The bacterial endotoxin test (BET) uses Limulus amebocyte lysate (LAL) extracted from horseshoe crab blood cells to detect endotoxins from gram-negative bacteria. The LAL contains enzymes that activate and coagulate in the presence of endotoxins, specifically lipopolysaccharides. To perform the BET, test samples are mixed with LAL and a positive control containing a known amount of endotoxin. If the LAL coagulates for the sample but not the negative control, endotoxins are present in the sample at a level depending on the dilution and lysate sensitivity. The test provides a quantitative measure of endotoxins to ensure safety for medical products.

2. The bacterial endotoxins test (BET)
is a test to detect or quantify
endotoxins from Gram-negative
bacteria using amoebocyte lysate
from the horseshoe crab (Limulus
polyphemus or Tachypleus
tridentatus).

3. Endotoxin:
● The toxic activity of LPS was first discovered and termed
"endotoxin" by Richard Friedrich Johannes Pfeiffer. which he
classified as a toxin that is released by bacteria into the surrounding
environment, and endotoxins, which he considered to be a toxin
kept "within" the bacterial cell and released only after destruction of
the bacterial cell wall.
● Subsequent work showed that release of LPS from gram
negative microbes does not necessarily require the destruction of the
bacterial cell wall, but rather, LPS is secreted as part of the normal
physiological activity of membrane vesicle trafficking in the form of
bacterial outer membrane vesicles (OMVs), which may also contain
other virulence factors and proteins.

4. ● The term 'endotoxin' is mostly used synonymously
with LPS, although there are a few molecules secreted
by other bacteria that are not related to LPS, such as
the so-called delta endotoxin proteins secreted by
Bacillus thuringiensis.
● LPS is the major component of the outer membrane of
Gram-negative bacteria, contributing greatly to the
structural integrity of the bacteria, and protecting the
membrane from certain kinds of chemical attack. LPS
also increases the negative charge of the cell membrane
and helps stabilize the overall membrane structure.

5. ● Lipopolysaccharides (LPS ) are large molecules
consisting of a lipid and a polysaccharide composed of
O-antigen, outer core and inner core joined by a
covalent bond; they are found in the outer membrane
of Gram-negative bacteria, and elicit strong immune
responses in animals.
● Endotoxin of bacteria consist of Lipopolysaccharide
(LPS), which is bound to protein and phospholipid.

7. Consequences of endotoxin contamination
• Fever
• Headache
• Chills
• Nausea/Vomiting
• Hypotension
• Acute lung injury
• Miscarriage
• Death

8. History of LAL Regulations
1912 Pyrogen test was performed by rabbit test
1941 Rabbit pyrogen test included in usp 12.
1973 FDA announced LAL as a biological product
1977 FDA described conditions for use of LAL as end product
test for human biological and medical devices.
1980 Federal register drafted guidelines for use of LAL test for
end product testing of human and animal injectable drug
products.

9. 1983 FDA determine the limit of endotoxin content based on
maximum human dose
1983 Federal registers list final guidelines on LAL testing
including chrom and turb.
1987 USFDA established guidelines for LAL testing of
pharmaceutical and medical devices
1991 Interim guidance – Kinetic LAL test kit
1995 USP 23 Bacterial endotoxin test
1998 European pharmacopeia BET
2000 Harmonized BET

10. Principle:
Gram-negative bacterial endotoxin catalyzes the
activation of a proenzyme in the LAL. The
initial rate of activation is determined by the
concentration of endotoxin present.
The activated enzyme (coagulase) hydrolyzes
specific bonds within a clotting protein
(coagulogen) also present in LAL. Once
hydrolyzed, the resultant coagulin self-
associates and forms a gelatinous clot.

12. ● The coagulation of Limulus amebocyte lysate
(LAL) is brought about by an enzymatic cascade,
which consists of three proenzymes, factor C,
factor B, and pro-clotting enzyme and one
clottable protein, coagulogen.
● LPS activates factor C, which in turn activates
factor B and proclotting enzyme, the latter
cleaving coagulogen to yield an insoluble gel and
releasing a soluble peptide C.

13. ● The resulting clotting enzyme is responsible for
anchoring the two peptide units in the coagulogen,
forming an insoluble gel.
● The rate of gelation was directly related to the
concentration of endotoxin.
● The catalytic nature of each activated enzyme in the
coagulation cascade serves in turn to amplify the next
step, resulting in a high sensitivity of LAL to LPS.

14. ● The coagulation chain described above is also activated
by the (1,3) -β-D-glucan, in this case through the
activation of factor G, which is another serine
protease pro-enzyme and which also leads to the
formation of the gel-forming enzyme which in turn
causes the gelation of coagulogen.
● Therefore, the β-D-glucan is considered to be an
interfering agent in the LAL test for the measurement
of endotoxins.

