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These methods make use of a linear regression of the log response with the log endotoxin concentration. To ascertain the precision or validity of the turbidimetric and chromogenic methods, preparatory tests are conducted to see that the criteria for the standard curve are valid and that the test solution does not interfere with (inhibit or enhance) the reaction.

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Using CSE, prepare solutions of not less than three endotoxin concentrations to obtain a linear standard curve. Carry out the procedure using at least two replicates of each standard endotoxin solution in accordance with the instructions of the lysate manufacturer (volume ratios, incubation times, temperature, pH, etc.).
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For validation of the test results, it must be demonstrated that the test preparation does not inhibit or enhance the test or otherwise interfere with the test. The validation must be repeated if the lysate vendor or the method of manufacture or formulation of the sample is changed. The initial dilution may be prepared using the following expression:

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Carry out the test in duplicate receptacles such as wells of a micro-titre plate. Into each chosen receptacle, add an appropriate volume of solution D (NC), standard CSE solutions in water BET (solution C), test solution (solution A) and solution B (PPC). Add the lysate and carry out the assay in accordance with the instructions given by the lysate manufacturer.
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Calculate the endotoxin concentration of solutions A and B from the regression equation obtained with solutions of series C. Calculate the mean percentage recovery of the added endotoxin by subtracting the mean endotoxin concentration in solution A from the mean endotoxin concentration in solution B.

The kinetic turbidimetric method is a photometric assay measuring the increase in turbidity caused by the reaction of the endotoxin with the lysate. The kinetic turbidimetric assay is a method measuring either the time (onset time) needed to reach a predetermined absorbance of the reaction mixture or the rate of turbidity development.

The kinetic chromogenic method is a photometric assay measuring the colour developed by the chromophore released from a chromogenic substrate by the reaction of the endotoxin with the lysate. The kinetic chromogenic assay is a method measuring either the time (onset time) needed to reach a predetermined absorbance of the reaction mixture or the rate of colour development.

The end-point chromogenic method is a photometric assay measuring the colour developed by the chromophore released from a chromogenic substrate by the reaction of the endotoxin with the lysate. The end-point assay is a method measuring the colour intensity at the end of an incubation period after the reaction is stopped by the addition of a suitable acid.

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**Preparation of the standard curve**

Using CSE, prepare solutions of not less than three endotoxin concentrations to obtain a linear standard curve. Carry out the procedure using at least two replicates of each standard endotoxin solution in accordance with the instructions of the lysate manufacturer (volume ratios, incubation times, temperature, pH, etc.).
The regression line must have a linearity with the coefficient of correlation, r, being not greater than -0.980 for the range of endotoxin concentrations.

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**Test for interfering factors**

For validation of the test results, it must be demonstrated that the test preparation does not inhibit or enhance the test or otherwise interfere with the test. The validation must be repeated if the lysate vendor or the method of manufacture or formulation of the sample is changed. The initial dilution may be prepared using the following expression:
Initial dilution =

__Endotoxin limit of the test solution__
C

C is the lowest CSE concentration of the standard curve expressed in EU/ml

Preparation of test solutions. Prepare solutions A to D as given below.

Solution A = Solution of the product under examination at the initial dilution (test solution).

Solution B = Test solution spiked with CSE at a concentration at or near the middle of the standard curve (PPC).

Solution C = Standard solutions of CSE in water BET covering the linear part of the standard curve.

Solution D = Water BET (NC).

The pH of the solutions must be in the range specified by the manufacturer of the lysate, usually between 6.0 and 8.0. Adjust the pH, if necessary, by addition of sterile 0.1 M hydrochloric acid BET, 0.1 M sodium hydroxide BET or a suitable buffer prepared with water BET.

Related: Bacterial Endotoxin Test (BET or LAL Test) Validation

Related: Bacterial Endotoxin Test (BET or LAL Test) Validation

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**Method:**

Carry out the test in duplicate receptacles such as wells of a micro-titre plate. Into each chosen receptacle, add an appropriate volume of solution D (NC), standard CSE solutions in water BET (solution C), test solution (solution A) and solution B (PPC). Add the lysate and carry out the assay in accordance with the instructions given by the lysate manufacturer.##
**Calculation and interpretation of results:**

Calculate the endotoxin concentration of solutions A and B from the regression equation obtained with solutions of series C. Calculate the mean percentage recovery of the added endotoxin by subtracting the mean endotoxin concentration in solution A from the mean endotoxin concentration in solution B.
The test for interfering factors is valid only if

(a) the negative control (solution D) does not yield a value higher than the limit for the value required in the description of the lysate employed;

(b) the CSE solutions of series C comply with the requirements given under Preparation of the standard curve;

(c) the mean percentage recovery of added endotoxin in solution B is between 50 per cent and 150 per cent.

If the mean percentage recovery is beyond the specified range, the interfering factors must be removed by the procedure described under the Gel-Clot Method.

Also see:

Also see:

**Method A. Kinetic Turbidimetric Method****Method B. Kinetic Chromogenic Method****Method C. End-Point Chromogenic Method**
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