The turbidimetric method measures increases in turbidity. Depending on the test principle used, this technique is classified as either endpoint-turbidimetric or kinetic-turbidimetric. The endpoint-turbidimetric technique is based on the quantitative relationship between the concentration of endotoxins and the turbidity (absorbance or transmission) of the reaction mixture at the end of an incubation period. The kinetic-turbidimetric technique is a method to measure either the onset time needed to reach a predetermined absorbance of the reaction mixture or the rate of turbidity development.
The chromogenic method measures the chromophore released from a suitable chromogenic peptide by the reaction of endotoxins with the LAL Reagent. Depending on the test principle employed, this technique is classified as either endpoint-chromogenic or kinetic-chromogenic. The endpoint-chromogenic technique is based on the quantitative relationship between the concentration of endotoxins and the release of chromophore at the end of an incubation period. The kinetic-chromogenic technique is a method to measure either the onset time needed to reach a predetermined absorbance of the reaction mixture or the rate of color development.
Preparatory Testing for the Photometric Techniques
To assure the precision or validity of the turbidimetric and chromogenic techniques, preparatory tests are conducted to verify that the criteria for the standard curve are valid and that the sample solution does not inhibit or enhance the reaction. Revalidation for the test method is required when conditions that are likely to influence the test result change.
Verification of Criteria for the Standard Curve
Using the Standard Endotoxin Solution, prepare at least three endotoxin concentrations to generate the standard curve. Perform the test using at least three replicates of each standard endotoxin concentration according to the manufacturer's instructions for the LAL Reagent (with regard to volume ratios, incubation time, temperature, pH, etc.). If the desired range in the kinetic methods is greater than two logs, additional standards should be included to bracket each log increase within the range of the standard curve. The absolute value of the correlation coefficient, |r|, must be greater than or equal to 0.980 for the range of endotoxin concentrations indicated by the manufacturer of the LAL Reagent.
Interfering Factors Test for the Photometric Techniques
Select an endotoxin concentration at or near the middle of the endotoxin standard curve. Prepare Solutions A, B, C, and D as shown in Table 4
. Perform the test on Solutions A, B, C, and D at least in duplicate following the instructions for the LAL Reagent used (with regard to volume of sample and LAL Reagent, volume ratio of sample to LAL Reagent, incubation time, etc.).
Table 4. Preparation of Solutions for the Inhibition/Enhancement Test for Photometric Techniques
||Solution to which Endotoxin is Added
||Number of Replicates
||not less than 2
||middle concentration of the standard curve
||not less than 2
||at least 3 concentrations (lowest concentration is
|LAL Reagent Water
||each not less than 2
||LAL Reagent Water
||not less than 2
Solution A: the sample solution may be diluted not to exceed MVD.
Solution B: the preparation under test at the same dilution as Solution A, containing added endotoxin at a concentration equal to or near the middle of the standard curve.
Solution C: the standard endotoxin at the concentrations used in the validation of the method described in Verification of Criteria for the Standard Curve under Preparatory Testing for the Photometric Techniques (positive control series).
Solution D: LAL Reagent Water (negative control).
Calculate the mean recovery of the added endotoxin by subtracting the mean endotoxin concentration in the solution (if any) from that containing the added endotoxin. In order to be considered free of interfering factors under the conditions of the test, the measured concentration of the endotoxin added to the sample solution must be within 50% to 200% of the known added endotoxin concentration after subtraction of any endotoxin detected in the solution without added endotoxin.
When the endotoxin recovery is out of the specified ranges, the interfering factors must be removed as described in the Interfering Factors Test for the Gel-Clot Techniques under Preparatory Testing for the Gel-Clot Techniques. Repeating the Interfering Factors Test for the Gel-Clot Techniques validates the treatment.
Calculation for the Photometric Techniques
Calculate the endotoxin concentration of each of the replicates of test Solution A using the standard curve generated by positive control series C. The test is not valid unless the following conditions are met: (1) the results of control series C comply with the requirements for validation defined under Verification of Criteria for the Standard Curve under Preparatory Testing for the Photometric Techniques; (2) the endotoxin recovery, calculated from the concentration found in Solution B after subtracting the endotoxin concentration found in Solution A is within 50 to 200%; and (3) the result of negative control series D does not exceed the limit of the blank value required in the description of the LAL Reagent used.
Interpretation of Results from the Photometric Techniques
In photometric assays, the preparation under test complies with the test if the mean endotoxin concentration of the replicates of Solution A, after correction for dilution and concentration, is less than the endotoxin limit for the product.