15. Reagents Used in BET test:
Lysate:
● The Danish-born pathologist Frederik
B. Bang reported that a bacterial
infection of Limulus polyphemus caused
fatal intravascular clotting and this
clotting could also be induced by a heat-
stable derivative of the bacterium.
● In 1964, Levin and Bang
demonstrated that extracts of the
amebocytes, but not the cell-free
hemolymph, would gel in the presence
of endotoxin

16. LAL
Limulus: Genera of Crab
Amebocyte: Crab Blood Cell from which active
component is derived
Lysate: Component is obtained by separating amebocyte
from the plasma and then lysing them

17. ● The reagent (lysate) used in the LAL test is of biological origin
and is extracted by the osmotic lysis of amebocytes found in the
intracellular fluid of the American horseshoe crab (Limulus
polyphemus). It is a complex mixture of different enzymes and co-
factors that form a clot through a cascade reaction when triggered by
the presence of endotoxins in the solution.
● The enzyme activity of this formulation (labeled as lysate
sensitivity, λ) is determined against a Reference Standard Endotoxin
(RSE) preparation supplied by the FDA. An assay is performed by
preparing a 2-fold dilution series made from 1 EU/mL of RSE. Since
the lysate potency is performed in 2-fold dilutions, e.g., the true
sensitivity of a labeled 0.125 EU/mL lysate lot may actually be 0.10
EU/mL.

18. ● LAL manufacturers supply Control Standard Endotoxin (CSE) whose potency
is determined against the RSE for every lot of lysate. Each lot-specific
combination of LAL and CSE must be characterized according to the method
being used and a specific RSE/CSE ratio (EU/ng value) cannot be assigned until
the testing is complete.
● Lyophilized (unreconstituted) Limulus Amebocyte Lysate should be stored
under refrigeration at 2-8°C. Care should be taken to avoid exposing the lysate to
temperatures in excess of 37°C. Lysate which has been exposed to prolonged
periods of temperatures above 37°C or to bright light may turn yellow and/or
become insoluble.
● Reconstituted lysate may be stored at 2-8°C for 24 hours. For longer storage,
reconstituted lysate can be stored at -20°C or colder. Freeze and thaw only once.
The lysate should be protected from exposure to light during storage. Use within
four weeks after reconstitution.

19. Control Standard Endotoxin:
● CSE is a highly purified lipopolysaccharide (LPS) preparation that is
reasonably free of detectable contaminants, particularly protein. It also contains
stabilizing fillers like human serum albumin, PEG, and starch, so that it performs
reliably and reproducibly when used in control and standard curves.
● A Standard Endotoxin Stock Solution is prepared from a USP Endotoxin
Reference Standard that has been calibrated to the current WHO International
Standard for Endotoxin.
● Endotoxin is expressed in Endotoxin Units (EU).
NOTE: One USP Endotoxin Unit (EU) is equal to one International Unit (IU) of
endotoxin.

20. ● Control Standard Endotoxin (CSE) is a widely used standard for
endotoxin testing. It is a purified extract from E. coli O113:H10, the
same strain used for the United States Pharmacopeia and the
European Pharmacopeia Reference Standard Endotoxin (RSE).
● Each vial when prepared according to the instructions below
provides the user with a Control Standard Endotoxin (CSE) whose
potency has been established using the current FDA Reference
Standard Endotoxin (RSE) and the enclosed lot of lysate.
● The appropriate RSE/CSE ratio and resultant CSE potency is
provided on the Certificate of Analysis.

21. Control Standard Endotoxin Preparation:
● Reconstitute by adding 1.0 ml of LAL Reagent Water warmed to room
temperature.
For example, if the value of the vial is 20 EU, when reconstituted with 1.0
ml water it will yield a concentration of 20 EU/ml.
● Shake vigorously as per COA at high speed on a vortex mixer.
● Lyophilized endotoxin is to be stored at 2–8°C. Reconstituted stock
endotoxin is stable for four weeks at 2–8°C. Prior to subsequent use, the
solution must be warmed to room temperature and vigorously vortexes for
15 minutes, because the endotoxin tends to attach to glass.
● This endotoxin is provided for the user’s convenience.

22. Preparation of CSE:
Step 1
Reconstitute Control Standard Endotoxin (CSE) with 1mL of LAL Reagent Water
(LRW).
Step 2
Vortex for 15 minutes or as per COA.
CSELRW
CSE

24. Step 3
Prepare a solution containing 1.0 EU/ml using the endotoxin potency identified on
the Certificate of Analysis (CoA).
0.1 mlCSE LRW
1.0 E
U/ml
1.9 ml

25. Step 4
Label tubes with the appropriate endotoxin concentration and add LRW to each.
Then prepare a series of endotoxin standards.
1EU/mL 0.5EU/mL 0.25EU/mL 0.125EU/mL 0.06EU/mL
0.5 ml0.5 ml0.5 ml
0.5 ml
1 min.
Vortex1 min.
Vortex
1 min.
Vortex
1 min.
Vortex
1 min.
Vortex
0.5 ml 0.5 ml 0.5 ml 0.5 ml
0.5 EU/ml
0.25 EU/
ml
0.1 EU/ml
LRW

26. Confirmation of Labelled Lysate Sensitivity:
● The test for confirmation of lysate sensitivity is to be 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.
● Confirm in four replicates the labelled sensitivity, λ, expressed in
IU/ml of the lysate prior to use in the test.
● Prepare standard solutions having four concentrations equivalent
to 2λ , λ, 0.5λ and 0.25λ by diluting the Standard Endotoxin Stock
Solution with water BET.

