8.3 Sample 2

X = Y + 0.026/1.019 Y = 0.335, X = 0.335 + 0.026/1.019, X = 0.354 EU/ml

Figure 3. Validation of standard curve.

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

## 8.4 Advantages of Gel Clot method

Gel Clot LAL provides a simple positive/negative result and is most often mentioned in pharmacopeial monographs as the official referee test.

This is very easy to perform.

This is not time consuming.

Accuracy is 100 percent.

The LAL Gel-Clot assay, gives a more quantitative measurement of endotoxin over a range of concentrations.

#### 8.5 Standard operating procedure

#### 8.5.1 Material

8. Results

R ≥ 0.98 R2 = 0.99 R = √R2 = 0.99

y = mx + c m = slop

c = y intercept y = mean absorbance Equation of straight line Y = 1.019X 0.026

and endotoxin concentration (Figure 3). R2 = coefficient of determination R = correlation coefficient

Growing and Handling of Bacterial Cultures

Equation of straight line

8.1 Rearranging the equation

X = Y + 0.026/1.019 m = slop = 1.019, C = y intercept = 0.026, Y = mean absorbance, X = endotoxin concentration

X = Y + 0.026/1.019

X = Y + 0.026/1.019

Y = 0.300, X = 0.300 + 0.026/1.019, X = 0.319EU/ml

Y = 0.335, X = 0.335 + 0.026/1.019, X = 0.354 EU/ml

8.2 Sample 1

8.3 Sample 2

Figure 3.

100

Validation of standard curve.

x = endotoxin concentration

Use Microsoft word for further calculations and results. Make standard curve

Gel Clot lysate for 20 test, Gel Clot standard 0.5 EU/Vial, LAL reagent water (LRW 50 ml).

#### 8.5.2 Reconstitution

Lysate: add 2 mL LRW and mix it slowly. Do not shake and avoid foaming. Transfer 0.1 ml in 20 test tubes. Store it at –degree (in freezer).

Standard: Add 2 mL of LRW in the vial and mix it well for 15 min. Store the vial at 2–8°C. Storage life is 15 days.

#### 8.5.3 Procedure

Take three test tubes and mark them as test, positive control and negative control [1].

Add your sample in test tube marked as sample. Add standard in test tube marked as Positive control. Add LRW in test tube marked as negative control. Incubate the test tubes at 37 + 2°C for 60 min. After an incubation, check for the gel by inverting the test tube. If the material remains firm in the bottom of the test tube, it means gel has formed. This positive if the material gets the flow down, it means gel has not formed. This means negative.

#### 8.5.4 For water for injection

Take similarly three test tubes as above and add water for injection (WFI) in test tube marked as sample. And proceed as above. The results should be as follows (Table 4):

#### 8.5.5 For product

We have to make dilution.


Table 4. Results shown sample pass or not.

Example: If the product endotoxin limit is 1 EU/ml, then we have to make the dilution as follows:

water into each of bottle no. 9 s and 10s. Ultimately, we will have three bottles 8, 9,

What Is Limulus Amebocyte Lysate (LAL) and Its Applicability in Endotoxin Quantification…

The pH of the sample is adjusted by pyrogen free 0.1 N NaOH or 0.1 N HCl. The

Arrange test tubes in two stands as under; stand 1—test tubes for sample and

Take 0.05 ml well-mixed sample into small test tubes. If required, make 1/10 dilution of the sample with Pyrogen free water as Below, Take 4.5 ml of pyrogen free water in the test tube. Then add 0.5 ml of well-mixed sample. Vortex mixing

Make a dilution of the endotoxin (concentration 0.470 EU/ml) according to the product limit. For making 0.235 EU/ml (if the product limit is 0.25) proceed as

Pour 0.05 ml of pyrogen free water (being used in the test) in small test tube as a

Place the tube stand for small test tubes (containing the tubes of blank, standard and diluted samples) in ice water bath or suitable ice water container. Add 0.05 ml of lysate to all of the tubes as soon as possible. Stir the contents of every tube soon

Soon after the addition of lysate, place the test tube rack in the incubator set at 32.5 + 2.5°C for 30 min. The tube rack can be placed in the water container placed in

Take 0.05 ml of the reconstituted endotoxin in the test tube after stirring. Add 0.05 ml of pyrogen free water and vortex to mix. Now the final dilution is

Take 0.05 ml of step 2 into a small test tube for further process.

after the addition of lysate for a few seconds. Avoid foaming.

and 10 s, which are used stepwise to block the reaction.

DOI: http://dx.doi.org/10.5772/intechopen.81331

pH of the sample should be between 6.0 and 8.0.

standard dilutions; stand 2—test tubes for reaction.

Take 0.1 ml into a small test tube for further process.

9. Pre-test preparations

9.1 pH of the sample

9.2 Test tubes settings

10.1 Sample preparations

10.2 Standard preparation

10. Procedure

for a few seconds.

follows;

0.235 EU/ml.

10.3 Blank preparation

blank for further process. Lysate addition

10.4 Incubation

the incubator.

103

Since we are using 0.25 EU/ml, this is called lambda. Divide the endotoxin limit of product with lambda

1/0.25 = 1:4

As per USP, we have to test 3 test as follows:


This means the product is passed.

#### 8.5.6 LAL test reagents (chromogenic method)

Chromogenic lysate [2], Respective endotoxin standard, Diazo coupling reagent (set of four bottles). Note: All reagents must be stored in refrigerator at 2–8°C.

#### 8.5.7 Preparation of acetic acid 0.8 m

Dissolve 45.6 ml of acetic acid in 1 liter of distilled water. The final concentration of acetic acid is 0.8 M. This solution can be stored for 3 months.

Remove the plastic cover. Wipe off with 70% alcohol around the rubber cap and top portion of every vial. Remove the aluminum cap with sterile and pyrogen free forceps and then cover with depyrogenated aluminum foil to avoid any Endotoxin contamination. (2.8 ml LAL water vial is provided with Endotoxin vial, concentration is mentioned on the label). Pour whole quantity of LAL water into the ET vial and cover with foil. Mix vigorously for at least 10 s by vortexer. During stirring solution must not touch the foil.

Storage: Store reconstituted Endotoxins solution at 4°C in a refrigerator for 14 days. The solution can also be stored at –20°C for a month. Avoid freezing during storage.

Note: Stir every time vigorously before use.

Toxicolor lysate

(Buffer vial 0.35 ml and LAL water are provided with Lysate. Sensitivity is mentioned on the certificate). After taking from the refrigerator, pour whole quantity of buffer and 0.35 ml LAL water into the lysate vial as soon as possible, covers with foil. Then quickly stir to dissolve. Avoid air bubbling during stirring. Place the vial in ice water bath for 2–3 min before use.

Note: Be sure that the reagent is completely dissolved. This reagent must be reconstituted just before use. The reagent is extremely sensitive and must be consumed at one time. Storage should be avoided, but can be stored at 20°C in 0.1 ml dispensed quantities in small test tubes. Use stored lysate if the color is not changed. Reconstituted lysate may only be deep frozen once.

#### 8.5.8 Diazo coupling reagent

Four bottles are provided with one set, marked as 7, 8, 9 and 10s respectively. Transfer whole quantity of bottle no. 7 s into bottle no. 8 s. Then add 12 ml distilled What Is Limulus Amebocyte Lysate (LAL) and Its Applicability in Endotoxin Quantification… DOI: http://dx.doi.org/10.5772/intechopen.81331

water into each of bottle no. 9 s and 10s. Ultimately, we will have three bottles 8, 9, and 10 s, which are used stepwise to block the reaction.
