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FEROZSONS LABORATORIES LIMITED

Product Analytical Method


PAM No.

PAM - 042 - 02

Effective Date
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1 of 41

Analytical Method of Rifaxa 200 mg Tablets


Title

Signature

Written by

Deputy Manager Quality Control

Reviewed &
Approved by

Manager Quality Control

Reviewed &
Approved by

Sr. Manager Quality Assurance

Authorized by

Director Quality Operations

Product Code No:

03 - 07 - 0054

Registration No:

068205

Shelf Life:

02 Years

Reference:

In house

Date

Declaration:
Each film coated tablet contains:
Rifaximin . 200 mg

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Analytical Method of Rifaxa 200 mg Tablets


PRODUCT SPECIFICATIONS AT TABLETTING MIXTURE STAGE:

Tests
Appearance

Specifications
Reddish granular, mixed powder.
Absorption spectrum obtained from sample preparation

Identification

and reference preparation shows absorption maximum at


wave length 235 nm.

Assay (Rifaximin)
Antimicrobial activity
(Microbial assay)

90 % 110 %
90 % 110 %

APPEARANCE:

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Analytical Method of Rifaxa 200 mg Tablets

1. Principle

Visual evaluation of color & shape.

2. Evaluation

The color must be as specified with no foreign matter.

3. Requirement

Reddish granular, mixed powder.

IDENTIFICATION (Rifaximin):

1. Principle

To identify Rifaximin.

2. Evaluation

The maximum absorbance of sample and reference standard


must be at wavelength maxima 235 nm.

3. Requirement

Absorption spectrum obtained from sample preparation and


reference preparation shows absorption maximum at wave
length 235 nm.

ASSAY (Rifaximin):
1. Principle

The quantitative analysis of Rifaximin.

2. Evaluation

The analysis of Rifaximin is as per in house method.

3. Requirement

90 % 110 %

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Analytical Method of Rifaxa 200 mg Tablets

4. Procedure

As under.

Apparatus:

Volumetric flasks

Spectrophotometer

Beaker

Mortar & pestle

Spatula

Analytical balance

Pipette 1 ml

Flask shaker

Funnel

Reagents:

Acetonitrile

Distilled water

Rifaximin (Working Standard)

Preparation of Diluent:
Mix distilled water and acetonitrile in ratio of 60: 40

Sample preparation:
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Analytical Method of Rifaxa 200 mg Tablets


Grind the granular mixed powder into finely powder. Transfer an accurately weighed portion
of the powder equivalent to 100 mg of Rifaximin in a 100 ml volumetric flask. Add
approximately 80 ml of diluent and shake for 40 minutes. Make volume with diluent. Filter
and discard first few ml of the filtrate. Dilute 1 ml of the filtrate to 100 ml with diluent .
Reference standard preparation:
Transfer an accurately weighed 100 mg of Rifaximin working standard in a 100 ml
volumetric flask and add about 80 ml diluent. Shake for 30 minutes to dissolve and make
volume up to the mark with diluent. Dilute 1 ml of this solution to 100 ml with diluent.

Procedure:
Measure the absorbance of the final solutions of sample and standard at 235 nm using
diluent as blank.

Calculations:
Absorbance of the sample
% age = ------------------------------------- x 100
Absorbance of the standard

ANTIMICROBIAL ACTIVITY MICROBIAL ASSAY:

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Analytical Method of Rifaxa 200 mg Tablets

1. Principle

Determination of potency of Rifaxa 200 mg Tablets.

2. Evaluation

Microbial analysis of Rifaxa 200 mg Tablets is as per WHO.


Disc Diffusion (Kirby-Bauer Method)

3. Requirement

90 % 110%.

4. Procedure

As under.

Apparatus & Materials:

Incubator

Autoclave

Face Mask

Sterile Forcep

Sterile Wire Loop

Dry Heat Sterilizer

Micropipette 20 L

Sterile Latex Gloves

Vernier Caliper / Ruler

Heat Resistant Gloves

Screw-capped Test Tubes

Plan Antibiotic Discs 6 mm

Bunsen burner / Sprit Lamp

Biological Safety Cabinet (B.S.C)

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Analytical Method of Rifaxa 200 mg Tablets


Culture Media & Reagents:

Tryptic Soy Broth

Specific ATCC Culture

Filtered 70% Isopropyl Alcohol spray bottle

0.5% MacFarland Assay / Turbidity Standard

Antibiotic Medium No. 11 / Mueller Hinton Agar

Commercially Prepared Specific Antibiotic Discs 6 mm

Sterile pH 7.2 Phosphate Buffer / Tryptic Soy Broth / Sterile Peptone Water

Preparation of Reference Standard Antibiotic Discs:


Antibiotic plain discs (having diameter of 6 mm) are used in this method and before
preparation of antibiotic disc, sterilize them by placing in dry heat oven at 100 C for 30
minutes 1 hour.
Standard preparation is as follow:
Standard Preparation:
Standard Disk Concentration = 40 g

Dilution Method (Lab prepared standard):


First Dilution = Weigh 100 mg accurately and dissolve it into 50 mL of filtered
acetonitrile and H2O (60 : 40).
Second Dilution = Then take 20 L and place on to sterile 6 mm disc to achieve
required centration on disc.