27. ● Mix a volume of the Lysate TS with an equal volume (such as
0.1mL aliquots) of one of the Standard Endotoxin Solutions in each
test tube.
● Incubate the reaction mixture for a constant period according to
the directions of the lysate manufacturer (usually at 37±1° for 60±2
min), avoiding vibration.
● To test the integrity of the gel, take each tube in turn directly from
the incubator, and invert it through about 180° in one smooth
motion.

28. ● If a firm gel has formed that remains in place upon inversion,
record the result as positive. A result is negative if an intact gel is
not formed.

29. ● The test is valid when the lowest concentration of the standard solutions shows
a negative result in all replicate tests.
● The endpoint is the smallest concentration in the series of decreasing
concentrations of standard endotoxin that clots the lysate.
● Determine the geometric mean endpoint by calculating the mean of the
logarithms of the endpoint concentrations of the four replicate series and then
taking the antilogarithm of the mean value, as indicated in the following formula:
geometric mean endpoint concentration = antilog (∑e/f)
where ∑e is the sum of the log endpoint concentrations of the dilution series used
f is the number of replicate test tubes.

30. ● The geometric mean endpoint concentration is the measured sensitivity of the lysate (in
EU/mL), this is not less than 0.5λ and not more than 2λ.
Lysate Sensitivity (λ) = 0.03 EU /ml
QuadraplicateResults
I II III IV
A
2 + + + +
+ + + +
/2 - - - -
/4
- - - -
END POINT 0.03 0.03 0.03 0.03

31. geometric mean = antilog (∑e/f)
= antilog (log (0.03)+log(0.03)+log(0.03)+log(0.03))
------------------------------------------
--------------------------
4
= antilog ( -1.52+ -1.52+ -1.52+ -1.52)
------------------------------------
4
= antilog (-1.52)
= 0.03

32. ● A positive test is characterized by the formation of solid gel which
remains intact after inversion. This should be observed in the
positive control vial and in the positive sample control vial.
● A negative test is characterized by the absence of solid clot after
inversion. This should be observed in the negative control vial. The
lysate may show an increase in turbidity or viscosity. This is
considered a negative result.
● A positive result should be observed with all Inhibit Control vials.
The absence of gel formation is indicative of product inhibition. If a
negative reaction is observe in the Inhibition Control vial with any
sample, then the samples test results are invalid.

33. Spiking Methods used in LAL Testing
● Double Strength Method or 50:50 Method
● Hot-Spike Method
Double Strength/Dilution Method or 50:50 Method
● Prepare double the strength test concentration or half of the test dilution of the
sample
● Prepare 4λ of CSE
● Dilute ½ Y (Y = Test Sample Dilution) with equal volume of LRW in Negative
Product Control (NPC)
● Dilute ½ Y (Y = Test Sample Dilution) with equal volume of 4λ in Positive Product
Control (PPC)

34. Example:
Product : ABCD Solution
Endotoxin limit : NMT 5.56 EU/mL
LAL sensitivity (l) = 0.125 EU/mL
MVD = 1:44
MVD/2 = 1:22
In order to test the sample at MVD or at Endotoxin Limit, prepare the solution at ½ MVD
i.e .1:22
Sample LRW Dilution
+
0.1 mL 2.1 mL 1:22

35. NPC = 50 µL LRW + 50µL Sample (1:22) + 100 µL LAL
PPC = 50µL Sample (1:22) + 50 µL 4l CSE + 100 µL LAL
If
then,
Endotoxin content of the sample = dilution factor x Lysate Sensitivity
= 44 x 0.125 EU/mL
= < 5.5 EU/mL

36. Hot-Spike Method
● Prepare test concentration or test dilution of the sample
● Prepare 20λ of CSE
● Take X product (where X = Test Sample Concentration) or Y (Y = Test Sample
Dilution) in Negative Product Control (NPC)
● Spike test sample with minimal but accurately measurable concentrated endotoxin
solution i.e. 10 µL of 20λ
Example :
Product : ACPD Solution
Endotoxin limit : NMT 5.56 EU/mL
Lysate sensitivity (l) = 0.125 EU/mL
MVD = 1:44

37. Sample Preparation
Sample LRW Dilution
0.1 mL 2.1 mL 1:22
Solution LRW Dilution
1 mL 1 mL 1:44

38. NPC = 100 µL Sample (1:44) + 100 µL LAL
PPC = 100 µL Sample (1:44) + 10 µL 20λCSE + 100 µL LAL
If
NPC = negative and PPC = positive,
Then
Endotoxin content of the sample = dilution factor x Lysate
Sensitivity
= 44 x 0.125 EU/mL
= < 5.5 EU/mL

39. Interference:
● Interference is defined as a significant difference
between the end points of positive water control
and positive product control using standard
endotoxin.
● This interference could be either inhibition wherein the
recovery of endotoxin is below than the expected or
enhancement wherein the recovery of endotoxin is
higher than expected.

