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107
Chapter 6
“BACTERIAL ENDOTOXINS QUANTIFICATION
IN VETERINARY INJECTIONS,
INTERMEDIATES, OIL BASED INJECTIONS
AND OTHER DRUGS”
108
6.1 Veterinary Drugs:
Introduction:
Like humans, animals also respond physiologically to the Endotoxins in the
same fashions to various primary and secondary responses upon
administration of the injectables. Same Endotoxins limits are applicable to
veterinary as of humans, based on the body weight. The bacterial Endotoxins
test to be passed as per the respective monograph before releasing the drug
into market but due to interfering factors in most of the cases there is a
probability of getting false positive results. So Non-Interfering Dilution (NID) to
be selected before validating a product. Four products were validated and
the NID factor among those veterinary injections is between + two fold.
For most of the new veterinary formulations the Endotoxin limits are not
available in pharmacopeia80, In that case the Endotoxin limit of such drugs
need to be calculated based on the body weight of the animal. It is prudent
to check clinical publications to determine if there are dosage patterns that
exceed the labeled dose range.
For example to new formulation name “X” with potency 500mg/mL.
Endotoxin limits was not available in pharmacopeia, wishing to analyze. The
“X” can be prescribed to hen, dog and buffalo. Lets quote an example, how
to calculate Endotoxin Limit to a new drug based on the body weight of the
animal.
Hen: Maximum allowable dosage per hour is 0.25mL (125mg).
Body weight is 1.5 Kg.
Dog: Maximum allowable dosage per hour is 2 mL (1000mg).
Body weight is 15 Kg.
Buffalo: Maximum allowable dosage per hour is 10 mL (2500 mg).
Body weight is 300 Kg.
The K/M formula
The K/M formula was presented in the FDA LAL-test Guideline (1987) as a way
to calculate the Endotoxin limit, as shown below.
Endotoxin Limit (EL) = K (tolerance limit)
M (maximum dose/kg) (hr.)
6.1 Veterinary Drugs
109
For Hen:
Endotoxin Limit (EL) = K (tolerance limit)
M (maximum dose/kg) (hr.)
= K 5Eu/Kg/hr .
125 mg/1.5/kg) (hr.)
=0.06 Eu/mg
For Dog:
Endotoxin Limit (EL) = K (tolerance limit)
M (maximum dose/kg) (hr.)
= K 5Eu/Kg/hr .
1000 mg/15/kg) (hr.)
=0.075 Eu/mg
For Buffalo:
Endotoxin Limit (EL) = K (tolerance limit)
M (maximum dose/kg) (hr.)
= K 5Eu/Kg/hr .
2500 mg/300/kg) (hr.)
=0.6 Eu/mg
Based on the body weight and the dosage of the drug the Endotoxin limit
varies. Lesser the body weight the limits are more stringent.
Materials and Methods:
Materials:
Lyophilized Limulus Amoebocyte Lysate of 0.125 sensitivity (LAL), Control
Standard Endotoxin 5 Eu/ng (CSE), LAL Reagent Water (LRW) of Endosafe US,
Depyrogenated (250°C for 30 min )10 X 75 mm assay tubes, 16X100 mm
dilution tubes ,pyrogen free Micropipette tips, vortex mixture, 1N NaoH, 1N
HCL, Meloxicam, Dexamethasone phosphate and Ivermectin were used for
determination of Endotoxin content by the gel clot technique.
The sensitivity of the Lysate (labeled 0.125 Eu/mL) was determined by using
known amount of E.coli Control Standard Endotoxin.
6.1 Veterinary Drugs
110
In the gel-clot techniques, the reaction end point is determined from dilutions
of the material under test in direct comparison with parallel dilutions or a
reference Endotoxin, and quantities of Endotoxins are expressed in Endotoxin
units69.
1. Preparation of Standard stock solution and standard solutions: The CSE
having a defined potency of 50 EU/Vial was reconstituted with 5ml of LRW
and mixed intermittently for 30 minutes using a vortex mixture and this
concentrate was used to prepare 2λ, λ, λ/2 & λ/4, where λ is the labeled
claim sensitivity of Lysate.
2. Preparation of sample solution: Test samples were diluted to the required
concentrations based on the formulae MVD. MVD is the maximum valid
dilution, which is allowable dilution of the specimen at which the Endotoxin
limit can be determined. The general equation to determine MVD is
MVD = (Endotoxin limit X Concentration of sample solution)/ (λ). Where E.L is
the Endotoxin limit of the test sample, which is specified in the individual
monograph in terms of volume or units of active drug (in EU/mg).
3. Meloxicam sample preparation: Potency= 5mg/mL , E.L= 10 Eu/mg , Lysate
sensitivity is 0.125 Eu/mL and MVD = 400.The following test dilutions are
prepared by 1:400 (0.01 mg/mL), 1:200 (0.03 mg/mL), 1:100 (0.05 mg/mL), 1:50
(0.10 mg/mL) & 1:25 (0.20 mg/mL).
4. Dexamethasone Phosphate sample preparation: Potency= 3.34 mg/mL,
E.L= 31.30 Eu/mg, Lysate sensitivity is 0.125 Eu/mL and MVD = 836. The following
test dilutions are prepared by 1:836 (0.004 mg/mL), 1:418 (0.008 mg/mL), 1:209
(0.016 mg/mL), 1:104 (0.033 mg/mL) & 1:52 (0.066 mg/mL).