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Analytical Method of Rifaxa 200 mg Tablets


01st Dilution

Final Conc.
6 mm Disc
20 L

100 mg : 50 mL

0.04 mg

Preparation of Sample Discs:


Antibiotic plain discs (having diameter of 6 mm) are used in this method and before
preparation of antibiotic disc, sterilize them by placing in dry heat oven at 100 C for 30
minutes 1 hour.
Sample preparation is as follow:
Dilution Method of Rifaxa 200 mg Tablets (Rifaximin)
Sample Preparation:
Standard Disk Concentration = 40 g

Dilution Method:
Grind the granular powder into fine powder and weigh accurately 330 mg of mixed
powder equivalent to 200 mg of Rifaximin.
First Dilution = Dissolve it into 100 mL of the mixture of filtered acetonitrile and H2O
(60 : 40).
Second Dilution = Then take 20 L and place on to sterile 6 mm disc.

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Analytical Method of Rifaxa 200 mg Tablets


01st Dilution

Final Conc.
6 mm Disc
20 L

200 mg : 100 mL

0.04 mg

Applying Disc Method:


After preparing microbial suspension, dip sterile swab to the bacterial suspension
tube.
Inoculate the plates by dipping a sterile swab into the inoculum, remove excess
inoculum by pressing and rotating the swab firmly against the side of the tube above
the level of the liquid.
Streak the swab all over the surface of the Mueller Hinton Agar Medium (M H Agar)
three times for making complete lawn of bacterial suspension (see Fig. 1 given
below), streaking the plate through an angle of 60 after each streaking stroke, and
finally, pass the swab round the edge of the agar surface.

Leave the inoculum to adjust & dry for 3 5 minutes (not more than 15 minutes) at
room temperature with the lid closed under the B.S.C.
After adjustment of inoculum with the agar, place antibiotic sample discs on the
inoculated plates using a sterile forcep along with standard antibiotic disc aseptically
under the B.S.C.
After placing each and every required disc, press gently with forcep to make sure
even contact with agar medium.

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Analytical Method of Rifaxa 200 mg Tablets


It is convenient to use a template (See Fig. 2a given below) to place the discs
uniformly.
Incubate the petri plate containing sample at the 30 35 C for 16 24 hours
(overnight) within 30 minutes after applying discs.
After incubation, observe the zone of inhibition around the sample and standard
antibiotic discs, and measure them with Vernier Caliper or with measuring scale (see
Fig. 3 given below) and report zones in millimeter (mm).
Use following formula to calculate the results in percentage;

%age

Zone of Sample (mm)

Zone of standard (mm)

100

Prepare duplicate discs and place on to the medium surface in Triangular Shape
(see Fig. 2b) / Linear Direction (see Fig. 2c given below).

Figure 1 Template for making bacterial lawn (90 mm Diameter of Plate).

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Analytical Method of Rifaxa 200 mg Tablets

Figure 2a Template for applying antibiotic discs (90 mm Diameter of Plate)

Figure 2b Template for applying antibiotic discs in Triangular Shape (90 mm Diameter of Plate)

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Analytical Method of Rifaxa 200 mg Tablets

Figure 2c Template for applying antibiotic discs in Linear Shape (90 mm Diameter of Plate)

Antibiotic Disc
Zone of Inhibition

Figure 3 Template for measuring zone of inhibition in 2 way directions

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form without the prior written permission from DQO Ferozsons Laboratories Limited.

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Analytical Method of Rifaxa 200 mg Tablets


PRODUCT SPECIFICATIONS AT CORE STAGE:

Tests
Appearance

Specifications
Reddish round, biconvex tablets with break line on one side
and plain other side.

Shape
Average Weight
Weight variation

330 mg 5 %
Minimum 18/20 + 5 %
Maximum 02/20 + 10 %

Thickness

4.80 mm 5.05 mm 5.30 mm

Hardness

100 N 125 N 150 N

Friability/ Breakage

NMT. 1.0 %/ Nil

Disintegration Time

NMT. 15.0 minutes

Dissolution

NLT 80 % (Q) of the labeled amount of Rifaximin is


dissolved.
Absorption spectrum obtained from sample preparation and

Identification

reference preparation shows absorption maximum at wave


length 235 nm.

Assay (Rifaximin)
Antimicrobial activity
(Microbial assay)

Reviewed & Approved by: Sr. MQA

90 % 110 %
90 % 110 %

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Laboratories Limited. It cannot be published or divulged for whatever purpose to unauthorized persons, or third parties, in any
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Analytical Method of Rifaxa 200 mg Tablets


APPEARANCE:
1. Principle

Visual evaluation of color & shape.

2. Evaluation

The color & shape must be as specified with no foreign matter.

3. Requirement

Reddish round, biconvex tablets with break line on one side


and plain other side.

AVERAGE WEIGHT:

1. Principle

Represents tablet weight in the batch formulation of product.

2. Evaluation

The average weight of tablets evaluated by the randomly


sampled 20 tablets and weigh on balance, and divide this
weight on the number of taken tablets.
Average weight = Weight of 20 tablets
20

3. Apparatus

Analytical balance.

4. Requirement

330 mg 5 %

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Analytical Method of Rifaxa 200 mg Tablets


CONTROL OF WEIGHTS:

1. Principle

Weigh the prescribed number of tablets individually on an


analytical balance and calculate the mean weight.
Calculate the percentage deviations of the actual mean weight
to the theoretical average weight.

2. Apparatus

Analytical balance.

3. Test performance

Sample size:
- 20 tablets per sample

4. Evaluation

The actual mean value should not deviate more than the
specified limit to the theoretical weight.
Weigh accurately 20 tablets and calculate average weight,
then weigh individually 20 tablets.
The difference in percentage from minimum weight to
maximum weight of individual tablets should not be more than
the specified limits.
Max. deviation = Max. tablet weight Avg. weight found
Avg. weight found
Min. deviation = Min. tablet weight Avg. weight found
Avg. weight found

5. Requirement

Minimum 18/20 + 5 %
Maximum 02/20 + 10 %

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Analytical Method of Rifaxa 200 mg Tablets


THICKNESS / DIAMETER:
1

Principle

Dimension determination of tablets.

