PHARMACOPOEIAL

METHODS OF ANALYSIS

Bacterial Endotoxin Test

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 BACTERIAL ENDOTOXIN TEST
(BET)
 The Bacterial Endotoxins Test (BET) is a test to detect or
quantify endotoxins from Gram-negative bacteria using
amoebocyte lysate from the horse shoe crab(Limulus
polyphemus or Tachypleus tridentatus).
 The quantities of endotoxins are expressed in defined
Endotoxin Units (EU).
 With the adoption of the second International Standard for
endotoxin by the Expert Committee on Biological Standards of
the World Health Organization, 1 EU = 1 IU.

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 IMPORTANCE OF BET
 Essential test for quality control of pharmaceutical
product
 Monitors product contamination during handling and
processing
 Complies the product standard as GMP , WHO , and
ISO regulation

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 METHODS AND BASIC PRINCIPLES

Gel Clot • Gel formation

Technique

Turbidometric • Development of turbidity after

Technique cleavage of an endogenous substrate

• Development of colour after cleavage

Chromogenic of synthetic peptide-chromogen
Technique complex

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 METHODS
USP IP BP
• Gel clot • Gel clot limit test • Gel clot limit
technique method test method
• Turbidometric • Semi quantitatie • Semi quantitatie
technique gel clot method gel clot method
• Kinetic
• Chromogenic • Kinetic
turbidometric
technique turbidometric • Kinetic
• Kinetic chromogenic
chromogenic • End point
• End point turbidometric
chromogenic • End point
chromogenic

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 Apparatus And Glasswares
• Depyrogenate all glassware and other heat-stable materials in
a hot air oven using a validated process.
• A commonly used minimum time and temperature is 30 min at
250°.
• If employing plastic apparatus, such as microplates and pipet
tips for automatic pipetters, use apparatus that is shown to be
free of detectable endotoxin and does not interfere in the test.

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 REAGENTS AND TEST SOLUTIONS
• Amoebocyte Lysate—A lyophilized product obtained from
the lysate of amoebocytes (white blood cells) from the
horseshoe crab (Limulus polyphemus or Tachypleus
Solutridentatus).
• Water for Bacterial Endotoxins Test (BET)—Water for
Injection or water produced by other procedures that shows
no reaction with the lysate employed, at the detection limit of
the reagent
• Lysate TS—Dissolved Amoebocyte Lysate in Water for BET,
or in a buffer by gentle stirring.

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 IMPORTANT TERMS
Maximum The maximum valid dilution is the maximum allowable
Valid dilution of a specimen at which the endotoxin limit can be
Dilution determined.
(MVD) MVD= Endotoxin limit × conc. of Sample Soln / λ

The endotoxin limit for parenteral drugs, defined on the

Endotoxin basis of dose, equals K/M2 ,where K is a threshold
pyrogenic dose of endotoxin per kg of bodyweight, and M
Limit
is equal to the maximum recommended bolus dose of
product per kg of body weight.

The labeled sensitivity in the gel-clot technique (EU/ml)

λ or the lowest concentration used in the standard
curve for the turbidimetric technique or chromogenic
technique.

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 Methods as per USP
1) Gel clot technique
 Based on the clotting of Lysate reagent in presence of
endotoxin
 Minimum concentration of Lysate reagent required for
formation of clot is lysate reagent sensitivity

PREPARATORY TESTING (as per IP,BP,USP)

 To ensure validity and accuracy of the test
 For confirming labeled lysate sensitivity and interfering
factors

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 Test for Confirmation of Labeled Lysate
Sensitivity
• Carried out when a new batch of lysate is used or when there is any
change in the test conditions that may affect the outcome of the test.

Prepare standard solutions having at least four concentrations

equivalent to 2λ, 1λ, 0.5λ, and 0.25λ by diluting the USP Endotoxin
RS with Water for BET.

Mix a volume of the Lysate TS with an equal volume of one of the

Standard Endotoxin Solutions

Incubate the reaction mixture usually at 37 ± 1° for 60 ± 2 min

Observe results,if gel is formed then consider it as positive,or else

negative.