5. Ivermectin sample preparation: Potency= 10 mg/mL, E.L= 5 Eu/mg, Lysate
sensitivity is 0.125 Eu/mL and MVD = 400. The following test dilutions are
prepared by 1:400 (0.025 mg/mL), 1:200 (0.05 mg/mL), 1:100 (0.10 mg/mL),
1:50 (0.20 mg/mL) & 1:25 (0.40 mg/mL).
6.1 Veterinary Drugs
111
Methods
Equal volume of test sample and LAL reagent is added in a
depyrogenated test tube of 10 X 75 mm and incubate this mixture at 37± 1°C
for 60±2 min. Then invert the tube by 180° and look for gel formation. If a gel
inside the test tube is able to maintain its integrity after inverting the tube to
180° then it is a positive reaction which indicates presence of Endotoxin in the
sample greater than the limit. Other than this any condition is considered as
negative which indicates absence of Endotoxin in the sample (lesser than the
lysate sensitivity).
Product Testing: For testing products equal volume of drug (sample) and LAL
reagent is taken and following tubes are prepared)
Negative Product Control (NPC) - Sample + LAL
Positive Product Control (PPC) - Sample + CSE (2λ) + LAL
Negative Water Control (NWC) - LRW + LAL
Positive Water Control (PWC) - LRW + CSE (2λ) + LAL
Majority of times it has been a common observation that if a product is tested
directly it inhibits the LAL test and thus shows interference
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.
Product Validation: Product needs to be validated before start for routine
testing. Validation is a test condition where an Endotoxin standard is detected
with the same efficiency in a test sample as it is in LRW. This validation study
consists of two different phases wherein in Phase I (Preliminary screening)
involve interference testing and Phase II consists of validation of product.
Significance of product validation is that it gives information on
whether there are any interfering factors in the drug product to the LAL test
and also it gives an idea of the approximate levels of Endotoxin content in the
drug product. It also covers manufacturing of product and formulation of
the product.
6.1 Veterinary Drugs
112
It is always advisable to carry out revalidation if product formulation is
changed and which is likely to affect the interference pattern of the product
for LAL test. Also revalidation is to be conducted for any product if there is any
change in manufacturing procedures or in vendor.
6.1 A: Meloxicam Preliminary Screening Test:
Product : Meloxicam
Concentration : 5mg/ml
Endotoxin Limit : 10 EU/mg
MVD : 400
Test Dilution : 1:25, 1:50, 1:100, 1:200 and 1: 400
Test Concentrations : 0.20mg/mL, 0.10mg/mL, 0.05mg/mL,
0.03mg/mL and 0.01mg/mL
Phase I: Preliminary Screening / interference Study25 (Cooper, 1990).
In this two identical series of product dilutions (two-fold dilutions), one
spiked with 2λ, and one left unspiked. The result of Phase I will tell you the
Non-Interfering Dilution (NID) of the product. The Non-Interfering Dilution (NID)
is the first set of PPC that shows a gel.
Test Dilutions /
Concentrations
NPC PPC
1:25 − − + +
1:50 − − + +
1:100 − − + +
1:200 − − + +
1:400 − − + +
++: Gel Formation − − : No Gel Formation
Conclusion: pH of the product was adjusted to 7-8 at initial dilution, carried
out screening and observed Non Interfering Dilution (NID) at 1/16
MVD (1:25). Non Interfering Concentration is 0.20mg/ mL.
Endotoxin content is < 10EU/ mg.
6.1 Veterinary Drugs
113
End-Product Endotoxins Test:
Product : Meloxicam
Preparation : Product Concentration : 5 mg / mL
Endotoxin Limit : 10 EU/mg
MVD : 400
Test Dilution : 1:50
Test Concentration : 0.10 mg/mL
Results :
Test Endotoxin
Concentration EU/mL.
NEG
Control
NEG
Product
Control
Test
End
point.
Log of
End
point.
Geometric Mean
=A log (log of End
point.)
Material 2λ λ ½λ ¼λ EU/mL
+ + - − − 0.125 -0.9030
+ + - − − 0.125 -0.9030
+ + - − 0.125 -0.9030
CSE
Water
Control
+ + - − 0.125 -0.9030
+ + + − − 0.0625 -1.2041
+ + + − − 0.0625 -1.2041
+ + - − − 0.125 -0.9030
Positive
Product
Control
+ + - − − 0.125 -0.9030
Antilog
(-3.612/4)
=Antilog –0.903
=0.125EU/mL
Antilog
(-4.2142/4)
=Antilog–1.05355
=0.088EU/mL
Interpretation :
Test results are : Valid
Comments : Product validation for Meloxicam was carried out at MVD/8
(1:50) (Test Concentration: 0.10mg/mL) after adjusting the pH81.
The GM of the end points in Endotoxin / LRW was 0.125EU/mL
and Endotoxin/product was 0.088 EU/mL which is in between 2λ
and ½ λ, which indicates no inhibition and enhancement at test
dilution MVD / 8 (1:50).
