin concentrations in the test sample solution and the test sample solution containing standard endotoxin as Ct and Cs, respectively. Then calculate the recovery (R) under the test conditions using the following equation:
When the endotoxin recovery is between 50% and 200%, the test sample solution is considered to be free from interference under the specified test conditions.
If the endotoxin recovery falls outside the specified range, the interfering factors should be removed using the methods described under the “Interference Test” for the gel-clot method. The interference test should then be repeated to verify the effectiveness of the treatment.
Perform the test according to the procedure described under “Interference Test by the Photometric Method.”
Use the standard curve generated from Solution C to calculate the endotoxin concentration in each replicate of Solution A.
(1) The results obtained for the series of Solution C must meet the requirements specified under “Standard Curve Reliability Test” in the photometric method preliminary test.
(2) The endotoxin recovery calculated after subtracting the endotoxin concentration of Solution A from that of Solution B must be within the range of 50%–200%.
(3) No endotoxin should be detected in Solution D (negative control) within the specified reaction time.
If the mean endotoxin concentration of the replicate tubes containing the test sample solution, multiplied by the dilution factor and concentration factor, is lower than the specified endotoxin limit, the test sample complies with the requirements. If the calculated value is higher than the specified endotoxin limit, the test sample does not comply with the requirements.
Note: In this test method, the term “tube” includes any other suitable reaction vessel, such as a well in a microplate.