2. Apparatus

Pharma test hardness and thickness tester

3. Method

Switch on the instrument. Display light on the instrument will


show as:
Nr

Thickness

Diameter

Hardness

00

00.00

00.00

00.00

To set value of thickness, Press thickness, enter required


value and then press * to set value.
To set value of diameter, Press diameter, enter required value
and then press * to set value
Place the sample into PTB thickness test station at right
direction.
Now press the START
All tests for thickness, diameter and hardness would be
performed simultaneously.
Take readings of five tablets and calculate the average.
(5 tablets to be checked and reading to be noted)
4. Equipment

It must meet the requirements that are drawn up for each


product.

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Analytical Method of Rifaxa 200 mg Tablets


5. Frequency

For each batch.

6. Requirement

Thickness: 4.80 mm 5.05 mm 5.30 mm


Diameter: 9.50 mm

HARDNESS:
1

Principle

Hardness determination of tablets.

2. Apparatus

Pharma test hardness and thickness tester

3. Method

Switch on the instrument. Display light on the instrument will


show as:
Nr

Thickness

Diameter

Hardness

00

00.00

00.00

00.00

To set value of hardness, Press hardness, enter required value


and then press * to set value.
Place the sample into PTB thickness test station at right
direction.
Now press the START
All tests for thickness, diameter and hardness would be
performed simultaneously
Take readings of five tablets and calculate the average.
(5 tablets to be checked and reading to be noted)
4. Equipment

It must meet the requirements that are drawn up for

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Analytical Method of Rifaxa 200 mg Tablets


each product.

5. Frequency

For each batch.

6. Requirement

Hardness: 100 N 150 N

FRIABILITY AND RESISTANCE TO ROLL WARE AND TARE CORE / TABLETS:

1. Principle

Sliding and dropping in a rotating drum.

2. Requirements

- Friability:

Not more than 1.0 %.

- Breakage: Nil.
3. Apparatus:

Friability test apparatus

4. Method

For tablets with a unit weight equal to or less than 650 mg,
take a sample of whole tablets corresponding as near as
possible to 6.5 g. For tablets with a unit weight of more than
650 mg, take a sample of 10 whole tablets. Dusted tablets and
placed in a drum. Close the drum and fix it on the shaft in such
a way that on rotating, the tablets are taken up by the concave
side of the blade. Allow the drum to rotate 100 times (or as per
specifications).
Place the contents of the drum on the sieve and examine the
tablets for breakage. Dust off the abrased material and sieve.

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Analytical Method of Rifaxa 200 mg Tablets


Accurately weigh the residue to within 10 mg and calculate the
friability. If the friability is greater than the requirement, the test
is complete; the requirement is not fulfilled.
If the requirement for friability is fulfilled and not more than
allowed tablets show breakage, the test is complete; the
requirements are fulfilled.
Evaluation and calculation:
- Friability: All parts of the tablets (abrased powder and
fragments) are considered as abrasion.

Percentage friability =

B-C

100

B
B

Weight (g) of the tablets

Weight (g) of the sieve residue.

100

Conversion factor to percent.

- Breakage: The following are considered as breakage:


- Broken tablets
- Tablets with damage edges, if their minimum diameter is
less than 4/5 of the declared diameter;
- Tablets with upper or lower sides split off (capping);
- Tablets with upper or lower sides partially split off (in the
case of scored tablets).
DISINTEGRATION TIME:
1. Apparatus

Disintegration Apparatus

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Analytical Method of Rifaxa 200 mg Tablets


2. Method

Fill sufficient water into the vessel so that during motion, the
gauze does not come within less than 2.5 cm of the surface of
the liquid warm the water to 37 + 2oC.
Place one tablet in each of the 6 glass tubes and operate the
apparatus and determine the time taken for each tablet to
disintegrate.

3. Requirement

Not more than 15 minutes


The requirement is fulfilled if all 6 tablets have disintegrated
in the prescribed time.
If 1 or 2 tablets fail to disintegrate completely, repeat the
test on 12 additional tablets. The requirement is met if not
less than 16 of the total of 18 tablets tested are
disintegrated.

DISSOLUTION:
1. Apparatus

Dissolution Apparatus # 2.

2. Method

Medium: 0.1 M Phosphate buffer pH 7.4


Preparation of 0.1 M Phosphate buffer pH 7.4:
Dissolve 15.6 gm of Sodium dihydrogen Phosphate and 4.5
gm of Sodium Lauryl sulphate in 100 ml of water. Check the
pH to be 7.4.
Dissolution conditions:

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Analytical Method of Rifaxa 200 mg Tablets


Volume: 1000 ml
Rpm: 75
Temperature: 37 + 2oC
Time: 45 minutes

Filled the 6 vessels of dissolution apparatus each vessel


contains 1000 ml medium. Maintain the said temperature of
the vessels medium.
Sample preparation:
Place 6 tablets individually in six dissolution vessels containing
1000 ml of Phosphate buffer pH 7.4 that has been equilibrated
to 37 C 2C. Take care to exclude air bubbles from the
surface of the tablet, start the apparatus immediately. Collect
the sample after the specified time (45 minutes), withdraw the
sample from zone midway between the surface of the medium
and top of the rotating paddle and not less than 1 cm from the
vessel wall and filter through 0.45 membrane filter and discard
the first few ml.
Dilute 5 ml of the filtrate to 100 ml with diluent.
Standard preparation:
Dissolve an accurately weighed 100 mg of Rifaximin working
standard in 100 ml volumetric flask in about 80 ml of diluent.
Shake for 10 minutes and dilute to volume with diluent. Further
dilute 1 ml of this solution to 100 ml with diluent.
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Analytical Method of Rifaxa 200 mg Tablets


Preparation of Diluent:
Mix distilled water and acetonitrile in ratio of 60: 40

Procedure:
Measure the absorbance of the final solutions of sample and
standard at 235 nm using distilled diluent as blank.
3. Requirement

NLT 80 % (Q) of the labeled amount of Rifaximin is


dissolved.