6.1 Veterinary Drugs
114
6.1 B: Dexamethasone Phosphate Preliminary Screening Test:
Product : Dexamethasone Phosphate
Concentration : 3.34 mg/ml
Endotoxin Limit : 31.30 EU/mg
MVD : 836
Test Dilution : 1:52, 1:104, 1:209, 1:418 and 1:836
Test Concentrations : 0.066mg/mL, 0.033mg/mL, 0.017mg/mL,
0.008mg/mL and 0.004mg/mL.
In this two identical series of product dilutions (two-fold dilutions), one spiked
with 2λ, and one left unspiked. The result of Phase I will tell you the Non-
Interfering Dilution (NID) of the product. The Non-Interfering Dilution (NID) is the
first set of PPC that shows a gel.
Test Dilutions /
Concentrations
NPC PPC
1:52 (0.066 mg/mL) + + + +
1:104 (0.033 mg/mL) − − + +
1:209 (0.016 mg/mL) − − + +
1:418 (0.008 mg/mL) − − + +
1:836 (0.004 mg/mL) − − + +
++: Gel Formation − − : No Gel Formation
Conclusion: pH of the product was adjusted to 7-8 at initial dilution, carried
out screening and observed Non Interfering Dilution (NID) at 1/8
MVD (1:104). Non Interfering Concentration is 0.033mg/ mL.
Endotoxin content is < 31.30EU/ mg.
6.1 Veterinary Drugs
115
End-Product Endotoxins Test:
Product : Dexamethasone Phosphate
Preparation : Product Concentration : 3.34 mg / mL
Endotoxin Limit : 31.30 EU/mg
MVD : 836
Test Dilution : 1:209
Test Concentration : 0.016 mg/mL
Results :
Test Endotoxin
Concentration EU/mL.
NEG
Control
NEG
Product
Control
Test
End
point.
Log of
End
point.
Geometric Mean
=A log (log of End
point.)
Material 2λ λ ½λ ¼λ EU/mL
+ + - − − 0.125 -0.9030
+ + - − − 0.125 -0.9030
+ + - − 0.125 -0.9030
CSE
Water
Control
+ + - − 0.125 -0.9030
+ + - − − 0.125 -0.9030
+ + + − − 0.0625 -1.2041
+ + - − − 0.125 -0.9030
Positive
Product
Control
+ + + − − 0.0625 -1.2041
Antilog
(-3.612/4)
=Antilog –0.903
=0.125EU/mL
Antilog
(-4.2142/4)
=Antilog–1.05355
=0.088EU/mL
Interpretation :
Test results are : Valid
Comments : Product validation for Dexamethasone Phosphate was carried
out at MVD/4 (1:209) (Test Concentration: 0.016mg/mL) after
adjusting the pH. The GM of the end points in Endotoxin / LRW
was 0.125EU/mL and Endotoxin/product was 0.088 EU/mL which
is in between 2λ and ½ λ, which indicates no inhibition and
enhancement at test dilution MVD/4 (1:209).
6.1 Veterinary Drugs
116
6.1 C: Ivermectin Preliminary Screening Test:
Product : Ivermectin
Concentration : 10 mg/ml
Endotoxin Limit : 5 EU/mg
MVD : 400
Test Dilution : 1:25, 1:50, 1:100, 1:200 and 1:400
Test Concentrations 0.40mg/mL, 0.20mg/mL, 0.10mg/mL, 0.05mg/mL
and 0.025mg/mL.
In this two identical series of product dilutions (two-fold dilutions), one spiked
with 2λ, and one left unspiked. The result of Phase I will tell you the Non-
Interfering Dilution (NID) of the product. The Non-Interfering Dilution (NID) is the
first set of PPC that shows a gel.
Test Dilutions /
Concentrations
NPC PPC
1:12 (0.83 mg/mL) − − + +
1:25 (0.40 mg/mL) − − + +
1:50 (0.20 mg/mL) − − + +
1:100 (0.10 mg/mL) − − + +
1:200 (0.05 mg/mL) − − + +
++: Gel Formation − − : No Gel Formation
Conclusion: pH of the product was adjusted to 7-8 at initial dilution, carried
out screening and observed Non Interfering Dilution (NID) at 1/16
MVD (1:12). Non Interfering Concentration is 0.40mg/ mL.
Endotoxin content is < 5EU/ mg.
6.1 Veterinary Drugs
117
End-Product Endotoxins Test:
Product : Ivermectin
Preparation : Product Concentration : 10 mg / mL
Endotoxin Limit : 5EU/mg
MVD : 400
Test Dilution : 1:50
Test Concentration : 0.40 mg/mL
Results :
Test Endotoxin
Concentration EU/mL.
NEG
Control
NEG
Product
Control
Test
End
point.
Log of
End
point.
Geometric Mean
=A log (log of End
point.)
Material 2λ λ ½λ ¼λ EU/mL
+ + - − − 0.125 -0.9030
+ + - − − 0.125 -0.9030
+ + - − 0.125 -0.9030
CSE
Water
Control
+ + - − 0.125 -0.9030
+ + - − − 0.125 -0.9030
+ + - − − 0.125 -0.9030
+ + - − − 0.125 -0.9030
Positive
Product
Control
+ + - − − 0.125 -0.9030
Antilog
(-3.612/4)
=Antilog –0.903
=0.125EU/mL
Antilog
(-3.612/4)
=Antilog –0.903
=0.125EU/mL
Interpretation :
Test results are : Valid
Comments : Product validation for Ivermectin was carried out at MVD/8
(1:50) (Test Concentration: 0.20mg/mL) after adjusting the pH.
