1. Purpose:-
It is established to
provide a procedure for the bacterial endotoxins testing in materials &
products by using end point chromogenic method.
2. Scope:-
This procedure is applicable
to those materials & products for which endotoxin testing is required.
3. Responsibilities:-
¹ Quality Control Manager
¹ Microbiologist
¹ Sr. Officer QC
4. Definitions:-
Bacterial
Endotoxins: Pyrogens are fever-producing materials that most often
originate from gram-negative bacterial cell walls, but can also originate as
leachates from some chemicals and materials. Pyrogens from bacterial cell walls
(the most commonly encountered type of pyrogen) are referred to as bacterial
endotoxin and are readily detected by Limulus Amebocyte Lysate (LAL) testing
systems.
What is LAL Test: Limulus amebocyte lysate (LAL)
is an aqueous extract of blood cells (amoebocytes)
from the horseshoe crab, Limulus polyphemus. LAL
reacts with bacterial endotoxin or lipopolysaccharide (LPS), which is a membrane
component of Gram negative bacteria. This reaction is the
basis of the LAL test, which is
used for the detection and quantification of bacterial endotoxins.
6. Description/Procedure:
Toxicolor Kit Components:
6.1 Endotoxins
Standard
6.1.1
One vial of endotoxin standard is
provided in the kit having potency 0.5EU/ml.
6.1.2
One vial of 2.8ml of LAL Reagent
Water is (LRW) also included in the kit for the reconstitution of standard.
6.1.3
Reconstitute the endotoxin
standard with 2.8ml of LRW for the preparation of 0.19EU/ml of endotoxin
standard.
6.1.4
Store reconstituted endotoxin solution
at 2-8˚C in a refrigerator
and don’t use this solution after 20% decline in absorbance compared with the
absorbance on first day of reconstitution.
6.2 Toxicolor Lysate (ES 24S
or 48S)
6.2.1
Reconstitute the test tube
containing lysate with 200µl of buffer provided in the kit. This lysate is
sufficient for four tests as 50µl is used per test.
6.2.2
Quickly stir to dissolve. Avoid
air bubbling or fuming during stirring.
6.2.3
Be sure that reagent is completely dissolved.
This reagent should be reconstituted just before use, however storage of
reconstituted lysate must be at -20 to -25˚C
in 50ul dispensed quantities in small test tubes. Use stored lysate if color is
not changed from white to yellowish.
6.3 Diazo Coupling Reagent
6.3.1
Four vials are provided in the kit
by the manufacturer named as 7s, 8s, 9s & 10s.
6.3.2
Transfer whole contents of the
vial 7s to vial 8s.
6.3.3
Add 12ml of distilled water in to
remaining vials number 9s & 10s.
6.3.4
So we come with three vials named
as 8s, 9s & 10s.
6.3.5
Store reconstituted vials at 2-8˚C in a refrigerator
6.4 TEST PROCEDURE
6.4.1
Adjust pH of the sample in between 5.0 – 7.0 with
Pyrogen free 0.1 N NaOH or 0.1 N HCl.
6.4.2
In case of dry powder injections,
make a 0.1% solution (50mg in 50ml Pyrogen free water). Fresh water from
distillation plant can be used for this purpose.
6.4.3
Mark the tubes as sample, standard
& blank.
6.4.4
Dispense 50ul test sample in tube
marked as sample, 50ul Pyrogen free water used during dilution / solution
preparation & 50ul of endotoxin standard in tube marked as standard.
6.4.5
Place the tube stand containing
tubes of sample, blank & standard in ice water bath for 5.0 minutes.
6.4.6
Add 50ul lysate into each tube of
sample, blank & standard.
6.4.7
Stir the contents of every tube
soon after the addition of lysate for few seconds.
6.4.8
Place the test tube rack in
incubator at 30 – 35˚C for
30 – 35 minutes.
6.4.9
After the completion of incubation
period place the test tube stand in ice water bath tray and add 0.25ml of diazo
coupling reagents one by one into each test tube from vial 8s, 9s & 10s.
Separate pipette must be used for dispensing of each solution.
6.4.10 Measure absorbance of the test sample and standard against blank
at 545±2nm.
Calculate endotoxins contents in the
sample according to following formula:
EU
/ ml = Potency of standard used ×
Absorbance of test sample
Absorbance of Standard
- Testing Frequency:-
Every third batch of each product or as and
when required in addition to bacterial endotoxin test by gel clot method.
8. Record:-
LAL TEST REPORT (End Point Chromogenic
Method)
9. References:-
Manufacturer’s Manual
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