1.1. To define a procedure and steps to be carried out bacterial endotoxin test
2. PRINCIPLE
2.1 It measures the concentration of bacterial endotoxins that may be present in the sample of the product to which the test is applied.
3. CONTROL STD. ENDOTOXIN
3.1 It is received Sales worth ……………..with the stated amount of Endotoxin Units present in one vial.
4. LAL REAGENT
4.1 Received from Sales worth with reported sensitivity of 0.125 EU/ml.
5. CONFORMITY OF SENSITIVITY OF LAL REAGENT
5.1 Confirm the sensitivity of LAL reagents using at least one vial of LAL reagent. Prepare a dilution of s, 0.5, and 0.25 Now it is 0.125 EU/ml. the test should be performed as follows :
Dilute CSE to have a dilution of 20EU/ml. now prepare stock solutions
4 = 0.1ml. CSE + 3.9 ml. of LRW ( Lal Reagent Water)
= 1ml.of 4 + 3ml. of LRW 5.2 Now take four test tubes in duplicate and follow the procedure as for testing samples.
|
Tube No.
|
LRW |
Stock soln. |
LAL REAGENT |
|
T1
|
------- |
100mcl of 4l |
100 mcl |
|
T2
|
50 mcl |
50mcl of 4l |
100 mcl |
|
T3
|
75 mcl |
25mcl of 4l |
100 mcl |
|
T4
|
87.5 mcl. |
12.5 mcl. of 4l |
100 mcl |
5.3 Interpretation: If the results found are
|
Trial |
Test tube no. 1 |
Test tube no. 2
|
|
T1 |
+ |
+
|
|
T2 |
+ |
+
|
|
T3 |
+ |
+
|
|
T4 |
+ |
-
|
Confirm the sensitivity of the LAL REAGENT.
6. TEST FOR INTERFERING FACTOR
6.1 For validation of test results, it must be demonstrated that the sample preparation does not inhibit or enhance the reaction or otherwise interfere with the test. The validation must be repeated if the lysate vendor or formulation of the sample is changed.
7. PREPARATION OF STAND SOLUTION
Prepare a stock solution for 4l and dilution as described earlier.
8. PREPARATION OF TEST DILUTION
8.1 Test Dilution
Limit: Not more than 1.0 E.U./mg.
Thiopental
Sodium : mvd: 1000
mg x
1.0 E.U./ml. = 8000
0.125 E.U./ml.
make dilution 20 mg. in 2 ml. WFI (1st dilution)
0.01 ml. and 1.99 ml. WFI. (2nd dilution)
0.01 ml. and 1.99 ml. WFI. (final dilution)
a) Oxytetracycline Inj. & Oxytetracycline V Bet C Injection.
Limit: Not more than 0.4 EU/mg
MVD = 0.4
EU/mg.x 50mg./ml. = 160
0.125 EU/ml.
make a dilution of 1 in 80 0.10ml. of Inj. +8.90 ml. of LRW
b) Genty Injection (Gentamicin Inj.)
Limit: Not more than 1.7 EU/mg
MVD = 1.7EU/mg x
40mg./ml. = 544
0.125 EU/ml.
make a dilution of 1 in 250
i.e. 0.025 ml. 12.475 ml. of LRW
c) Water for Inj.
Limit: Not more than 0.25 EU/ml
MVD =
0.25EU/ml. = 2
0.125EU/ml.
make dilution 1 in 1 i.e.use as such.d) Pyrimine-12 Inj. : MVD = 0.4 EU/mg. x 50mg./ml. =160
0.125 EU/ml.
8.2 NOW TESTS AS FOLLOWS IN DUPLICATE
|
|
LRW |
DILUTIONS |
LAL REAGENT |
|
NWC |
100mcl |
-------- |
100mcl |
|
PWC |
50mcl |
50mcl of 4l |
100mcl |
|
NPC |
50mcl |
50mcl of test dilution+ 50mcl of 4l |
100mcl |
Note: Take these solns. in 10mm x 75mm depyrogenated tubes
9.0 PROCEDURE
Mix gently and place in the incinerator at 37 Deg. + 1 Deg. undisturbed for 60 + 2 minutes. Remove tubes gently and examine the contents carefully. A positive reaction is characterized by the formation of a firm gel that retains its integrity when inverted through 180 Deg. A negative formation of a viscous gel that does not maintain its integrity. Record such results as negative.
10. INTERPRETATION OF RESULTS
10.1 The sample complies with the test if ppc is +ve, PWC is positive and negative controls are negative. The test is invalid if ppc is negative or if any negative control is positive if ppc is positive and PWC, NPC are positive and negative blank is negative then the product fails the test.
11. PRECAUTIONS TO BE TAKEN
11.1 Use depyrogenated apparatus.
11.2 Use lower dilutions first then move towards higher dilutions.
11.3 Tube from the heating blocks should be then out gently.
END OF DOCUMENT
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