The GM of the end points in Endotoxin / LRW was 0.125EU/mL
and Endotoxin/product was 0.125 EU/mL which is in between 2λ
and ½ λ, which indicates no inhibition and enhancement at test
dilution MVD/8 (1:50).
118
6.2 Intermediates
The schematic drug manufacturing process initiate with the starting raw
material proceeded with intermediated/stages and ends with the final
formulation. In order to comply the Endotoxin limit of the final drug product
the level of the Endotoxins in the raw materials used or intermediates formed
to be controlled so that the Endotoxin content in the final drug product was
controlled. It is always advisable to check intermediates and key starting raw
materials for Endotoxins and controlled at lower levels than the final drug
product in which they are used. So that the final drug product will pass the
bacterial Endotoxins test.
Materials and Methods:
Materials:
Lyophilized Limulus Amoebocyte Lysate of 0.125 sensitivity (LAL), Control
Standard Endotoxin 5 Eu/ng (CSE), LAL Reagent Water (LRW) of Endosafe US,
Depyrogenated (250°C for 30 min )10 X 75 mm assay tubes, 16X100 mm
dilution tubes ,pyrogen free Micropipette tips, vortex mixture, 1N NaoH, 1N
HCL and Sodium Bicarbonate. were used for determination of Endotoxin
content by the gel clot technique.
The sensitivity of the Lysate (labeled 0.125 Eu/mL) was determined by using
known amount of E.coli Control Standard Endotoxin.
In the gel-clot techniques, the reaction end point is determined from dilutions
of the material under test in direct comparison with parallel dilutions or a
reference Endotoxin, and quantities of Endotoxins are expressed in Endotoxin
units.
1. Preparation of Standard stock solution and standard solutions: The CSE
having a defined potency of 50 EU/Vial was reconstituted with 5ml of LRW
and mixed intermittently for 30 minutes using a vortex mixture and this
concentrate was used to prepare 2λ, λ, λ/2 & λ/4, where λ is the labeled
claim sensitivity of Lysate.
2. Preparation of sample solution: Test samples were diluted to the required
concentrations based on the formulae MVD. MVD is the maximum valid
dilution, which is allowable dilution of the specimen at which the Endotoxin
limit can be determined. The general equation to determine MVD is
6.2 Intermediates
119
MVD = (Endotoxin limit X Concentration of sample solution)/ (λ). Where E.L is
the Endotoxin limit of the test sample, which is specified in the individual
monograph in terms of volume or units of active drug (in EU/mg).
3. Sodium Bicarbonate sample preparation: Potency = 0.5952 mEq/mL, E.L= 5
mEq/mL , Lysate sensitivity is 0.125 Eu/mL and MVD = 20 .The following test
dilutions are prepared by 1:20, 1:10, 1:5 & 1:2.5).
Methods
Equal volume of test sample and LAL reagent is added in a
depyrogenated test tube of 10 X 75 mm and incubate this mixture at 37± 1°C
for 60±2 min. Then invert the tube by 180° and look for gel formation. If a gel
inside the test tube is able to maintain its integrity after inverting the tube to
180° then it is a positive reaction which indicates presence of Endotoxin in the
sample greater than the limit. Other than this any condition is considered as
negative which indicates absence of Endotoxin in the sample (lesser than the
lysate sensitivity).
Product Testing: For testing products equal volume of drug (sample) and LAL
reagent is taken and following tubes are prepared)
Negative Product Control (NPC) - Sample + LAL
Positive Product Control (PPC) - Sample + CSE (2λ) + LAL
Negative Water Control (NWC) - LRW + LAL
Positive Water Control (PWC) - LRW + CSE (2λ) + LAL
6.2 Intermediates
120
6.2 A: Sodium Bicarbonate Preliminary Screening Test:
Product : Sodium Bicarbonate
Concentration : 0.5952 mEq/mL
Endotoxin Limit : 5 EU/mEq
Molecular weight of Sodium Bicarbonate (NaHCO3) = 23 + 1 +12 + 48 = 84
1 Molar = 84 gms in 1000 mL = 84 mg/mL
1 millimole = 0.084 mg/mL
1 gm equivalent = Molarity / no. of replaceable H ions per liter.
= 84 mg/mL
1 / 1000 mL
1 milli equivalent = milli Molarity / no. of replaceable H ions per liter
= 0.084 mg/mL
1 / 1000 mL
= 0.084 mg x 1000
= 84mg /mL
Potency of Product = 50 mg/mL
= (50/84) mEq/mL
= 0.5952 mEq/mL
MVD = Potency of a product x Endotoxin limit / Lysate sensitivity
= 0.5952 mEq/mL x 5 EU/mEq
0.125 EU/mL
= 23.8
MVD : 23.8
It is recommended to take MVD as 20.
6.2 Intermediates
121
MVD = 20
MVD/2 =10
MVD/4 = 5
MVD/8 = 2.5
Test Dilutions: 1:2.5, 1:5, 1:10 and 1: 20
Test Dilutions / Concentrations NPC PPC
1:2.5 (0.0625 mEq/mL) − − + +
1:5 (0.1250 mEq/mL) − − + +
1:10 (0.2501 mEq/mL) − − + +
1:20 (0.5002 mEq/mL) − − + +
++: Gel Formation − − : No Gel Formation
Conclusion: Carried out screening and observed Non Interfering Dilution
(NID) at 1/8 MVD (1:2.5). Non Interfering Concentration is (0.0625
mEq/mL). Endotoxins content is < 5u/ mEq.