40. Two classes of interference are considered here:
1. inhibition
2. enhancement
Inhibition : Inhibition occurs when a material interferes with the
ability of the LAL reagent to react with endotoxin, causing
underestimation of the amount of endotoxin present.
Tests must be properly controlled so that inhibition is detected
if it occurs. Appropriate controls provide assurance that negative
results are due to absence of endotoxin, not to inhibition.

41. Enhancement : Enhancement is interference that
increases the sensitivity of the assay, resulting in
overestimation of the endotoxin concentration of
the sample.
Enhancement is much less dangerous than
inhibition. It could result in the inability to release a
product that should have passed, but it does not
result in a threat to public health and safety.

42. Interference: Causes and Solutions
pH :
The LAL reaction consists of a series of enzymatic reactions in
which serine proteases, each with a pH optimum, cleave their
respective substrates. Consequently, it is critical that the pH of the
reaction mixture of product and LAL reagent be in the range
specified in the product insert.
The harmonized pharmacopeia endotoxins test require pH is in the
range of 6.0 - 8.0. However, it is quite possible that the pH of the
product alone will be outside the 6.0 - 8.0 range, but that the pH of
the reaction mixture will be within it because of the buffering
capacity of the LAL reagent.

43. The pH of the reaction mixture may be outside the specified range
for undiluted product, but the pH of a dilution of product (not to
exceed the MVD) can be within the range.
For example, if the pH of the reaction mixture is out of range for an
undiluted product with an MVD of 1:200, but it meets specification
at 1:20, then the product can be validated and tested at 1:40 or 1:50.
There is no need to adjust the pH
If pH problems are not overcome by dilution, use Tris Buffer to
reconstitute the LAL reagent as stipulated in the product insert and
then check pH of the reaction mixture.

44. In samples for which the buffer does not resolve the problem, the pH
of the sample (or of a sample dilution) can be adjusted by adding a
solution of acid or base (HCl or NaOH).
Prepare solutions from concentrated HCl or NaOH pellets with LAL
reagent water (LRW). Before conducting any LAL tests, perform
titrations to determine the appropriate volume and concentration of
acid or base solution to bring the pH into range.
The volume of added NaOH or HCl should not change the sample
volume by more than 10%.

45. Divalent Cations :
Divalent cations influence both the reactivity of endotoxin and the
LAL reaction. Cations are attracted to and neutralize the negative
charge of endotoxin, allowing increased aggregation size and
decreasing activity/potency, which is observed as inhibition.
Divalent cations are also required for optimal LAL sensitivity, but
excess concentrations inhibit the reaction.
Dilution is the usual solution to this problem. When interference by
salts (or other small molecules) cannot be overcome by dilution,
endotoxin can be separated from interfering substances by
ultrafiltration.

46. Chelating Agents :
Materials which bind (chelate) divalent cations, such as EDTA
(ethylene diamine tetraacetic acid) and heparin, can reduce the
aggregation state of endotoxin. This results in increased reactivity,
which is observed as enhancement.
Once again, interference is usually overcome by dilution of the
sample.
Extractables:
Extractables from plastics can interfere with LAL tests. Water
soluble substance extracted from polypropylene tubes that was
highly inhibitory.

47. Lipids and Liposomes: Some drug products are dissolved
in oils, such as sesame oil, while others are enclosed in
liposomes. Perhaps surprisingly, some products dissolved
in oils can be diluted in water (LRW) and tested in the
normal way, as endotoxin may partition into the aqueous
phase during dilution. Verify (and validate) this by spiking
the product with endotoxin and demonstrate recovery. In
other cases, a surfactant may be used to disperse a lipid,
e.g. 0.01% sodium desoxycholate (which is slightly
inhibitory). Piluso and Martinez(11) report that sodium
dodecyl sulfate (SDS) or CHAPS may be used to disrupt
liposomes to facilitate their testing.

48. Other Strategies for Overcoming Interference
Ultrafiltration can be used to separate lower
molecular weight interfering substances from
endotoxin in the sample. One product, the Sartorius
Ultrasart® D-20, in which the membrane has a 20
kD molecular weight cutoff, is designed
specifically for this purpose and has been used
effectively in laboratory.

49. The Three Essentials Steps for Endotoxin Testing
● Establish endotoxin limits for pharmaceutical and
medical devices.
● Establish procedures for validating the use of the BET
in your laboratory.
● Establish procedures for conducting routine testing.

