2. Applications of the LAL test in the pharmaceutical industry

Among the most well-known and important applications of the LAL test are the ones related to the pharmaceutical industry. It can be said that the most common pyrogens in pharmaceutical products are endotoxins, which is why the pyrogen tests on rabbits have been replaced by the LAL test according to the recommendations of the international pharmacopeia. One of the reasons that has made the LAL test prevail in the pharmaceutical industry is the careful avoidance by the LAL manufacturers of bringing harm to live animals during both production and testing. It is important to clarify that the crabs, from which part of the hemolymph used for the LAL test was extracted, are returned to alive to their natural habitat with no lasting problems after the extraction.

#### 2.1 Limulus amebocyte lysate (LAL) test types

Gel Clot technique: based on gel formation [4].

### 3. Methods to determine the pyrogen in pharma products

Chromogenic method: based on the producing color after cleavage of a synthetic peptide-chromogen complex.

Turbidimetric method: based on forming turbidity after cleavage of an endogenous substrate.

End point method: 0.005 endotoxins units (EU) per ml.

Kinetic method: 0.001 endotoxins units (EU) per ml.

Kinetic method: time taken to reach a specific absorbance at 405 nm (onset time) is determined. The assay requires specialized instrumentation. Take optical density readings at regular intervals. The greatest sensitivity, λ, of lysate is 0.001 EU/ml.

Endpoint chromogenic method [5]: the released amount of pNA can be calculated after a fixed incubation period. A standard curve, consisting of measured optical density plotted against known standard endotoxin concentration. Later used to determine concentrations in the product. The greatest sensitivity, λ, is 0.005 EU/ml UV/visible spectrophotometers.

#### 4. Test performance

Add volume of lysate to a volume of product dilution. Incubating the reaction mixture at 37.5°C. Endotoxin in the reaction would activate the LAL reagent. Cleave 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 concentrations in product specimens.

7.4 Lysate storage conditions

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

7.5 Control standard endotoxins (CSE)

stopper and place it in a cold place aseptically for reuse.

tion if not used immediately, vortex the vial for 30 s prior to use.

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

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

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

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

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 solu-

CSE + lysate Incubation time (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

Stop the reaction by adding 50% acetic acid. Add 0.025 ml (25 μl) read the

Sample + lysate Incubation (min)

50 μl of sample + 50 μl 30

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

37°C cause rapid deterioration, loss of sensitivity and distinct yellowing.

7.4.1 Lyophilized lysate

7.5.1 Mixing and incubation

7.5.2 Mixing and incubation

Dilution mixing and incubation time.

Table 1.

7.6.1 Read the test

97

optical density (OD) at 405 nm read the test.

7.6 Stop reaction solution preparation

## 5. Materials and equipment

10 75 mm fully depyrogenated borosilicate glass culture tubes (Associates of Cape Cod, Inc. catalog numbers TB050).

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.

#### 6. Chemicals and reagents

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 (NEDA)), LRW.

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.