6.2 Intermediates
122
End-Product Endotoxins Test79:
Product : Sodium Bicarbonate
Preparation : Product Concentration : 0.5952 mEq/mL
Endotoxin Limit : 5 EU/mEq
MVD : 23.8
Test Dilution : 1:5
Test Concentration : 0.1250 mEq/mL
Results : Test Endotoxin
Concentration EU/mL.
NEG
Control
NEG
Product
Control
Test
End
point.
Log of
End
point.
Geometric Mean
=A log (log of End
point.)
Material 2λ λ ½λ ¼λ EU/mL
+ + - − − 0.125 -0.9030
+ + - − − 0.125 -0.9030
+ + - − 0.125 -0.9030
CSE
Water
Control
+ + - − 0.125 -0.9030
+ + - − − 0.125 -0.9030
+ + - − − 0.125 -0.9030
+ + - − − 0.125 -0.9030
Positive
Product
Control
+ + - − − 0.125 -0.9030
Antilog
(-3.612/4)
=Antilog –0.903
=0.125EU/mL
Antilog
-3.612/4)
=Antilog –0.903
=0.125EU/mL
Interpretation :
Test results are : Valid
Comments : Product validation for Sodium Bicarbonate was carried out at
¼ MVD (1:5) (Test Concentration: 0.1250 mEq/mL) after adjusting the pH. The
GM of the end points in Endotoxin / LRW was 0.125EU/mL and
Endotoxin/product was 0.125 EU/mL which is in between 2λ and ½ λ, which
indicates no inhibition and enhancement at test dilution MVD / 4 (1:5).
123
6.3 Oil Based Injections:
Materials and Methods:
The prototypic examples of Endotoxins are lipopolysaccharides (LPS) or Lipo-
oligo-Saccharides (LOS) found in the outer membrane of various gram-
Negative bacteria and are an important cause of their ability to cause fever,
a lowering of blood pressure and activation of inflammation and
coagulation. Endotoxins are the large part responsible for the dramatic
clinical manifestations of infections with Pathogenic gram negative bacteria.
In pharmaceutical production, it is necessary to remove all traces of
Endotoxins from drug product containers as even small amounts of Endotoxins
will cause illness in humans.
A very sensitive assay for detecting presence of Endotoxins is the Limulus
Amoebocyte Lysate assay, utilizing blood from Horseshoe crab. Very low
amounts of LPS can cause coagulation of lysate due to a powerful
amplification through Enzymatic cascade.
Even though Lysate is very sensitive in Endotoxins detection but in case of
Hormones and Oil based products, we have observed false negative results
due to non availability of the Endotoxins to the Lysate. It was observed that
vigorous vortexing of the test sample in LRW with valid dilution will disperse the
Endotoxins into the aqueous media and thus available to lysate. We have
cross checked the technique by spiking known concentration of Endotoxins
into the sample and achieved 100% recovery upon analysis after vigorous
vortexing.
Lyophilized Limulus Amoebocyte Lysate of 0.0625 & 0.0312 sensitivity
(LAL), Control Standard Endotoxin 5 Eu/ng (CSE), LAL Reagent Water (LRW) of
Endosafe US, Depyrogenated (250°C for 30 min) 10 X 75 mm assay tubes,
16X100 mm dilution tubes, pyrogen free Micropipette tips, vortex mixture, 1N
NaoH, 1N Hcl, Calcium gluconate Injection & Amosil (Silicon Oil) were used for
determination of NID by gel clot technique.
The sensitivity of the Lysate (labeled 0.0625 & 0.0312) was determined
by using known amount of E.coli Control Standard Endotoxin.
6.3 Oil Based Injections
124
In the gel-clot techniques, the reaction end point is determined from dilutions
of the material under test in direct comparison with parallel dilutions or a
reference Endotoxin, and quantities of Endotoxins are expressed in Endotoxin
units.
1. Preparation of Standard stock solution and standard solutions: The CSE
having a defined potency of 50 EU/Vial was reconstituted with 5ml of LRW
and mixed intermittently for 30 minutes using a vortex mixture and this
concentrate was used to prepare 2λ, λ, λ/2 & λ/4, where λ is the labeled
claim sensitivity of Lysate.
2. Preparation of sample solution: Test samples were diluted to the required
concentrations based on the formulae MVD. MVD is the maximum valid
dilution, which is allowable dilution of the specimen at which the Endotoxin
limit can be determined. The general equation to determine MVD is
MVD = (Endotoxin limit X Concentration of sample solution)/ (λ).
Where E.L is the Endotoxin limit of the test sample, which is specified in the
individual monograph in terms of volume or units of active drug (in EU/mg).
3. Calcium gluconate Injection sample preparation: Potency=0.85 mg/ml,
E.L=0.17 EU/m, Lysate sensitivity is 0.625 Eu/mL and MVD = 2. The following test
dilutions are prepared by 1:1 (0.85 mg/ml) & 1:2 (0.42mg/mL).