50. Establishment of endotoxin limits
● The endotoxin limit for active substances administered
parenterally, defined on the basis of dose, is equal to:
EL = K/M
K = threshold pyrogenic dose of endotoxin per kilogram of body
mass in a single hour period,
M =maximum recommended dose of product per kilogram of
body mass in a single hour period.
● The endotoxin limit for active substances administered
parenterally is specified in units such as IU/ml, IU/mg, IU/Unit
of biological activity, etc.,

51. ● Note: The endotoxin limit depends on the product and its route
of administration
● For intravenous route: K = 5 IU endotoxin per kg body weight
● For intrathecal route K = 0.2 IU endotoxin per kg body weight
● For radiopharmaceutical products not administered intrathecally, the
endotoxin limit is calculated as 175 EU/V, where V is the maximum
recommended dose in mL
● For intrathecally administered radiopharmaceuticals, the endotoxin limit is
obtained by the formula 14 EU/V
● For formulations (usually anticancer products) administered on a per square
meter of body surface, the formula is K/M, where K = 100 EU/m2 and M is
the maximum dose/m2

52. Example:
Product: Piperacillin and tazobactam for Injection 2.25 g
Endotoxin limit calculation:
K = 5 EU/kg
M= 64.28 mg/kg/hr (4500mg per 70kg/hr) = 4500mg/70kg/hr = 64.28 mg/kg/hr
BET Limit = K/M
= 5 EU/kg/hr = 0.077 EU/mg ≈ 0.07 EU/mg
64.28 mg/kg/hr
BET Limit = 0.07 EU/mg.

53. Establish endotoxin limit For Medical Devices
● Level of endotoxin on medical devices are obtained by an
extraction procedure. This involves soaking a number of devices
in an extracting fluid normally Lal reagent water
EL = K X N/V
K= 20 EU/ device, for cerebrospinal device K= 2.15
EU/device
N = Number of device: if batch size is 30-100 test 3 samples,
batch size is more than 100 test 3% and up to maximum of 10
V = Total volume of extraction solution

54. Example:
Product: Coronary stents
Number of devices (N) : 3
K: 20 EU/ device
Rinse Volume (V) : 60 ml
EL = K X N/V = 20 x 3/ 60
= 1 EU/ mL

55. Maximum Valid Dilution (MVD):
Maximum valid dilution is the maximum allowable dilution of a
sample at which the endotoxin limit can be determined. It applies to
injections or to solution for parenteral administration in the form of
constituted or diluted for administration, or, where applicable, to the
amount of drug by weight if the volume of the dosage form for
administrated could be varied.
MVD = (endotoxin limit × concentration of Sample Solution)/(λ)
The endotoxin limit for parenteral drugs is specified in the individual
monograph in units such as EU/mL, EU/mg, EU/Unit of biological
activity

56. Concentration of Sample Solution:
mg/mL: in the case of endotoxin limit specified by weight (EU/mg)
Units/mL: in the case of endotoxin limit specified by unit of
biological activity (EU/Unit)
mL/mL: when the endotoxin limit is specified by volume (EU/mL).
λ: the labeled sensitivity in the Gel-Clot Technique (EU/mL) or the
lowest concentration used in the standard curve for the Turbidimetric
Technique or Chromogenic Technique

57. Example
Product name : Gentamycin Sulphate
Concentration : 40 mg/mL
Endotoxin Limit : 0.71 EU/mg
Lysate Sensitivity (λ) : 0.125 EU/mL
MVD =40 mg/mL x 0.71 EU/mg
-------------------------------
0.125 EU/mL
= 1: 227≈ 224

59. PRODUCT INHIBITION:
● The Limulus Amebocyte Lysate reaction is enzyme mediated and,
as such, has an optimal pH range, and specific salt and divalent
cation requirements.
● Occasionally test samples may alter these optimal conditions to
an extent that the lysate is rendered insensitive to endotoxin.
● Negative results with samples which inhibit the LAL test do not
necessarily indicate the absence of endotoxin. Initially, each type of
sample should be screened for product inhibition.

60. ● Prepare a series of two-fold dilutions of endotoxin in LAL
Reagent Water and a similar series of endotoxin dilutions using
sample as diluent. Assay each series in parallel using standard
procedures. At the end of the incubation period, record positive and
negative results and calculate the geometric mean endpoint for both
series of endotoxin dilutions.
● Products are said to be free of product inhibition if the geometric
mean endpoint of endotoxin in product is within 1/2 to 2 times the
labeled lysate sensitivity. See the following example.

61. Product Inhibition Testing:
Sr.No 0.25 0.125 0.06 0.03
1 + + - -
2 + + + -
3 + + - -
4 + + - -
geometric mean endpoint = 0.10 EU/ml
non-inhibitory
Water for Injection:
Labelled Lysate sensitivity: 0.125 EU/mL

62. Sr.No 0.25 0.125 0.06 0.03
1 + - - -
2 + + - -
3 + + - -
4 + + - -
geometric mean endpoint = 0.15 EU/ml
non-inhibitory
in Product A

63. in Product B:
Sr.No 0.25 0.125 0.06 0.03
1 - - - -
2 - - - -
3 + - - -
4 + - - -
geometric mean endpoint = 0.15 EU/ml
inhibitory

64. ● The easiest way to overcome product inhibition is through
dilution.
● The dilution factor must be taken into account when calculating
the total endotoxin concentration in a test sample.
● As a quick screen to determine a non-inhibitory dilution of
product, prepare a series of increasing dilutions of the product
containing an endotoxin spike equal in concentration to twice the
lysate sensitivity.
● Assay each spiked product dilution using standard procedures.
Positive results indicate when product inhibition has been overcome.