The endpoint is the smallest concentration in the series of

decreasing concentrations of standard endotoxin that clots the10
lysate.
 Calculation
• Calculate the average of the logarithms of the lowest
concentration of endotoxin in each series of dilutions for which
a positive result is found.
• The geometric mean end-point concentration is the measured
sensitivity of the lysate in EU/ml, which is calculated using the
following expression:
Geometric mean end-point concentration = antilog (Ʃe/f)
Where,
Ʃe = sum of the log end-point concentrations of the series of
dilutions used;
f = number of replicate test-tubes.
• Lysate sensitivity must lie between 0.5λand 2λ.

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 Test for Interfering Factors
•For validation of the test results it must be demonstrated that the test
preparation does not inhibit or enhance the reaction or otherwise interfere
with the test.  

Solution A • Solution of the sample at a dilution at or below

MVD (test solution)

Solution B • Test solution spiked with indicated CSE

concentrations (Positive Pdt Control; PPC)

Solution C • Standard solution with indicated CSE

concentrations in water BET

Solution D • Water BET (Negative Control; NC)

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 Method
Into each tube, add an appropriate volume of negative control
(NC), standard CSE solutions in water BET, test solution and
positive product control (PPC).

Mix the sample-lysate mixture gently and place in an incubating

device

Incubate each tube at 37º ± 1º undisturbed for 60 ± 2 minutes.

Observe results

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 Quantitative Test
• The test quantifies bacterial endotoxins in Sample Solutions by
titration to an endpoint.
• Prepare solutions A, B, C, and D as shown in previous table.
• Test these solutions by following the procedure in Preparatory
Testing, Test for Confirmation of Labeled Lysate Sensitivity.

Interpretation:
The test is considered valid when the following three
conditions are met
1.Both replicates of negative control Solution D are negative
2.Both replicates of positive product control Solution B are positive
3. The geometric mean endpoint concentration of Solution C is in
the range of 0.5λto 2λ.

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 Photometric Quantitative Techniques
Turbidimetric Technique
• This technique is a photometric assay measuring increases in
reactant turbidity.
Quantitative relationship
Endpoint- between the concentration of
turbidimetric endotoxins and the turbidity of
Assay the reaction mixture at the end
of an incubation period
Turbidimetric
Assay
Measures either the time
Kinetic- needed to reach a prede-
turbidimetric termined absorbance or
Assay transmission of the reaction
mixture, or the rate of turbidity
development.

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 Chromogenic Technique
• Technique is an assay to measure the chromophore released
from a suitable chromogenic peptide by the reaction of
endotoxins with lysate.

Based on the quantitative

Endpoint- relationship between the concen-
chromogenic tration of endotoxins and the
Assay release of chromophore at the end
of an incubation period.
Chromogeni
c Assay
Kinetic- Method to measure either the time
chromogeni needed to reach a predetermined
c assay absorbance of the reaction mixture,
or the rate of color development.

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 METHODS AS PER IP AND BP

• Gel-Clot Methods
• Methods A and B depend on the formation of a firm gel when a
solution containing bacterial endotoxins is incubated after mixing
with the lysate.
• Sensitivity of the lysate and Test for interfering factors are
performed as per USP.

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 Method A. Gel-Clot Limit Test Method:
• Prepare the solutions and dilutions with water BET.
• If necessary, adjust the pH of the solution under examination to 6.0 to 8.0
using sterile 0.1M hydrochloric acid BET, 0.1M sodium hydroxide BET.
• Prepare the sample solution at any dilution at or below MVD(Maximum Valid
Dilution).
• Use water BET as negative control (NC) and two positive controls.

• One of the positive controls consists of the CSE(Control Standard

Endotoxin) at a concentration of 2λ and the other consists of the test solution
spiked with CSE to give a concentration of 2λ (Positive Product Control)

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 • Solution of the sample at a dilution at or
Solution A below MVD (test solution)
• Test solution spiked with indicated CSE
Solution B concentrations (Positive Pdt Control; PPC)
• Standard solution with indicated CSE
Solution C concentrations in water BET

Solution D • Water BET (Negative Control; NC)

• Method:
• Into each tube, add an appropriate volume of solution D (NC), standard
CSE solutions in water BET (solution C), test solution (solution A) and
solution B (PPC).
• Add the lysate and carry out the assay in accordance with the instructions
given by the lysate manufacturer.

