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This article explains about LAL test method /procedure used to test bacterial contamination of pharmaceutical products especially parenteral preparations.


LAL test aim is to measure quantitatively the amount of bacterial endotoxins in a given sample of parenteral.


The solution of endotoxins containing preparation is added to the lysate derived from heamolymph cells of horseshoe crab (limulus polymhemus). The result of the reaction is turbidity or precipitation or gelation of the mixture. This is used as a quantitative measure to estimate the endotoxin content. The rate of reaction depends upon conc. of endotoxins, pH, temperature and presence of clotting enzyme system and clottable proteins from lysate.
The quantities of endotoxins are expressed in defined Endotoxin Units (EU). Also 1 EU is equal to 1 IU.
The endotoxin limit for a given test preparation is calculated from the expression K/M; where M is maximum dose administered to adult per kg per hour. The value for K is 5.0 EU/Kg for pareneteral preparations, and it is 0.2 EU/Kg for intrathecal preparations.


1. Endotoxin reference standard (ERS): It is the freeze dried purified endotoxin of Escherichia coli, which is calibrated in Endotoxin units (EU) by comparison with the International standard.
2. Control Standard Endotoxin (CSE):CSE is suitably standardized against the ERS which is used for routine bacterial endotoxin testing.
3. Water BET: Water that gives a negative result under the conditions prescribed in the test for bacterial endotoxins on the preparation under examination.
4. 0.1 M HCl BET: Prepared from hydrochloric acid using BET with pH adjusted to 6.0- 8.0 with o.1 M sodium hydroxide BET. It also gives a negative result under the conditions of the test.
5. 0.1 M NaOH BET:Prepared from sodium hydroxide using water BET with pH adjusted to 6.0- 8.0 with 0.1 M hydrochloric acid BET. It gives a negative result under conditions of the test.
6. Lysate: Lysate is the reconstituted lysate of amoebocytes of either of the species of horseshoe crabs; Limulus polyphemus or Tachypleus tridentatus.


Both method A and B depend upon formation of firm gel formed from reaction of bacterial endotoxin with Lysate on incubation. Method A is a limit test wherein both the replicate solutions of preparation under examination must contain endotoxin in the concentration less than the endotoxin limit concentration specified in monograph. Method B determines the endotoxin concentration semi quantitatively in the preparation under examination.
Sensitivity of Lysate:
Sensitivity of the lysate is determined for every batch of lysate used. One vial from the batch of lysate must be used to evaluate the sensitivity. For the test, prepare a series of dilutions of CSE as 2l, l, 0.5l, 0.25 l; where l is the labeled sensitivity in EU per ml. Perform the test as per method in duplicate. Include a negative control using water BET. At least one dilution in each series must give a negative result. Calculate the average of the logarithm of the lowest concentration of endotoxin in each series of the dilutions for which positive results is found. The geometric mean end point concentration is measured sensitivity of the lysate in EU per ml which is calculated as:
Geometric mean end point concentration = antilog (?e/f)
Method A:
Prepare a series of test solutions with water BET. Adjust the pH to 6.0-8.0 using sterile 0.1 M HCl BET and 0.1 M NaOH BET.
Water BET is used as negative control and two positive controls are to be used.
One positive control is CSE at 2 l concentration.
Second positive control is test solution spiked with CSE to give 2 l concentrations (PPC)
Method: Procedure is same as under test for interfering factors. Perform in duplicate.
Interpretation of results: The given sample complies with endotoxin test if the positive product control gives positive result and the negative as well as the test solution gives negative result.
The test is not valid if the product positive control is negative or the negative control is positive or both of these conditions occur.
Retest: If the positive test is found for one of the test in the duplicate, the test is repeated as above.
Method B:
Prepare a series of test solutions in the concentrations of MVD, 0.5 MVD, and 0.25 MVD. Additionally prepare series of test solutions spiked with 2 l CSE each (PPC)
Method: Procedure is same as under test for interfering factors. Perform in duplicate.
Calculation and interpretation of result: For the series of test solutions, determine the lowest concentration or the highest dilution giving positive reaction. Multiply this factor with lambda to obtain endotoxin concentration of the product.
E.g.: If the MVD = 8 and positive reaction is obtained at 0.25 MVD and l = 0.125 EU per ml,
Endotoxin concentration = 8x0.25x0.125
= 0.25 EU per ml.
The product meets the requirements of the test if the endotoxin concentration is less than the limit prescribed in the given monograph.