65. Methods - Bacterial Endotoxin Test
Gel Clot Technique: based on gel formation.
Turbidimetric Method: based on development of turbidity
after cleavage of an endogenous substrate.
Chromogenic Method: based on development of colour
after cleavage of synthetic peptide chromogen complex.

66. Gel-clot Method:
● The gel-clot method is the simplest and most widely used LAL test. The
reaction in the test tube is essentially the same as that in nature when a horseshoe
crab is injured.
● The gel-clot technique is used for detecting or quantifying endotoxins based on
clotting of the lysate reagent in the presence of endotoxin. The minimum
concentration of endotoxin required to cause the lysate to clot under standard
conditions is the labeled sensitivity of the lysate reagent.
● Gel-clot reagent is labelled with a sensitivity(A.) which is the lowest endotoxin
concentration to cause a clot to form under standard conditions.

67. ● The accepted error of the method is plus or minus two fold dilution. Therefore, a
reagent with a sensitivity of 0.125 EU/mI may not clot at this concentration in
some tests. However, it should always clot at 0.25 EU/mI. Similarly, the reagent
may clot at 0.125EU/mI and at 0.06EU/mI, but it should never clot at 0.03 EU/mI.
Because of the twofold error of the method, positive controls and positive product
controls (spiked sample) are always at a concentration of 2λ (twice the labeled
sensitivity).
● The gel-clot test yields a binary result, either positive or negative. A tube is
scored as positive (+) if the clot withstands 180° inversion without breaking. All
other conditions are scored as negative (-), even if the clot almost remains intact
but then collapses.

68. ● The endotoxin concentration of the original sample is calculated by multiplying
the reagent sensitivity by the dilution factor at the endpoint.
● Gel-Clot reagent is available in a range of sensitivities. In order to decide which
sensitivity to use, a number of factors must be considered.
● First, determine the endotoxin levels or limits to be detected. Clearly the reagent
must be sufficiently sensitive to at least detect the limits. Also consider the type of
sample and, if possible, the likelihood of it interfering with the test. A greater
sensitivity will give increased scope for dilution to overcome interferences.
● Ultimately, the sample must be tested to assure that it does not interfere with the
test at a dilution at which the endotoxin limit can be detected.

69. ● The gel-clot method includes a limit test (qualitative) and an endotoxin standard
series is prepared using four concentrations of CSE that bracket the lysate
sensitivity (λ).
● It is run at least once a day with the first set of tests and repeated if there is any
change in lysate lot, endotoxin lot, or test conditions during the day.
Please refer to Appendix C: 1987 FDA Guidelines or the USP <85> Bacterial
Endotoxins Test for detailed procedures 5.
● The gel-clot test is valid when the end-point of the endotoxin standard series is
within a + two-fold dilution of the labeled lysate sensitivity (indicating the inherent
2-fold error/variability in the test).
● The test is performed in duplicate. In all gel-clot assays, the PPC must always be
positive (form a clot) to be valid.

70. Test for Interfering Factors:
● Usually prepare solutions (A–D) as shown in Table 1, and perform the
inhibition/ enhancement test on the Sample Solutions at a dilution less than the
MVD, not containing any detectable endotoxins, operating as described for Test
for Confirmation of Labeled Lysate Sensitivity.
● The geometric mean endpoint concentrations of Solutions B and C are
determined using the formula described in the Test for Confirmation of Labeled
Lysate Sensitivity.
● The test is considered valid when all replicates of Solutions A and D show no
reaction and the result of Solution C confirms the labeled sensitivity. If the
sensitivity of the lysate determined in the presence of Solution B is not less than
0.5λ and not greater than 2λ, the Sample Solution does not contain factors that
interfere under the experimental conditions used. If the sample under test does not
comply with the test at a dilution less than the MVD, repeat the test using a greater
dilution, not exceeding the MVD.

71. ● The use of a more sensitive lysate permits a greater dilution of the sample to be
examined, and this may contribute to the elimination of interference. Interference
may be overcome by suitable treatment such as filtration, neutralization, dialysis,
or heating.
●To establish that the chosen treatment effectively eliminates interference without
loss of endotoxins, perform the assay described above using the preparation to be
examined to which Standard Endotoxin has been added and which has then been
submitted to the chosen treatment.
● The test for interfering factors must be repeated when any condition changes
that is likely to influence the result of the test.

72. Solution Endotoxin
Concentration/
Solution to Which
Endotoxin
Is Added
Diluent Dilution
Factor
Endotoxin
Concentration
Number
of
Replicates
Aa None/Sample Solution — — — 4
Bb 2λ/Sample Solution
Sample
Solution 1 2λ 4
2 1λ 4
4 0.5λ 4
8 0.25λ 4
Cc 2λ/Water for BET
Water for
BET 1 2λ 2
2 1λ 2
4 0.5λ 2
8 0.25λ 2
Dd None/Water for BET — — — 2
Table 1. Preparation of Solutions for the
Inhibition/Enhancement Test for Gel-Clot Techniques

73. Solution A: A Sample Solution of the preparation under test that is
free of detectable endotoxins.
Solution B: Test for interference.
Solution C: Control for labeled lysate sensitivity.
Solution D: Negative control of Water for BET.

