Endotoxin Detection by End-Point Chromogenic Method

Preparation of test solutions

Unless otherwise prescribed, prepare the solutions to be employed in the test using water BET. If necessary, adjust the pH of the solution under examination to 6.0 to 8.0 using sterile 0.1M hydrochloric acid BET, 0.1M sodium hydroxide BET or a suitable buffer prepared with water BET.

Prepare the test solution at a suitable dilution

Prepare a reagent blank and not less than three dilutions of CSE in water BET to prepare a linear standard curve. Use water BET as a negative control and one positive control. The positive control consists of the test solution spiked with CSE to give an endotoxin concentration at the middle or below the middle point of the standard curve (PPC).

Method

Carry out the procedure described under Test for interfering factors. The chromogenic substrate and lysate are added to the solution and incubated for the recommended time. Stop the reaction and measure the absorbance at the wavelength specified by the lysate manufacturer.

Perform the linear regression analysis of the absorbance on the endotoxin concentration using standard statistical methods (method of least squares is usually suitable). Do not average the absorbance values of the replicates of each standard before performing the linear correlation regression analysis. Determine the endotoxin concentration of the test solution from the standard curve.
Related: Bacterial Endotoxin Test (BET or LAL Test) Validation

Interpretation of results

The assay is valid only if
(a) the standard curve is linear for the range of CSE concentrations used;
(b) the coefficient of correlation, r, is not less than 0.980;
(c) the mean percentage recovery of the added endotoxin in positive product control is between 50 percent and 150 percent.

The product under examination meets the requirements of the test if the mean endotoxin content of the replicates, after correction for dilution and concentration, is less than the endotoxin limit stated in the individual monograph.
Also see: Hold Time Study of Prepared Control Standard Endotoxin (CSE)