4. Calculation

Absorbance of sample
%age = -------------------------------- x 100
Absorbance of standard

IDENTIFICATION (Rifaximin):

1. Principle

To identify Rifaximin.

2. Evaluation

The maximum absorbance of sample and reference standard


must be at wavelength maxima 235 nm.

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Analytical Method of Rifaxa 200 mg Tablets


3. Requirement

Absorption spectrum obtained from sample preparation and


reference preparation shows absorption maximum at wave
length 235 nm.

ASSAY (Rifaximin):
1. Principle

The quantitative analysis of Rifaximin.

2. Evaluation

The analysis of Rifaximin is as per in house method.

3. Requirement

90 % 110 %

4. Procedure

As under.

Apparatus:

Volumetric flasks

Spectrophotometer

Beaker

Mortar & pestle

Spatula

Analytical balance

Pipette 1 ml

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Analytical Method of Rifaxa 200 mg Tablets

Flask shaker

Funnel

Reagents:

Acetonitrile

Distilled water

Rifaximin (Working Standard)

Preparation of Diluent:
Mix distilled water and acetonitrile in ratio of 60: 40
Sample preparation:
Weigh and finely powder not less than 20 tablets. Transfer an accurately weighed portion of
the powder equivalent to 100 mg of Rifaximin in a 100 ml volumetric flask. Add
approximately 80 ml of diluent and shake for 40 minutes. Make volume with diluent. Filter
and discard first few ml of the filtrate. Dilute 1 ml of the filtrate to 100 ml with diluent .
Reference standard preparation:
Transfer an accurately weighed 100 mg of Rifaximin working standard in a 100 ml
volumetric flask and add about 80 ml diluent. Shake for 30 minutes to dissolve and make
volume up to the mark with diluent. Dilute 1 ml of this solution to 100 ml with diluent.

Procedure:
Measure the absorbance of the final solutions of sample and standard at 235 nm using
diluent as blank.

Calculations:
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Analytical Method of Rifaxa 200 mg Tablets

Absorbance of the sample


% age = ------------------------------------- x 100
Absorbance of the standard

ANTIMICROBIAL ACTIVITY MICROBIAL ASSAY:


1. Principle

Determination of potency of Rifaxa 200 mg Tablets.

2. Evaluation

Microbial analysis of Rifaxa 200 mg Tablets is as per WHO.


Disc Diffusion (Kirby-Bauer Method)

3. Requirement

90 % 110%.

4. Procedure

As under.

Apparatus & Materials:

Incubator

Autoclave

Face Mask

Sterile Forcep

Sterile Wire Loop

Dry Heat Sterilizer

Micropipette 20 L

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Analytical Method of Rifaxa 200 mg Tablets

Sterile Latex Gloves

Vernier Caliper / Ruler

Heat Resistant Gloves

Screw-capped Test Tubes

Plan Antibiotic Discs 6 mm

Bunsen burner / Sprit Lamp

Biological Safety Cabinet (B.S.C)

Culture Media & Reagents:

Tryptic Soy Broth

Specific ATCC Culture

Filtered 70% Isopropyl Alcohol spray bottle

0.5% MacFarland Assay / Turbidity Standard

Antibiotic Medium No. 11 / Mueller Hinton Agar

Commercially Prepared Specific Antibiotic Discs 6 mm

Sterile pH 7.2 Phosphate Buffer / Tryptic Soy Broth / Sterile Peptone Water

Preparation of Reference Standard Antibiotic Discs:


Antibiotic plain discs (having diameter of 6 mm) are used in this method and before
preparation of antibiotic disc, sterilize them by placing in dry heat oven at 100 C for 30
minutes 1 hour.
Standard preparation is as follow:
Standard Preparation:
Standard Disk Concentration = 40 g

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Analytical Method of Rifaxa 200 mg Tablets

Dilution Method (Lab prepared standard):


First Dilution = Weigh 100 mg accurately and dissolve it into 50 mL of filtered
acetonitrile and H2O (60 : 40).
Second Dilution = Then take 20 L and place on to sterile 6 mm disc to achieve
required centration on disc.

01st Dilution

Final Conc.
6 mm Disc
20 L

100 mg : 50 mL

0.04 mg

Preparation of Sample Discs:


Antibiotic plain discs (having diameter of 6 mm) are used in this method and before
preparation of antibiotic disc, sterilize them by placing in dry heat oven at 100 C for 30
minutes 1 hour.
Sample preparation is as follow:
Dilution Method of Rifaxa 200 mg Tablets (Rifaximin)
Sample Preparation:
Standard Disk Concentration = 40 g

Dilution Method:

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Analytical Method of Rifaxa 200 mg Tablets


Crush 20 tablets and weigh accurately 330 mg of fine powder equivalent to 200 mg
of Rifaximin.
First Dilution = Dissolve it into 100 mL of the mixture of filtered acetonitrile and H2O
(60 : 40).
Second Dilution = Then take 20 L and place on to sterile 6 mm disc.