4. Amosil (Silicon Oil) sample preparation: Potency=1 ml/ml, E.L=0.5 EU/ml,
Lysate sensitivity is 0.312 Eu/mL and MVD = 16. The following test dilutions are
prepared by 1:16, 1:8, 1:4 & 1:2.
Results and discussions:
Phase I: Preliminary Screening / interference Study25
In this two identical series of product dilutions (two-fold dilutions), one spiked
with 2λ, and one left unspiked. The result of Phase I will tell you the Non-
Interfering Dilution (NID) of the product. The Non-Interfering Dilution (NID) is
the first set of PPC that shows a gel.
6.3 Oil Based Injections
125
Calcium gluconate Injection: Inhibition at 1:1
Sample Dilution 1:1 1:2
Unspiked -- --
Spiked -- ++
Table:1 In the above assay is showing interference (Inhibition) at 1:1 dilution even in the spiked samples with known concentration of controlled standard Endotoxin. Amosil (Silicon Oil): Inhibition at 1:2
Sample Dilution 1:2 1:4 1:8 1:16
Unspiked -- ++ -- --
Spiked -- ++ ++ ++
Table:2 In the above assay is showing interference (Inhibition) at 1:2 dilution even in the spiked samples with known concentration of controlled standard Endotoxin. Calcium gluconate Injection: NID-1:1(1:2 selected for validation)
Sample Dilution 1:1 1:2
Unspiked -- --
Spiked ++ ++
Table:3 The sample was vorted vigoursly for 20 min, after vortexing the product is showing NID at 1:1 dilution. Amosil (Silicon Oil): NID-1:8 (1:16 selected for validation)
Sample Dilution 1:2 1:4 1:8 1:16
Unspiked ++ ++ -- --
Spiked ++ ++ ++ ++
Table:4 The sample was vorted vigoursly for 20 min, after vortexing the product is
showing NID at 1:8 dilution.
This assay shows no inhibition from 1:1dilution onwards in Calcium gluconate
Injection and 1:8 Amosil (Silicon Oil) and the spike recovery from the NID
onwards. It is advisable to validate the product next to MVD to take care of
any batch to batch variation during regular production in the
pharmaceutical industries.
6.4 Other Drug Molecules
126
Phase II: Validation of Product
For validation, test and compare two identical series of Endotoxin dilutions
bracketingλ; One prepared in LRW and another prepared in product diluted
to the proposed test dilution. Here dilution selected for validation is refer table
1. (Hot spike method).
Phase: II Results:
Calcium gluconate Injection Amosil (Silicon Oil)
Repl
icat
es
0.
125
Eu/m
L
0.
0625
Eu
/mL
0.
0312
Eu
/mL
0.
0156
Eu
/mL
0.
0625
Eu
/mL
0.
0312
Eu
/mL
0.
0156
Eu
/mL
0.
0078
Eu
/mL
1 + + - - + + - - 2 + + - - + + - - 3 + + - - + + - - 4 + + - - + + - -
Table: 5 Endotoxin/product; Negative product control: --; Geometric Mean = 0.0625
EU/ml for Calcium gluconate Injection & 0.0312 Eu/ml for Amosil (Silicon Oil).
Assay results of the products after spiking with known concentration of
Endotoxin are presented in Table 5.
Replicates 0.125 Eu/mL 0.0625 Eu/mL 0.0312 Eu/mL 0.0156 Eu/mL 1 + + - - 2 + + - - 3 + + - - 4 + + - -
Table: 6 Endotoxin/LRW; Negative product control: --; Geometric Mean = 0.0625 EU/ml. Assay results of label claim sensitivity of the lysate are presented in Table 3.
Replicates 0.0625 Eu/mL 0.0312 Eu/mL 0.0156 Eu/mL 0.0078 Eu/mL 1 + + - - 2 + + - - 3 + + - - 4 + + - -
Table: 7 Endotoxin/LRW; Negative product control: --; Geometric Mean = 0.0312 EU/ml.
Assay results of label claim sensitivity of the lysate are presented in Table 7.
Successful validation requires that both series confirm label claim (Geometric
mean) within +/- one two-fold dilution.
127
6.4 Other Drug Molecules:
Introduction:
During the research along with various drug substances, Intermediates, Drug
products and medical devices, we have studied the behavior of the other
drug products like anti-cancer drugs e.t.c in this chapter. The bacterial
Endotoxins test to be passed as per the respective monograph before
releasing these drugs into market but due to interfering factors in most of the
cases there is a probability of getting false positive results.
Materials and Methods:
Materials:
Lyophilized Limulus Amoebocyte Lysate of 0.125 sensitivity (LAL), Control
Standard Endotoxin 5 Eu/ng (CSE), LAL Reagent Water (LRW) of Endosafe US,
Depyrogenated (250°C for 30 min )10 X 75 mm assay tubes, 16X100 mm
dilution tubes ,pyrogen free Micropipette tips, vortex mixture, 1N NaoH, 1N
HCL, Gentamycin sulphate, Rantidine Hcl, Ringer lactate, Ondansetron Hcl
Injection, Insulin, Insugen N, Diclofenac sodium, Aristoneurol Injection and
CB-12 Injection were used for determination of Endotoxin content by the gel
clot technique.
The sensitivity of the Lysate (labeled 0.125 Eu/mL) was determined by using
known amount of E.coli Control Standard Endotoxin.
