7.6.6 Minimum valid concentration (MVC)

MVC = λ/endotoxin limit

λ: the labeled lysate sensitivity in the gel-clot technique (IU/ml) or the lowest concentration used in the standard curve for the turbidimetric or chromogenic techniques.

Sample 1 Endotoxin limit: 0.5 EU/ml Concentration of sample: 100 mg/ml λ: 0.06 EU/ml MVD = 0.5 EU/ml 100 mg/ml/0.06 EU/ml MVD = 833

Add 1 ml of sample 1 in to 832 ml of LRW. Prepare sample 2 in using the same method.

What Is Limulus Amebocyte Lysate (LAL) and Its Applicability in Endotoxin Quantification… DOI: http://dx.doi.org/10.5772/intechopen.81331

7.6.7 Preparation of CSE dilutions

Using 10-fold and 2-fold dilution methods prepare the following dilutions of control standard endotoxins (CSE)

1. 0.5 EU/ml

(sodium nitrite reconstituted with dilute HCL). Add 0.05 ml (50 μl) of solution 2 (ammonium sulfamate). Add 0.05 ml (50 μl) of solution 3 (NEDA) use new pipette tip agitate the plate to mix. Full color (magenta) should develop immediately. Read

Positive control must be included to verify that it is appropriate to use the parameters of a previous (archived) standard curve to calculate endotoxin

Prepare sample solutions by dissolving or diluting drugs (pH 6.0–8.0). The pH may be adjusted by the use of acid, base, or suitable buffers as recommended. Do not exceed the MVD or MCV while making dilutions and adjusting the pH.

MVD = (endotoxin limit concentration of sample solution)/(λ)

Endotoxin limit given in USP, concentration of a sample of the label, λ: the labeled lysate sensitivity in the gel-clot technique (IU/ml) or the lowest concentration used in the standard curve for the turbidimetric or chromogenic techniques.

λ: the labeled lysate sensitivity in the gel-clot technique (IU/ml) or the lowest concentration used in the standard curve for the turbidimetric or chromogenic

Add 1 ml of sample 1 in to 832 ml of LRW. Prepare sample 2 in using the same

LRW negative controls should be included with each test

1: Equation of straight line (results)

x = endotoxin concentration,

7.6.5 Maximum valid dilution (MVD)

7.6.6 Minimum valid concentration (MVC)

Concentration of sample: 100 mg/ml

MVD = 0.5 EU/ml 100 mg/ml/0.06 EU/ml

MVC = λ/endotoxin limit

Endotoxin limit: 0.5 EU/ml

techniques. Sample 1

method.

98

λ: 0.06 EU/ml

MVD = 833

c = y-intercept and y = mean absorbance

Example calculation

7.6.4 Sample preparation

the test at 540–550 nm.

Growing and Handling of Bacterial Cultures

7.6.2.1 Make standard curve

7.6.2 Positive control

concentrations.

7.6.3 Negative controls

y = mx + c m = slop

X = y-c/m


Reconstitute the lysate with 3.2 ml of buffer provided with it. Follow the standard procedure for reconstitution.

7.6.7.1 Mixing and incubation

Stop reaction.

For sample 1 and sample 2:

Stop the reaction by adding 50% acetic acid. Add 0.025 ml (25 μl) (Tables 2 and 3).


#### Table 2.

Different dilution of CSE and lysate.


#### Table 3.

Make two replicates of each CSE and sample preparation to reduce any errors.