01st Dilution

Final Conc.
6 mm Disc
20 L

200 mg : 100 mL

0.04 mg

Applying Disc Method:


After preparing microbial suspension, dip sterile swab to the bacterial suspension
tube.
Inoculate the plates by dipping a sterile swab into the inoculum, remove excess
inoculum by pressing and rotating the swab firmly against the side of the tube above
the level of the liquid.
Streak the swab all over the surface of the Mueller Hinton Agar Medium (M H Agar)
three times for making complete lawn of bacterial suspension (see Fig. 1 given
below), streaking the plate through an angle of 60 after each streaking stroke, and
finally, pass the swab round the edge of the agar surface.

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Analytical Method of Rifaxa 200 mg Tablets


Leave the inoculum to adjust & dry for 3 5 minutes (not more than 15 minutes) at
room temperature with the lid closed under the B.S.C.
After adjustment of inoculum with the agar, place antibiotic sample discs on the
inoculated plates using a sterile forcep along with standard antibiotic disc aseptically
under the B.S.C.
After placing each and every required disc, press gently with forcep to make sure
even contact with agar medium.
It is convenient to use a template (See Fig. 2a given below) to place the discs
uniformly.
Incubate the petri plate containing sample at the 30 35 C for 16 24 hours
(overnight) within 30 minutes after applying discs.
After incubation, observe the zone of inhibition around the sample and standard
antibiotic discs, and measure them with Vernier Caliper or with measuring scale (see
Fig. 3 given below) and report zones in millimeter (mm).
Use following formula to calculate the results in percentage;

%age

Zone of Sample (mm)

Zone of standard (mm)

100

Prepare duplicate discs and place on to the medium surface in Triangular Shape (see Fig.
2b) / Linear Direction (see Fig. 2c given below).

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Analytical Method of Rifaxa 200 mg Tablets

Figure 1 Template for making bacterial lawn (90 mm Diameter of Plate).

Figure 2a Template for applying antibiotic discs (90 mm Diameter of Plate)

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Analytical Method of Rifaxa 200 mg Tablets

Figure 2b Template for applying antibiotic discs in Triangular Shape (90 mm Diameter of Plate)

Figure 2c Template for applying antibiotic discs in Linear Shape (90 mm Diameter of Plate)

Antibiotic Disc
Zone of Inhibition

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Analytical Method of Rifaxa 200 mg Tablets

Figure 3 Template for measuring zone of inhibition in 2 way directions

PRODUCT SPECIFICATIONS AT COATED STAGE:

Tests
Appearance

Specifications
An orange color, round biconvex film coated tablets with break
line on one side and plain other side.

Shape
Average Weight

343 mg 5 %
Minimum 18/20 + 5 %

Weight variation
Maximum 02/20 + 10 %
Disintegration Time

NMT. 30 minutes

Dissolution

NLT 80% (Q) of the labeled amount of Rifaximin is


dissolved.

Identification

The retention time of the major peak in the chromatogram of


the sample preparation corresponds to that in the
chromatogram of the standard preparation, as obtained in

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Analytical Method of Rifaxa 200 mg Tablets


assay.
Assay (Rifaximin)
Antimicrobial activity
(Microbial assay)

90 % 110 %
90 % 110 %

APPEARANCE:
1. Principle

Visual evaluation of color & shape.

2. Evaluation

The color must be as specified with no foreign matter.

3. Requirement

An orange color, round biconvex film coated tablets with break


line on one side and plain other side.

AVERAGE WEIGHT:
1. Principle

Represents tablet weight in the batch formulation of product.

2. Evaluation

The average weight of tablets evaluated by the randomly


sampled 20 tablets on balance, and divide this weight on the
number of taken tablets.

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Analytical Method of Rifaxa 200 mg Tablets

Average weight = Weight of 20 tablets


20
3. Apparatus

Analytical balance.

4. Requirement

343 mg 5 %

CONTROL OF WEIGHTS:

1. Principle

Weigh the prescribed number of tablets individually on an


analytical balance and calculate the mean weight.
Calculate the percentage deviations of the actual mean weight
to the theoretical average weight.

2. Apparatus

Analytical balance.

3. Test performance

Sample size:
- 20 tablets per sample

4. Evaluation

The actual mean value should not deviate more than the
specified limit to the theoretical weight.
Weigh accurately 20 tablets and calculate average weight,
then weigh individually 20 tablets.
The difference in percentage from minimum weight to
maximum weight of individual tablets should not be more than
the specified limits.

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Analytical Method of Rifaxa 200 mg Tablets


Max. deviation = Max. tablet weight Avg. weight found
Avg. weight found
Min. deviation = Min. tablet weight Avg. weight found
Avg. weight found
5. Requirement

Minimum 18/20 + 5 %
Maximum 02/20 + 10 %

DISINTEGRATION TIME:

1. Apparatus

Disintegration Apparatus

2. Method

Fill sufficient water into the vessel so that during motion, the
gauze does not come within less than 2.5 cm of the surface of
the liquid warm the water to 37 + 2oC.
Place one tablet in each of the 6 glass tubes and operate the
apparatus and determine the time taken for each tablet to
disintegrate.

3. Requirement

Not more than 30 minutes


The requirement is fulfilled if all 6 tablets have disintegrated in
the prescribed time.
If 1 or 2 tablets fail to disintegrate completely, repeat the
test on 12 additional tablets. The requirement is met if not
less than 16 of the total of 18 tablets tested are
disintegrated.

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Analytical Method of Rifaxa 200 mg Tablets

DISSOLUTION:

1. Apparatus

Dissolution Apparatus # 2.