In the gel-clot techniques, the reaction end point is determined from dilutions
of the material under test in direct comparison with parallel dilutions or a
reference Endotoxin, and quantities of Endotoxins are expressed in Endotoxin
units.
1. Preparation of Standard stock solution and standard solutions: The CSE
having a defined potency of 50 EU/Vial was reconstituted with 5ml of LRW
and mixed intermittently for 30 minutes using a vortex mixture and this
concentrate was used to prepare 2λ, λ, λ/2 & λ/4, where λ is the labeled
claim sensitivity of Lysate.
2. Preparation of sample solution: Test samples were diluted to the required
concentrations based on the formulae MVD. MVD is the maximum valid
dilution, which is allowable dilution of the specimen at which the Endotoxin
limit can be determined. The general equation to determine MVD is
128
MVD = (Endotoxin limit X Concentration of sample solution)/ (λ). Where E.L is
the Endotoxin limit of the test sample, which is specified in the individual
monograph in terms of volume or units of active drug (in EU/mg).
3.Gentamycin Sulphate sample preparation: Batch No: GMS-0708,
Potency=80mg/2mL , E.L=1.67 Eu/mg, Lysate sensitivity is 0.125 Eu/mL and
MVD = 520. The following test dilutions are prepared by 1:520 (0.07 mg/mL),
1:260 (0.15 mg /mL), 1:130 (0.30 mg/mL), 1:65 (0.61 mg /mL) & 1:32 (1.25 mg
/mL).
4.Rantidine Hcl sample preparation: Batch No: RNH-0809, Potency=27.9
mg/mL , E.L= 7 Eu/mg , Lysate sensitivity is 0.125 Eu/mL and MVD = 1562. The
following test dilutions are prepared by 1:1560 (0.02 mg/mL), 1:780 (0.04
mg/mL), 1:390 (0.07 mg/mL) 1:195 (0.14 mg/mL) & 1:97 (0.29 mg/mL).
5.Ringer Lactate sample preparation: Batch No: RRL-0908, Potency= 1 mL/mL,
E.L=0.5 Eu/mL , Lysate sensitivity is 0.125 Eu/mL and MVD = 4. The following test
dilutions are prepared by 1:4, 1:2 & 1:1
6. Ondansetron Hcl Inj : Batch No: ONH-1008, Potency= 2 mg/mL, E.L=9.9
Eu/mg , Lysate sensitivity is 0.125 Eu/mL and MVD = 158. The following test
dilutions are prepared by 1:152 (0.013 mg/mL), 1:76 (0.026mg/mL), 1:38
(0.052mg/mL), 1:19 (0.105mg/mL) & 1:9.5 (0.210mg/mL).
7. Insulin sample preparation: Batch No: ISL-1108, Potency= 40 IU/ mL, E.L=
80Eu/100 = 0.8 Eu/IU, Lysate sensitivity is 0.125 Eu/mL and MVD = 256. The
following test dilutions are prepared by 1:256 (0.15 IU/mL), 1:128 (0.31 IU/mL),
1:64 (0.62 IU/mL), 1:32 (1.25 IU/mL) & 1:16 (2.5 mg/mL).
8. Insugen N (NPH) sample preparation: Batch No: ISN-1108, Potency= 40 IU/
mL, E.L= 80Eu/100 = 0.8 Eu/IU, Lysate sensitivity is 0.125 Eu/mL and MVD = 256.
The following test dilutions are prepared by 1:256 (0.15 IU/mL), 1:128 (0.31
IU/mL), 1:64 (0.62 IU/mL), 1:32 (1.25 IU/mL) & 1:16 (2.5 mg/mL).
6.4 Other Drug Molecules
129
9. Diclofenac Sodium sample preparation: Batch No: DCS-0109, Potency=25
mg/ mL, E.L= 4.60 Eu/mg , Lysate sensitivity is 0.125 Eu/mL and MVD = 920. The
following test dilutions are prepared by 1:920 (0.02 mg/mL), 1:460 (0.05
mg/mL), 1:230 (0.10 mg/mL), 1:115 (0.21 mg/mL) & 1:57 (0.44 mg/mL).
10. Aristoneurol Injection (Vitamin B1-B6-B12 Injection) sample preparation:
Batch No: ANI-0209, Potency= 1mL/ mL, E.L=116 Eu/mg , Lysate sensitivity is
0.125 Eu/mL and MVD = 928. The following test dilutions are prepared by
1:928, 1:464, 1:232, 1:116 & 1:58
11. CB - 12 Injection (CB-12 Injection consists of Ascorbic acid (Vitamin C))
sample preparation: Batch No: CBI-0209, Potency= 150 mg/ 1.5mL, E.L=1.2
Eu/mg, Lysate sensitivity is 0.125 Eu/mL and MVD = 960. The following test
dilutions are prepared by 1:960 (0.10 mg/mL), 1:480 (0.20 mg/mL), 1:240 (0.41
mg/mL), 1:120 (0.83 mg/mL) & 1:60 (1.66 mg/mL).
