7. Quality control steps or test procedure

#### 7.1 Specimen collection and preparation

Collect aseptically containers that are free of detectable endotoxins in depyrogenated glassware apparatus.

#### 7.2 pH of the specimen

The pH must be 6–8. Adjust the pH of the product specimen with dilute HCl, NaOH, or buffer (free of endotoxins). Dilute concentrated HCl or NaOH with LRW. Use a volume that will not lead to significant dilution of the test specimen. Dilution (LRW) alone can overcome the issue sometimes.

#### 7.3 Method of lysate reconstitution

Gently tap the vial of lysate. Loose material fall to the bottom. Break the vacuum by lifting the gray stopper. Do not contaminate the mouth of the vial. Remove and discard the stopper. Start the reconstituted lysate with 3.2 ml buffer. Avoid vigorous mixing that may cause excessive foaming and a loss of sensitivity. Wrap the vials with parafilm and store in a cold place (2–8°C) when not in use and use within 8 h of reconstitution.

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

## 7.4 Lysate storage conditions

## 7.4.1 Lyophilized lysate

small chromogenic peptides and liberates pNA. pNA, color is yellow and absorbs light at 405 nm. For samples that absorb at 405–410 nm, Diazo-coupling agent modification may be used. In this method, pNA reacted with nitrite in hydrochloric acid, ammonium sulfamate and N-(1-naphthyl)-ethylenediamine (NEDA). Absorbs at a range between 540 and 550 nm. A standard curve is used to establish concen-

10 75 mm fully depyrogenated borosilicate glass culture tubes (Associates of

Optical reader is capable of reading at 405 nm, or at 540–550 nm for the diazo method. Incubator is able to maintaining 37 1°C. A water bath can be used for the endpoint test tube method. Both devices should have a uniform heat distribution. Test tube racks to hold the tubes and/or incubate dilution and reaction tubes. Micropipettes or disposable pipette tips free of interfering endotoxins and glucans are recommended. Vortex-type mixer, Para film (American National Can™) and hot-air oven with the capacity to heat to at least 250°C for depyrogenation of glassware.

Limulus amebocyte lysate (LAL), LAL reconstitution buffer, control standard endotoxins (CSE), solution 1 (nitrite), solution 1A (0.1 N hydrochloric acid), solution 2 (ammonium sulfamate), solution 3 (N-(1-naphthyl)-ethylenediamine

The endotoxins limit for USP/BP sterile WFI is only 0.25 EU/ml; therefore, sterile WFI may contain detectable endotoxins and be unsuitable for use. Use certified LRW to make dilutions of standards, and to prepare positive controls.

Collect aseptically containers that are free of detectable endotoxins in

The pH must be 6–8. Adjust the pH of the product specimen with dilute HCl, NaOH, or buffer (free of endotoxins). Dilute concentrated HCl or NaOH with LRW. Use a volume that will not lead to significant dilution of the test specimen.

Gently tap the vial of lysate. Loose material fall to the bottom. Break the vacuum by lifting the gray stopper. Do not contaminate the mouth of the vial. Remove and discard the stopper. Start the reconstituted lysate with 3.2 ml buffer. Avoid vigorous mixing that may cause excessive foaming and a loss of sensitivity. Wrap the vials with parafilm and store in a cold place (2–8°C) when not in use and use within 8 h

trations in product specimens.

Growing and Handling of Bacterial Cultures

5. Materials and equipment

6. Chemicals and reagents

7. Quality control steps or test procedure

Dilution (LRW) alone can overcome the issue sometimes.

7.1 Specimen collection and preparation

depyrogenated glassware apparatus.

7.3 Method of lysate reconstitution

7.2 pH of the specimen

of reconstitution.

96

(NEDA)), LRW.

Cape Cod, Inc. catalog numbers TB050).

This is relatively well stable and, if stored properly, will retain full activity through the expiration date on the label. Store the product at 2–8°C. Excess temperature over 37°C cause rapid deterioration, loss of sensitivity and distinct yellowing.

### 7.5 Control standard endotoxins (CSE)

Each vial of control standard endotoxins (CSE) contains 10 ng of endotoxins. Reconstitute CSE with the volume mentioned on the Certificate of Analysis (CA, which gives the potency of the CSE). Gently knocks the vial of control standard endotoxins (CSE) to cause loose material to fall to the bottom. Break the vacuum by lifting the gray stopper. Do not contaminate the mouth of the vial. Remove the stopper and place it in a cold place aseptically for reuse.

Reconstitute CSE with the volume specified on the Certificate of Analysis (CA, which gives the potency of the CSE) and as directed in the package insert. Place the stopper. Vortex the vial for 40–60 s to form a homogenous mixture. Discard solution if not used immediately, vortex the vial for 30 s prior to use.

#### 7.5.1 Mixing and incubation

Read the tubes UV/visible spectrophotometers (Table 1).


#### Table 1.

Dilution mixing and incubation time.

#### 7.5.2 Mixing and incubation

Stop the reaction by adding 50% acetic acid. Add 0.025 ml (25 μl) read the optical density (OD) at 405 nm read the test.


#### 7.6 Stop reaction solution preparation

#### 7.6.1 Read the test

Reconstitute vial 1 with entire contents of vial, reconstitute vial 2 with 4 ml of water, reconstitute vial 3 with 4 ml of water. Add 0.05 ml (50 μl) of solution 1

(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 the test at 540–550 nm.

7.6.7 Preparation of CSE dilutions

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

control standard endotoxins (CSE)

dard procedure for reconstitution.

For sample 1 and sample 2:

7.6.7.1 Mixing and incubation

Different dilution of CSE and lysate.

1. 0.5 EU/ml

2. 0.25 EU/ml

3. 0.125 EU/ml

4.0.0625 EU/ml

Stop reaction.

Table 2.

Table 3.

99

Using 10-fold and 2-fold dilution methods prepare the following dilutions of

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

Reconstitute the lysate with 3.2 ml of buffer provided with it. Follow the stan-

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

CSE + lysate Incubation (min)

 μl of 0.50 EU/ml + 50 μl 30 μl of 0.250 EU/ml + 50 μl 30 μl of 0.125 EU/ml + 50 μl 30 μl of 0.0625 EU/ml + 50 μl 30 μl of sample 1 + 50 μl 30 μl of sample 2 + 50 μl 30

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