PHOTOMETRIC QUANTITATIVE TECHNIQUES
This technique is a photometric assay measuring increases in reactant turbidity. On the basis of the particular assay principle employed, this technique may be classified as either an endpoint-turbidimetric assay or a kinetic-turbidimetric assay. The endpoint-turbidimetric assay 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 assay is a method to measure either the time (onset time) needed to reach a predetermined absorbance or transmission of the reaction mixture, or the rate of turbidity development. The test is carried out at the incubation temperature recommended by the lysate manufacturer (which is usually 37 ± 1).
This technique is an assay to measure the chromophore released from a suitable chromogenic peptide by the reaction of endotoxins with lysate. On the basis of the particular assay principle employed, this technique may be classified as either an endpoint-chromogenic assay or a kinetic-chromogenic assay. The endpoint-chromogenic assay 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 assay is a method to measure either the time (onset time) needed to reach a predetermined absorbance of the reaction mixture, or the rate of color development. The test is carried out at the incubation temperature recommended by the lysate manufacturer (which is usually 37 ± 1).
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 interfere with the test. Validation for the test method is required when conditions that are likely to influence the test result change.
Assurance of Criteria for the Standard Curve The test must be carried out for each lot of lysate reagent. Using the Standard Endotoxin Solution, prepare at least three endotoxin concentrations within the range indicated by the lysate manufacturer to generate the standard curve. Perform the assay using at least three replicates of each standard endotoxin concentration according to the manufacturer's instructions for the lysate (volume ratios, incubation time, temperature, pH, etc.). If the desired range is greater than two logs in the kinetic methods, additional standards should be included to bracket each log increase in 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 set up.
Test for Interfering Factors 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, according to the instructions for the lysate employed, for example, concerning volume of Sample Solution and Lysate TS, volume ratio of Sample Solution to Lysate TS, incubation time, etc.
Table 4. Preparation of Solutions for the Inhibition/Enhancement Test for Photometric Techniques
The test is considered valid when the following conditions are met.
1. The absolute value of the correlation coefficient of the standard curve generated using Solution C is greater than or equal to 0.980.
2. The result with Solution D does not exceed the limit of the blank value required in the description of the lysate reagent employed, or it is less than the endotoxin detection limit of the lysate reagent employed.
Calculate the mean recovery of the added endotoxin by subtracting the mean endotoxin concentration in the solution, if any (Solution A, Table 4), from that containing the added endotoxin (Solution B, Table 4). In order to be considered free of factors that interfere with the assay 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 range, the Sample Solution under test is considered to contain interfering factors. Then, repeat the test using a greater dilution, not exceeding the MVD. Furthermore, interference of the Sample Solution or diluted Sample Solution not to exceed the MVD may be eliminated by suitable validated treatment such as filtration, neutralization, dialysis, or heat treatment. To establish that the chosen treatment effectively eliminates interference without loss of endotoxins, perform the assay described above, using the preparation to be examined to which Standard Endotoxin has been added and which has then been submitted to the chosen treatment.
Follow the procedure described for Test for Interfering Factors under Preparatory Testing, immediately above.
Calculate the endotoxin concentration of each of the replicates of Solution A, using the standard curve generated by the positive control Solution C. The test is considered valid when the following three requirements are met.
1. The results of the control Solution C comply with the requirements for validation defined for Assurance of Criteria for the Standard Curve under Preparatory Testing.
2. The endotoxin recovery, calculated from the concentration found in Solution B after subtracting the concentration of endotoxin found in Solution A, is within the range of 50% to 200%.
3. The result of the negative control Solution D does not exceed the limit of the blank value required in the description of the lysate employed, or it is less than the endotoxin detection limit of the lysate reagent employed.
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.