Results and discussions:
Gentamycin Sulphate: NID-1:32 (1:65 selected for validation)
Sample Dilution 1:32 1:65 1:130 1:260 1:520
Unspiked -- -- -- -- --
Spiked ++ ++ ++ ++ ++
Rantidine Hcl: NID-1:97 (1:195 selected for validation)
Sample Dilution 1:97 1:195 1:390 1:780 1:1560
Unspiked -- -- -- -- --
Spiked ++ ++ ++ ++ ++
Ringer Lactate NID-1:1 (1:2 selected for validation)
Sample Dilution 1:1 1:2 1:4
Unspiked -- -- --
Spiked ++ ++ ++
6.4 Other Drug Molecules
130
Ondansetron Hcl Injection : NID-1:9.5 (1:19 selected for validation)
Sample Dilution 1:9.5 1:19 1:38 1:76 1:152
Unspiked -- -- -- -- --
Spiked ++ ++ ++ ++ ++
Insulin: NID-1:32 (1:64 selected for validation)
Sample Dilution 1:16 1:32 1:64 1:128 1:256
Unspiked ++ -- -- -- --
Spiked ++ ++ ++ ++ ++
Insugen N (NPH): NID-1:32 (1:64 selected for validation)
Sample Dilution 1:16 1:32 1:64 1:128 1:256
Unspiked ++ -- -- -- --
Spiked ++ ++ ++ ++ ++
Diclofenac Sodium: NID-1:57(1:115 selected for validation)
Sample Dilution 1:57 1:115 1:230 1:460 1:920
Unspiked -- -- -- -- --
Spiked ++ ++ ++ ++ ++
Aristoneurol Injection: Vitamin B1-B6-B12 Injection)
NID-1:58 (1:116 selected for validation)
Sample Dilution 1:58 1:116 1:232 1:464 1:928
Unspiked -- -- -- -- --
Spiked ++ ++ ++ ++ ++
CB - 12 Injection: NID-1:240 (1:480 selected for validation)
Sample Dilution 1:60 1:120 1:240 1:480 1:960
Unspiked ++ ++ -- -- --
Spiked ++ ++ ++ ++ ++
Table: 1
This assay shows no inhibition from 1:32 in Gentamycin Sulphate, 1:97 in
Rantidine Hcl, 1:1 in Ringer Lactate,1:9.5 in Ondansetron Hcl Injection, 1:32 in
Insulin, 1:32 in Insugen N, 1:57 in Diclofenac Sodium, 1:58 in Aristoneurol
6.4 Other Drug Molecules
131
Injection and 1:240 in CB-12 Injection and the spike recovery from the NID
onwards. It is advisable to validate the product next to MVD to take care of
any batch to batch variation during regular production in the
pharmaceutical industries.
Phase II: Validation of Product
For validation, test and compare two identical series of Endotoxin dilutions
bracketingλ; One prepared in LRW and another prepared in product diluted
to the proposed test dilution. Here dilution selected for validation is refer table
1. (Hot spike method).
Phase: II Results:
Gentamycin Sulphate Rantidine Hcl
Repl
icat
es
0.
25
Eu/m
L
0.
125
Eu/m
L
0.
0625
Eu
/mL
0.
0312
Eu
/mL
0.
25
Eu/m
L
0.
125
Eu/m
L
0.
0625
Eu
/mL
0.
0312
Eu
/mL
1 + + - - + + - -
2 + + - - + + - -
3 + + - - + + - -
4 + + - - + + - -
Ringer Lactate Ondansetron Hcl Injection
Repl
icat
es
0.
25
Eu/m
L
0.
125
Eu/m
L
0.
0625
Eu
/mL
0.
0312
Eu
/mL
0.
25
Eu/m
L
0.
125
Eu/m
L
0.
0625
Eu
/mL
0.
0312
Eu
/mL
1 + + - - + + - -
2 + + - - + + - -
3 + + - - + + - -
4 + + - - + + - -
6.4 Other Drug Molecules
132
Insulin & Insugen N (NPH) Diclofenac Sodium
Repl
icat
es
0.25
Eu
/mL
0.
125
Eu/m
L
0.
0625
Eu
/mL
0.
0312
Eu
/mL
0.
25
Eu/m
L
0.
125
Eu/m
L
0.
0625
Eu
/mL
0.
0312
Eu
/mL
1 + + - - + + - -
2 + + - - + + - -
3 + + - - + + - -
4 + + - - + + - -
Aristoneurol Injection
(Vitamin B1-B6-B12 Injection) CB - 12 Injection
Repl
ica
tes
0.
25
Eu/m
L
0.
125
Eu/m
L
0.
0625
Eu
/mL
0.
0312
Eu
/mL
0.
25
Eu/m
L
0.
125
Eu/m
L
0.
0625
u/
mL
0.
0312
Eu
/mL
1 + + - - + + - -
2 + + - - + + - -
3 + + - - + + - -
4 + + - - + + - -
Table: 2 Endotoxin/product; Negative product control: --; Geometric Mean =
0.125 EU/ml.
Assay results of the products after spiking with known concentration of
Endotoxin are presented in Table 2.
Replicates 0.25 Eu/mL 0.125 Eu/mL 0.0625 Eu/mL 0.0312 Eu/mL
1 + + - -
2 + + - -
3 + + - -
4 + + - -
Table: 3 Endotoxin/LRW; Negative product control: --; Geometric Mean = 0.125
EU/ml.
Assay results of label claim sensitivity of the lysate are presented in Table 3.
Successful validation requires that both series confirm label claim (Geometric
mean) within +/- one two-fold dilution. Validation is conducted at this dilution
on three batches of product.
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