What are the advantages of LAL Test over Rabbits Test ?

1. LAL test is used to quantify the endotoxin count where as rabbit test is done to detect the presence of pyrogens.
2. Less time consuming (within 1 hr)
3. Simple and Economic.


Indian Pharmacopeia, 2007, Volume 1, Published by The Indian Pharmacopeia Commission, Ghaziabad; Tests for pyrogens 2.2.3

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About the Author

Santosh kumar. JH's picture

Key Skills:-

Lean Methodology

Six Sigma Expertise

Formulation Operations


Prof. J. Vijaya Ratna's picture

Dear Santosh Bring out the advantages of LAL test over the Rabbits test. Can you add an image that can increase the impact of the write up? Vijaya Ratna
Santosh kumar. JH's picture

I had added the advantages of LAL test over Rabbits test.... I will try to make a image of my own and upload it as soon as possible.
Prof. J. Vijaya Ratna's picture

Dear Santosh In doing the LAL test, we are giving less suffering to the animals. WE take the crab, collect some blood from it and then leave it again in its water. In the rabbits test, we have to maintain the rabbits in a certain way, then give them injections and then record rectal temperatures for a certain time in a certain way and so on. It is a lot of trouble to the animals. So from ethical point of view also LAL test is better. Please read up on these points and add to advantages. Vijaya Ratna
Mrudula Boyapati's picture

hii sir... I heard this test while listening to test for pyrogens and also that rabbits cannot be used frequently and a wash out period is required and that too as it is involving animals, there are many protection acts and restrictions to be followed but inorder to obtain the reagent do we need to destroy the organism...? are there any other tests to obtain the quantitative analysis of endotoxins..? regards MRUDULA
Santosh kumar. JH's picture

Dear Mrudula, Rabit test is done to detect the presence of Pyrogens. Although their are concerns of ethics of using animals, In rabbit test we are not killing rabbits we are just detecting their body temperature rise so it is not a serious issue. Similarly in the preparation of LAL reagent we are just extracting the blood from horse shoe crab without killing them and then centrifuging it to collect the cell mass, these cells are then placed in distilled water which makes the cells swell and burst, releasing the chemicals from the cell which is then purified and finally freeze died. Well, the other methods for quantifying endotoxin content beside gel cloth technique are: Kinetic Turbidimetric analysis, Kinetic Chromogenic analysis, End point Chromogenic analysis, All these tests also uses LAL reagent.....
Anonymous's picture

Hi Santosh.....

The material really is helpful, but I have my doubts regarding the efficiency of LAL. I have refered to other articles on LAL where they have stated that the method is capable of detecting and measuring free endotoxins only and fails at detecting endotoxins bound to organic and inorganic substrates. Besides that, the technique is incapable of detecting pyrogens other than endotoxins. So could you please brief us on any alternate available techniques, or has there been any advancements that cover those limitations.


Ph. Bhagavan P S RPh's picture

Late 1990s. It was a big relief to hear that an alternate at last has been found out for Pyrogen test and rabbits could be spared. But we couldnt immediately shift over due to procedural issues in Government and subsequently the strict enforcement of schedule M updated consequent to NHRC direction, all the IV fluid mfg units under Hospital pharmacy had to be closed. Well, having experienced the difficulty in maintaining  the rabit columns I feel In-vitro method is a boon and least messy. LAL test is unquestionably advantages over the Rabit test and any issues with respect to reliability, extension of reads on the parameter, short comings if any etc should be addressed to drive the issue forward. Good blog. thank you

Bhagavan P.S. B Pharm

Rtd. Dy Dir.(Pharmacy),Govt of Karnataka, India

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