2. Method

Medium: 0.1 M Phosphate buffer pH 7.4

Preparation of 0.1 M Phosphate buffer pH 7.4:


Dissolve 15.6 gm of Sodium dihydrogen Phosphate and 4.5
gm of Sodium Lauryl sulphate in 100 ml of water. Check the
pH to be 7.4.
Dissolution conditions:
Volume: 1000 ml
Rpm: 75
Temperature: 37 + 2oC
Time: 45 minutes

Filled the 6 vessels of dissolution apparatus each vessel


contains 1000 ml medium. Maintain the said temperature of
the vessels medium.
Sample preparation:
Place 6 tablets individually in six dissolution vessels containing
1000 ml of Phosphate buffer pH 7.4 that has been equilibrated
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Analytical Method of Rifaxa 200 mg Tablets


to 37 C 2C. Take care to exclude air bubbles from the
surface of the tablet, start the apparatus immediately. Collect
the sample after the specified time (45 minutes), withdraw the
sample from zone midway between the surface of the medium
and top of the rotating paddle and not less than 1 cm from the
vessel wall and filter through 0.45 membrane filter and discard
the first few ml.
Dilute 5 ml of the filtrate to 100 ml with diluent.
Standard preparation:
Dissolve an accurately weighed 100 mg of Rifaximin working
standard in 100 ml volumetric flask in about 80 ml of diluent.
Shake for 10 minutes and dilute to volume with diluent. Further
dilute 1 ml of this solution to 100 ml with diluent.
Preparation of Diluent:
Mix distilled water and acetonitrile in ratio of 60: 40
Procedure:
Measure the absorbance of the final solutions of sample and
standard at 235 nm using distilled diluent as blank.
3. Requirement

NLT 80 % (Q) of the labeled amount of Rifaximin is


dissolved.

4. Calculation

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Absorbance of sample

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Analytical Method of Rifaxa 200 mg Tablets


%age = -------------------------------- x 100
Absorbance of standard

IDENTIFICATION (Rifaximin):
1. Principle

To identify Rifaximin.

2. Evaluation

The retention time of the major peak in both sample and


reference standard must be same.

3. Requirement

The retention time of the major peak in the chromatogram of


the sample preparation corresponds to that in the
chromatogram of the standard preparation, as obtained in
assay.

ASSAY (Rifaximin):
1. Principle

The quantitative analysis of Rifaximin.

2. Evaluation

The analysis of Rifaximin is as per In house method.

3. Requirement

90 % 110 %

4. Procedure

As under.

Apparatus:
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Analytical Method of Rifaxa 200 mg Tablets

A liquid chromatograph equipped with variable UV/Visible detector and integrator.

Volumetric Flasks 100 ml, 50 ml and 25 ml

Beaker 100 ml

Funnel

Filter paper

Mortar and Pestle

Pipettes 5 ml with pipette filler

Flask shaker

Analytical balance

Spatula

Watch glass

Reagents:

Acetonitrile

Methanol

Ammonium Acetate

Distilled water

Rifaximin working Standards.

Preparation of standard solution:


Accurately weigh and dissolve 100 mg of Rifaximin working standard in about 10 ml
methanol in 100 ml volumetric flask. Shake till it is completely dissolved; make volume to
100 ml with same solvent. Dilute 5 ml of this solution to 100 ml with mobile phase to get the
final concentration of 0.05 mg/ml.

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Analytical Method of Rifaxa 200 mg Tablets


Preparation of sample solution:
Weigh and finely powder not less than 20 tablets. Transfer an accurately weighed portion of
the powder, equivalent to 100 mg of Rifaximin to 100 ml volumetric flask with the help of
about 50 ml methanol. Shake for about 15 minutes. Make volume to 100 ml with methanol.
Filter and discard first few ml of the filtrate and dilute 5 ml of this solution to 100 ml with
mobile phase to get the final concentration equivalent to that of standard preparation.
PREPARATION OF MOBILE PHASE:
Mix 0.05 M Ammonium Acetate with acetonitrile in ratio 15 : 85 v/v, filter through 0.45 , and
degas.
Chromatographic conditions:

Injection Size

20 l

Flow Rate

1.0 ml / min

Column

C-18, BDS, 250 mm x 4.6 mm, 5

Wavelength

236 nm

Procedure:
Condition the column by running mobile phase and checking the UV value of the UV
detector.
When the UV detector became stable, separately inject equal volumes of 20 l of standard
preparation and the sample preparation into the chromatograph, record the chromatograms
and measure the peak responses.

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Analytical Method of Rifaxa 200 mg Tablets


Calculations:

Peak response of sample


%age = ------------------------------------- x 100
Peak response of standard

ANTIMICROBIAL ACTIVITY MICROBIAL ASSAY:


1. Principle

Determination of potency of Rifaxa 200 mg Tablets.

2. Evaluation

Microbial analysis of Rifaxa 200 mg Tablets is as per WHO.


Disc Diffusion (Kirby-Bauer Method)

3. Requirement

90 % 110%.

4. Procedure

As under.

Apparatus & Materials:

Incubator

Autoclave

Face Mask

Sterile Forcep

Sterile Wire Loop

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Analytical Method of Rifaxa 200 mg Tablets

Dry Heat Sterilizer

Micropipette 20 L

Sterile Latex Gloves

Vernier Caliper / Ruler

Heat Resistant Gloves

Screw-capped Test Tubes

Plan Antibiotic Discs 6 mm

Bunsen burner / Sprit Lamp

Biological Safety Cabinet (B.S.C)

Culture Media & Reagents:

Tryptic Soy Broth

Specific ATCC Culture

Filtered 70% Isopropyl Alcohol spray bottle

0.5% MacFarland Assay / Turbidity Standard

Antibiotic Medium No. 11 / Mueller Hinton Agar

Commercially Prepared Specific Antibiotic Discs 6 mm

Sterile pH 7.2 Phosphate Buffer / Tryptic Soy Broth / Sterile Peptone Water

Preparation of Reference Standard Antibiotic Discs:


Antibiotic plain discs (having diameter of 6 mm) are used in this method and before
preparation of antibiotic disc, sterilize them by placing in dry heat oven at 100 C for 30
minutes 1 hour.
Standard preparation is as follow:

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Analytical Method of Rifaxa 200 mg Tablets


Standard Preparation:
Standard Disk Concentration = 40 g

Dilution Method (Lab prepared standard):


First Dilution = Weigh 100 mg accurately and dissolve it into 50 mL of filtered
acetonitrile and H2O (60 : 40).
Second Dilution = Then take 20 L and place on to sterile 6 mm disc to achieve
required centration on disc.