74. Limit Test:
Procedure: Prepare Solutions A, B, C, and D as shown in Table 2, and perform the test on
these solutions following the procedure above for Preparatory Testing, Test for
Confirmation of Labeled Lysate Sensitivity.
Table 2. Preparation of Solutions for the Gel-Clot Limit Test
Solution*
Endotoxin Concentration/
Solution to Which
Endotoxin Is Added
Number of
Replicates
A None/Diluted Sample Solution 2
B 2λ/Diluted Sample Solution 2
C 2λ/Water for BET 2
D None/Water for BET 2
* Prepare Solution A and the positive product control Solution B using a dilution not
greater than the MVD and treatments as described for the Test for Interfering Factors in
Preparatory Testing. The positive control Solutions B and C contain the Standard
Endotoxin Solution at a concentration corresponding to twice the labeled lysate sensitivity.
The negative control Solution D consists of Water for BET.

75. Interpretation:
● The test is considered valid when both replicates of Solutions B and C are
positive and those of Solution D are negative.
● When a negative result is found for both replicates of Solution A, the
preparation under test complies with the test.
● When a positive result is found for both replicates of Solution A, the preparation
under test does not comply with the test.
● When a positive result is found for one replicate of Solution A and a negative
result is found for the other, repeat the test.
● In the repeat test, the preparation under test complies with the test if a negative
result is found for both replicates of Solution A. The preparation does not comply
with the test if a positive result is found for one or both replicates of Solution A.
However, if the preparation does not comply with the test at a dilution less than the
MVD, the test may be repeated using a greater dilution, not exceeding the MVD.

76. Troubleshooting Guide for Gel Clot Assay
A. Endotoxin Mixing:
● Endotoxins that are used as controls with LAL tests are very large, highly
purified molecules that have unique properties affecting their behaviour in aqueous
solutions. A portion of the molecule is very water-soluble and another part is not.
For this reason, endotoxin molecules in solution tend to aggregate. When a
solution containing endotoxin is withdrawn, such as when making serial dilutions
for endotoxin testing, the endotoxin must be well dispersed or the portion
withdrawn may not contain the proper amount of endotoxin. Thorough mixing of
endotoxin and accurate transfer throughout a dilution series are best accomplished
by observing the points noted below.

77. 1. Make endotoxin dilutions in increments no greater than 1:10. This will increase
the chance of obtaining the correct amount of endotoxin in the withdrawn portion
and decrease the extent to which it will have to be dispersed when it is diluted.
2. Adhere strictly to the agitation and vortex mixing times specified for each step in
the endotoxin dilution instructions. Vortex mixing should be done at the “high”
speed setting in order to disperse endotoxin aggregates. The aliquot for subsequent
dilution should be withdrawn immediately after mixing, and the endotoxin sample
should be thoroughly mixed just prior to use.
NOTE: It is not recommended to vortex for more than 30 minutes in one 8 hour
period to avoid damage to the endotoxin and loss of potency.
B. Pipetting Accuracy
Pipetting accuracy is exceptionally important during every phase of LAL testing.
Ensure you are adding the proper amounts of material by utilizing high quality
graduated pipettes and pipette tips and the proper technique for the pipette you
have chosen. Pipettes should be recalibrated annually at a minimum.

78. C. Conditions for Incubation of LAL Test Tubes:
• Check the temperature of the water bath or heating block.
• The LAL reaction with endotoxin is temperature dependent and the
specified standard conditions for the test require one hour incubation
at 37°C ±1°C.
• The critical temperature is that of the material inside the reaction
tube. An accurate check can be made by placing a calibrated
thermometer inside a 75 x 10 mm test tube containing just enough
water to cover the thermometer bulb.

79. • Place the tube and thermometer in a test tube incubation rack or a
heating block and observe the temperature over a one-hour period.
Check the location of the water bath or heating block.
• While the gel is forming, it is quite fragile and may be irreversibly
broken by mechanical shock or vibration. Small motions, including
closing a door or drawer in your work bench or a centrifuge running
can cause sufficient vibration to affect the development of the clots.
• Generally speaking, any motion of the incubating test tubes must
be regarded as a potential problem source. Agitating or circulating
water baths must not be used for this reason.

80. D. Test Results
A positive result is a firm gel that maintains its integrity through inversion to 180°.
When reading test results after the one-hour incubation period, gently withdraw
each tube and invert it 180° in one smooth motion. Do not stop at 45° or 90° to
look before continuing inversion, and be mindful of adjacent tubes when removing
them from the heat block or tube rack.
E. Test Reagent Handling
Before reconstitution, the PYROGENT™ Gel Clot LAL reagent is very stable but should
be stored in the dark at 2 – 8°C (note expiration date). The lysate reagent, when
reconstituted, is a buffered solution containing enzymes and other proteins from the
Limulus amebocyte. Preparation, handling and storage are critical and should be
approached keeping the following points in mind:

