Code : APL/MIB/STP-012
ARBRO PHARMACEUTICALS Issue No. : 04
PRIVATE LIMITED Effective Date : 15-Jun-2019
Review Date : 14-Jun-2022
Analytical Division, 4/9, Kirti Nagar, Industrial Area New Delhi - 110015
STANDARD TESTING PROCEDURE
TITLE : MICROBIOLOGICAL ASSAY OF AMIKACIN SULFATE BY TUBIDITY
AS PER IP
OWNER(S): SECTION IN CHARGE
DOCUMENT APPROVAL
AUTHORITY NAME JOB TITLE SIGNATURE DATE
Author Mr. Pratee Tyagi Junior Microbiologist
Process Owner Dr. Anurup Adak Section In charge
Deputy Quality
Reviewer Ms. Shikha Pandey
Manager
Approver Dr. Saurabh Arora Executive Director
DOCUMENT DISTRIBUTION
COPY NO. COPY HOLDER LOCATION
5 Analytical Division (Quality Assurance) 4/9, Kirti Nagar Industrial Area
5/3 Analytical Division (Microbiology Section) 4/9, Kirti Nagar Industrial Area
CROSS REFERENCES:
APL/QA/SOP-062 Standard operating procedure for SOPs & STPs
APL/QA/SOP-073 Awareness/ Training on Documents
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Code : APL/MIB/STP-012
ARBRO PHARMACEUTICALS Issue No. : 04
PRIVATE LIMITED Effective Date : 15-Jun-2019
Review Date : 14-Jun-2022
MICROBIOLOGICAL ASSAY OF AMIKACIN SULFATE BY TUBIDITY AS PER IP
CONTENTS
0.0 VERSION CONTROL HISTORY------------------------------------------------------------------------------2
1.0 OBJECTIVE---------------------------------------------------------------------------------------------------------3
2.0 SCOPE--------------------------------------------------------------------------------------------------------------- 3
3.0 RESPONSIBILITY AND ACCOUNTABILITY--------------------------------------------------------------3
4.0 REQUIREMENTS------------------------------------------------------------------------------------------------- 4
4.1 TEST ORGANISMS ---------------------------------------------------------------------------------------------4
4.2 STANDARD AND TEST DILUTION -------------------------------------------------------------------------4
4.3 SAMPLE PREPARATION--------------------------------------------------------------------------------------4
4.4 PROCEDURE-----------------------------------------------------------------------------------------------------5
4.5 ESTIMATION OF POTENCY---------------------------------------------------------------------------------5
5.0 ABBREVIATIONS-------------------------------------------------------------------------------------------------6
0.0 VERSION CONTROL HISTORY
Date Issue No. CCR Change description / reason / supersedes
24-Sep-2012 01 Nil New document
24-Sep-2015 02 Nil Periodic Review
21-Feb-2016 03 Management/ Company converted from Public Limited
CCP/APL/ to Private Limited
QA/QM-
001/1640
12-Jun-2019 04 Nil Periodic Review
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Code : APL/MIB/STP-012
ARBRO PHARMACEUTICALS Issue No. : 04
PRIVATE LIMITED Effective Date : 15-Jun-2019
Review Date : 14-Jun-2022
MICROBIOLOGICAL ASSAY OF AMIKACIN SULFATE BY TUBIDITY AS PER IP
1.0 OBJECTIVE
The purpose of this standard testing procedure is to describe the methodology for
Microbiological assay of Amikacin sulfate by Turbidity.
2.0 SCOPE
It is the policy of Arbro Pharmaceuticals Private Limited that a written procedure shall be
followed for Microbiological assay of Amikacin sulfate by Turbidity.
3.0 RESPONSIBILITY AND ACCOUNTABILITY
Persons along with their responsibilities are given below:
Sr. No. Designation Responsibility and Accountability
Trainee Analyst/ Jr. Analyst/
01 To prepare and follow the STP.
Analyst/ Sr. Analyst
02 Section In charge To follow and ensure the implementation of the STP
03 Technical Manager To ensure the implementation of the STP
04 Deputy Quality Manager To ensure the implementation of the STP
05 Executive Director To ensure the implementation of the STP
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Code : APL/MIB/STP-012
ARBRO PHARMACEUTICALS Issue No. : 04
PRIVATE LIMITED Effective Date : 15-Jun-2019
Review Date : 14-Jun-2022
MICROBIOLOGICAL ASSAY OF AMIKACIN SULFATE BY TUBIDITY AS PER IP
4.0 REQUIREMENTS
4.0.1 Micropipettes (1ml, 0.1ml)
4.0.2 Volumetric Flasks (100ml)
4.0.3 Conical Flasks
4.0.4 Test Tubes
4.0.5 Test Tubes Stand
4.0.6 Antibiotic Assay Media-3 (Himedia M042-500G)
4.0.7 BOD Incubator 32.5±1°C
4.0.8 Culture Staphylococcous aureus ATCC29737.
4.0.9 Weighing Balance
4.0.10 Spectrophotometer
4.0.11 Measuring Cylinder (100 & 250 ml)
4.0.12 Graduated Pipette (10ml)
4.0.13 Bulb Pipette (5 & 2ml)
4.1 TEST ORGANISMS
Maintain the test organism on slants of Nutrient Agar.
4.2 STANDARD & TEST DILUTION
Weigh equivalent 100mg of Amikacin in 100ml volumetric flask make up volume with water.
Dilute10ml of this solution to 100ml with water(s). Further dilute the resulting solution in
following manner.
DILUTIONS
6.4 ml of (S) to 100 ml (With Water 6.4mcg/ml) ---------S1
8.0 ml of (S) to 100 ml (With Water 8.0mcg/ml) ---------S2
10.0 ml of (S) to 100 ml (With Water 10.0mcg/ml) --------S3
12.5 ml of (S) to 100 ml (With Water 12.5mcg/ml) --------S4
15.6 ml of (S) to 100 ml (With Water 15.6mcg/ml) --------S5
4.3 SAMPLE PREPARATION
Transfer an accurately measured quantity of the test sample equivalent to 250mg of
Amikacin and dissolve in water to produce 250ml. Dilute 10ml of this solution to 100ml with
water. Further dilute 10ml of this resulting solution to 100ml with water i.e (10mcg/ml)
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Code : APL/MIB/STP-012
ARBRO PHARMACEUTICALS Issue No. : 04
PRIVATE LIMITED Effective Date : 15-Jun-2019
Review Date : 14-Jun-2022
MICROBIOLOGICAL ASSAY OF AMIKACIN SULFATE BY TUBIDITY AS PER IP
PROCEDURE
Add culture suspension of staphylococcus aureus (0.1ml per 100ml) to the sterilized
antibiotic assay media -3. Place 1 ml of each concentration of standard solution (S1 to S5)
and of the samples solution (T) in each of the tubes in duplicates. Add to each tube 9ml of
nutrient medium previously seeded with test organism.
At the same time, prepare three control tube, one containing the inoculated culture media
(culture control), and another identical with it but treated immediately with 0.5ml of dilute
formaldehyde solution (Blank), add a third containing uninoculated culture media.
Place all the tubes in an incubator maintain them at 32.5°C for three – four hrs. After
incubation add 0.5 ml of dilute formaldehyde solution to each tube. Measure the growth of
the test organism by determining the absorbance at about 530nm of each of the solution in
the tubes against the blank.
4.5 ESTIMATION OF POTENCY
Average the reading of the absorbance of 5 standard and test solution. The straight
response line is drawn through the points plotted for highest and lowest absorbance;
obtained by means of the following expressions:
L= 3a + 2b + c-e H= 3e + 2d + c-a
5 5
Where L = the calculated absorbance for the lowest concentration of the standard curve
response line.
H = the calculated absorbance for the highest concentration of the standard curve response
line.
a, b, c, d, e = average absorbance values for the standard solution, lowest to highest
concentration, respectively.
From the response line read the concentration of samples corresponding to the values of
Absorbance. Multiply the concentration by the appropriate dilution factor to obtain the
antibiotic content of the sample.
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ARBRO PHARMACEUTICALS Issue No. : 04
PRIVATE LIMITED Effective Date : 15-Jun-2019
Review Date : 14-Jun-2022
MICROBIOLOGICAL ASSAY OF AMIKACIN SULFATE BY TUBIDITY AS PER IP
5.0 ABBREVIATIONS
APL : Arbro Pharmaceuticals Private Limited
SOP : Standard Operating Procedure
QA : Quality Assurance
STP : Standard Testing Procedure
BOD : Biochemical Oxygen Demand
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