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 Interpretation

• The product under examination complies with the bacterial

endotoxin test if the positive product control is positive and the
negative control as well as the test solutions are negative.
• The test is not valid if the positive product control is negative or if
the negative control is positive.
• Retests : If a positive result is found for one of the test solution
duplicates and a negative result for the other, the test may be
repeated as described above.
• The results of the retest should be interpreted as for the initial test.
• If the results are still inconclusive, the sample fails the test.

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 Method B. Semi-Quantitative Gel-Clot Method
Preparation of test solutions
• Prepare test solutions at concentrations of MVD, 0.5 MVD, 0.25
MVD or any other appropriate dilutions relative to the dilution at
which the test for interfering factors was completed.
• Additionally, prepare a similar series of test solutions spiked with
2λ of CSE each (PPC).
Method. Carry out the procedure on the test solutions in duplicate as
described under Test for interfering factors.
Calculation and interpretation of results :To calculate the
endotoxin concentration in the product, determine for the series of
test solutions the lowest concentration or the highest dilution giving
a positive (+) reaction.
• Multiply this dilution factor with λ to obtain the endotoxin
concentration of the product.

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 CONT…
• For instance, if MVD is equal to 8 and the positive reaction
was obtained at 0.25 MVD and λ was equal to 0.125 EU/ml,
the endotoxin concentration in the test solution will be 8 ×
0.25 × 0.125 = 0.25 EU/ml.
• If none of the dilutions of the series gives a positive reaction,
the endotoxin concentration will be less than the value
obtained by multiplying the lowest dilution factor with λ Or If
all the dilutions of the series give a positive reaction, the
endotoxin concentration will be more than the value obtained
by multiplying the highest dilution factor with λ.
Calculate the endotoxin content of the product under
examination from the endotoxin concentration.

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 Quantitative Methods
The quantitative methods include
 Kinetic Turbidimetric Method (Method C),
 Kinetic Chromogenic Method (Method D) and
 End-Point Chromogenic Method (Method E)
• These methods make use of an appropriate regression
analysis of the log response with the log endotoxin
concentration.

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 • Photometric assay measuring the increase
Kinetic Turbidimetric in turbidity caused by the reaction of the
Method endotoxin with the lysate.

• Method measuring either the time (onset

Kinetic Turbidimetric time) needed to reach a predetermined
Assay absorbance of the reaction mixture or the
rate of turbidity development

• Photometric assay measuring the colour

Kinetic Chromogenic developed by the chromophore released
Method from a chromogenic substrate by the
reaction of the endotoxin with the lysate.

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 • Method measuring either the time (onset
Kinetic Chromogenic time) needed to reach a predetermined
Assay absorbance of the reaction mixture or the
rate of colour development.

• Photometric assay measuring the colour

End-point developed by the chromophore released
chromogenic from a chromogenic substrate by the
method reaction of the endotoxin with the lysate.

• Method measuring the colour intensity at

the end of an incubation period after the
End-point Assay reaction is stopped by the addition of a
suitable acid.

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 Cont…
• Method C, Method D ,Method E have same methods for praparation of
solutions and analysis BUT only for method E procedure is different.
• METHOD E
• Carry out the procedure described under Test for interfering factors.
• The chromogenic substrate and lysate are added to the solution and
incubated for the recommended time.
• Stop the reaction and measure the absorbance at the wavelength
specified by the lysate manufacturer.
• Perform the linear regression analysis of the absorbance on the
endotoxin concentration using standard statistical methods (method of
least squares is usually suitable).
• Do not average the absorbance values of the replicates of each
standard before performing the linear correlation regression analysis.
• Determine the endotoxin concentration of the test solution from the
standard curve.

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 REFERENCES

• INDIAN PHARMACOPOEIA, VOLUME 1

• UNITED STATES PHARMACOPOEIA , VOLUME1

• BRITISH PHARMACOPOEIA, VOLUME 4

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