01st Dilution

Final Conc.
6 mm Disc
20 L

100 mg : 50 mL

0.04 mg

Preparation of Sample Discs:


Antibiotic plain discs (having diameter of 6 mm) are used in this method and before
preparation of antibiotic disc, sterilize them by placing in dry heat oven at 100 C for 30
minutes 1 hour.
Sample preparation is as follow:
Dilution Method of Rifaxa 200 mg Tablets (Rifaximin)
Sample Preparation:
Standard Disk Concentration = 40 g
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Analytical Method of Rifaxa 200 mg Tablets

Dilution Method:
Crush the 20 tablets and weigh accurately 343 mg of fine powder equivalent to 200
mg of Rifaximin.
First Dilution = Dissolve it into 100 mL of the mixture of filtered acetonitrile and H2O
(60 : 40).
Second Dilution = Then take 20 L and place on to sterile 6 mm disc.

01st Dilution

Final Conc.
6 mm Disc
20 L

200 mg : 100 mL

0.04 mg

Applying Disc Method:


After preparing microbial suspension, dip sterile swab to the bacterial suspension
tube.
Inoculate the plates by dipping a sterile swab into the inoculum, remove excess
inoculum by pressing and rotating the swab firmly against the side of the tube above
the level of the liquid.
Streak the swab all over the surface of the Mueller Hinton Agar Medium (M H Agar)
three times for making complete lawn of bacterial suspension (see Fig. 1 given

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Analytical Method of Rifaxa 200 mg Tablets


below), streaking the plate through an angle of 60 after each streaking stroke, and
finally, pass the swab round the edge of the agar surface.
Leave the inoculum to adjust & dry for 3 5 minutes (not more than 15 minutes) at
room temperature with the lid closed under the B.S.C.
After adjustment of inoculum with the agar, place antibiotic sample discs on the
inoculated plates using a sterile forcep along with standard antibiotic disc aseptically
under the B.S.C.
After placing each and every required disc, press gently with forcep to make sure
even contact with agar medium.
It is convenient to use a template (See Fig. 2a given below) to place the discs
uniformly.
Incubate the petri plate containing sample at the 30 35 C for 16 24 hours
(overnight) within 30 minutes after applying discs.
After incubation, observe the zone of inhibition around the sample and standard
antibiotic discs, and measure them with Vernier Caliper or with measuring scale (see
Fig. 3 given below) and report zones in millimeter (mm).
Use following formula to calculate the results in percentage;

%age

Zone of Sample (mm)

Zone of standard (mm)

100

Prepare duplicate discs and place on to the medium surface in Triangular Shape (see Fig.
2b) / Linear Direction (see Fig. 2c given below).

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Figure 1 Template for making bacterial lawn (90 mm Diameter of Plate).

Figure 2a Template for applying antibiotic discs (90 mm Diameter of Plate)

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Figure 2b Template for applying antibiotic discs in Triangular Shape (90 mm Diameter of Plate)

Figure 2c Template for applying antibiotic discs in Linear Shape (90 mm Diameter of Plate)

Antibiotic Disc
Zone of Inhibition

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Figure 3 Template for measuring zone of inhibition in 2 way directions

PRODUCT SPECIFICATIONS AT BEFORE FINAL RELEASE STAGE:

Tests
Appearance

Specifications
An orange color, round biconvex film coated tablets with break
line on one side and plain other side.

Shape
Average Weight

343 mg 5 %
Absorption spectrum obtained from sample preparation and

Identification

reference preparation shows absorption maximum at wave


length 235 nm.

Assay (Rifaximin)

90 % 110 %

Microbial Limit Test

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Analytical Method of Rifaxa 200 mg Tablets


Total Aerobic Microbial
Count (TAMC)

Not more than 1,000 CFU per g of tablet.

Total Yeast Mold Count


(TYMC)

Not more than 100 CFU per g of tablet.

Bile-Tolerant GramNegative Bacteria

Should be absent.

Escherichia coli

Must be absent.

Staphylococcus aureus

Must be absent.

Pseudomonas
aeruginosa

Must be absent.

Salmonella Species

Must be absent.

Clostridia Species

Must be absent.

Candida albicans

Must be absent.

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APPEARANCE:
1. Principle

Visual evaluation of color & shape.

2. Evaluation

The color must be as specified with no foreign matter.

3. Requirement

An orange color, round biconvex film coated tablets with break


line on one side and plain other side.

AVERAGE WEIGHT:
1. Principle

Represents tablet weight in the batch formulation of product.

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2. Evaluation

The average weight of tablets evaluated by the randomly


sampled 20 tablets on balance, and divide this weight on the
number of taken tablets.
Average weight = Weight of 20 tablets
20

3. Apparatus

Analytical balance.

4. Requirement

343 mg 5 %

IDENTIFICATION (Rifaximin):

1. Principle

To identify Rifaximin.

2. Evaluation

The maximum absorbance of sample and reference standard


must be at wavelength maxima 235 nm.

3. Requirement

Absorption spectrum obtained from sample preparation and


reference preparation shows absorption maximum at wave
length 235 nm.