81. 1. The water used to rehydrate the PYROGENT™ Gel Clot LAL reagent should
be LAL Reagent Water (LRW). Water for Injection (WFI) is generally not
acceptable as it is not tested to the proper endotoxin concentrations for endotoxin
content nor has it been tested for assay performance compatibility. Furthermore,
LRW manufactured by other endotoxin detection companies may not be
compatible with the LAL gel clot reagents. If it is necessary to use reagent water
other than a Lonza product, it must be thoroughly validated prior to use in testing
samples.
2. Pipetting technique must be aseptic and apyrogenic. Even when rehydration
water is pyrogen-free, it can easily become contaminated unless appropriate
precautions are observed. Aseptic and apyrogenic techniques consist of precautions
to avoid the entry of gross amounts of bacterial and/or endotoxin contamination. A
great deal of endotoxin may be present on surfaces (hands, glassware, etc.) even
when few or no viable bacteria are found. For this reason, labware must be treated
to remove endotoxin and handled carefully to prevent contamination.

82. 3. Rehydrated PYROGENT™ Gel Clot LAL reagent may be stored for as long as
24 hours, but it must be kept at 2 – 8°C. The lysate should not be pre-incubated in
the LAL test tubes before adding samples to them, as this may cause sensitivity
loss in the PYROGENT™ Gel Clot LAL reagent.
If rehydrated lysate must be stored for a longer period (up to four weeks), it must
be kept frozen at –10°C or below and checked to ensure the lysate is frozen solid
in the tubes. Many frost-free freezers have surface heating cycles that may
periodically warm articles in contact with the heated surfaces. Repeated freezing
and thawing of the lysate will result in deterioration well before four weeks have
passed.
4. Lyophilized E. coli endotoxin should be stored at 2 – 8°C. Endotoxin solutions
are generally very stable upon storage except when highly diluted. This is related
to the solubility characteristics discussed earlier. Rehydrated E. coli endotoxin
should be stored refrigerated only at 2 – 8°C and not be frozen. The stock solution
kept for up to four weeks. Further dilutions of E. coli endotoxin may begin to lose
potency after a few days.

83. The storage stability of dilute endotoxins may be related to the cleanliness of the
glassware in which they are kept, as endotoxin can bind on irregular or dirty glass
surfaces. Since some types of plastic have been shown to have an affinity for
endotoxin, the use of plastic containers to prepare and store endotoxin must be
approached with caution. The precautions for technique, water and pipettes
discussed previously (for the lysate rehydration) also apply to endotoxin
rehydration.
5. Diluents other than water or saline may also cause inhibition. When all water
controls behave as expected in the LAL test but a specific sample fails to give
positive results when prepared to contain the specified endotoxin concentrations,
true inhibition has been recognized
6. Handling of samples is as critical as the handling of reagents, and cleanliness is
important to ensure the use of aseptic and apyrogenic techniques. Always check
the product for inhibition as some products, especially members of the small
volume parenteral family, may contain substances at great enough concentrations
to inhibit the LAL gelation reaction.

84. In addition, the pH of the sample to be tested should be checked and should be in
the range of 6.0 – 8.0. A pH outside of this range is one of the most common
causes of product inhibition in the LAL test.
The PYROGENT™ Gel Clot LAL reagent does have a certain amount of
buffering capacity, but it must be assured that a product, when added to the
PYROGENT™ Gel Clot LAL reagent, does not exceed that capacity. An easy way
to determine whether or not a given product will require pH adjustment is simply
add a volume of the product in question to an equal volume of rehydrated
PYROGENT™ Gel Clot LAL reagent and check the pH to determine if it falls
between 6.0 and 8.0. Remember that because of PYROGENT™ Gel Clot LAL
reagent’s buffering capacity, there is no need to adjust the pH of WFI or Saline for
Injection USP. If a pH adjustment is necessary, Lonza Tris Buffer (Lonza Catalog
No: S50-642) is available for this use. Tris Buffer is a convenient way to adjust
pH when the buffering capacity of the sample will allow it.

85. Category II: Problem Sources
• The majority of Category II problems is caused by failure to observe proper handling
technique, failure to use adequate equipment preparation procedures, and/or use of
contaminated water in the test.
• The extreme sensitivity of LAL to endotoxin is regarded as its greatest virtue, but this
sensitivity can also be a source of problems when careless technique is used.
• Care in preparation and handling of PYROGENT™ Gel Clot LAL reagent and test
samples is essential. Any surface or substance that could come into contact with either the
LAL or the sample must be free of endotoxins. This need was discussed previously and is
re-emphasized here. Remember that endotoxin is virtually everywhere in nature.
As a rule, when testing parenteral pharmaceutical products, if a firm gel appears in the LAL
test, it must be assumed that endotoxin is present.
• Use of a negative control as part of the test will help you determine if positive results are
obtained due to endotoxin in the test sample or to inadvertent contamination.
• During inhibition/enhancement testing, another indication of contamination may appear
when you have prepared a series of dilutions of endotoxin in a product and another series of
endotoxin in LRW. If the endpoint of the LAL assay of endotoxin in product is more than a
two-fold dilution lower in concentration than the endpoint of the water-endotoxin dilution
series, contamination of the product might be suspected. Further testing is necessary to
exclude enhancement.