ASSAY (Rifaximin):

1. Principle

The quantitative analysis of Rifaximin.

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2. Evaluation

The analysis of Rifaximin is as per in house method.

3. Requirement

90 % 110 %

4. Procedure

As under.

Apparatus:

Volumetric flasks

Spectrophotometer

Beaker

Mortar & pestle

Spatula

Analytical balance

Pipette 1 ml

Flask shaker

Funnel

Reagents:

Acetonitrile

Distilled water

Rifaximin (Working Standard)

Preparation of Diluent:
Mix distilled water and acetonitrile in ratio of 60: 40

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Sample preparation:
Weigh and finely powder not less than 20 tablets. Transfer an accurately weighed portion of
the powder equivalent to 100 mg of Rifaximin in a 100 ml volumetric flask. Add
approximately 80 ml of diluent and shake for 40 minutes. Make volume with diluent. Filter
and discard first few ml of the filtrate. Dilute 1 ml of the filtrate to 100 ml with diluent .
Reference standard preparation:
Transfer an accurately weighed 100 mg of Rifaximin working standard in a 100 ml
volumetric flask and add about 80 ml diluent. Shake for 30 minutes to dissolve and make
volume up to the mark with diluent. Dilute 1 ml of this solution to 100 ml with diluent.

Procedure:
Measure the absorbance of the final solutions of sample and standard at 235 nm using
diluent as blank.

Calculations:
Absorbance of the sample
% age = ------------------------------------- x 100
Absorbance of the standard

MICROBIAL LIMIT TEST:


1. Principle

Microbiological analysis of the Rifaxa 200 mg Tablets.

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2. Evaluation

Microbiological analysis of the Rifaxa 200 mg Tablets is as per


USP.

3. Requirement

Tests meet the General Chapter <61> and <62> of USP.


Limit: As per COA.

4. Procedure

As under.

Apparatus & Materials:

Oven

Incubator

Autoclave

Sterile latex gloves

Sterile lint free duster

Sterile filtration assembly

L shaped sterile glass rod

Sterile screw-capped glass bottles 250 mL

Filtered 70% Isopropyl Alcohol spray bottle

Sterile petri dishes/plates (9 cm in diameter)

Sterile micro tips having 1 mL capacity with micropipette/1 mL sterile pipette

Culture Media:

Columbia Agar

Peptone Water

Cetrimide Agar

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Tryptic Soy Agar

Tryptic Soy Broth

MacKonkey Agar

MacKonkey Broth

Mannitol Salt Agar

Violet Red Bile Agar

Sabouraud Dextrose Agar

Sabouraud Dextrose Broth

Reinforced Clostridial Medium

Xylose Lysine Deoxycholate Agar

Enterobacteriaceae Enrichment Broth

Rappaport Vassiliadis Salmonella Broth

Preparation of Sample:
Dissolve (usually a 1:10 dilution is prepared) 10 g of Rifaxa 200 mg Tablets in Sterile
Peptone Buffered Solution / Sterile Tryptic Soy Broth. If necessary, adjust the pH to 6 8.
Further dilutions, where necessary, are prepared with the same diluent.
Neutralization of Antimicrobial Activity:
Add 1 mL Polysorbate 80 to the diluent and sterile it before use, it is used for inactivation of
antibiotic properties of the sample.
Total Aerobic Microbial Count (TAMC):
Pipette 0.5 1 mL of the prepared sample into each of two Sterile Tryptic Soy Agar
containing petri-dishes of 90 mm diameter. Spread the sample volume with the help of L

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shaped sterile glass rod. Cover the petri-dishes and incubate at 30 35 C for 3 days in
inverted position.
Total Yeast / Mold Count (TYMC):
Pipette 0.5 1 mL of the prepared sample into each of two Sterile Sabouraud Dextrose Agar
containing petri-dishes of 90 mm diameter. Spread the sample volume with the help of L
shaped sterile glass rod. Cover the petri-dishes and incubate at 20 25 C for 5 days in
inverted position.
TEST FOR SPECIFIED MICRO-ORGANISMS:
Bile-Tolerant Gram-Negative Bacteria:

Sample Preparation and Pre-incubation:


Dissolve 10 g of sample in Sterile Tryptic Broth and make up the volume to 100 mL
with the same diluent, shake well and incubate at 20 25 C for a time sufficient to
resuscitate the bacteria but not sufficient to encourage multiplication of the bacteria
(usually 2 hours but not more than 5 hours).

Test for Absence:


Inoculate 10 mL the volume corresponding to 1 g of the product containing Tryptic
Soy Broth to 100 mL Sterile Enterobacteriaceae Enrichment Mossel Broth. Incubate
at 30 35 C for 24 hours.

Subculturing:
After completion of 24 hours incubation time, subculture a small aliquot (0.5 1 mL)
from mossel broth to Violet Red Bile Agar plates in duplicate. Incubate at 30 35 C
for 24 hours.

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Analytical Method of Rifaxa 200 mg Tablets

Interpretation:
The product complies with the test if there is no growth of colonies.

Test for Escherichia coli:

Sample Preparation and Pre-incubation:


Prepare a sample using a 1 in 10 dilution of not less than 10 g of Rifaxa 200 mg
Tablets in 100 mL Tryptic Soy Broth, mix well and incubate at 30 35 C for 24
hours.

Subculturing:
Shake the bottle of sample containing Tryptic Soy Broth, transfer 1 mL of Tryptic Soy
Broth to 100 mL of MacKonkey Broth and incubate at 42 44 C for 24 hours.
After completion of 24 hours of incubation time, subculture a small aliquot from
MacKonkey Broth to MacKonkey Agar plates in duplicate. Incubate at 30 35 C for
24 48 hours.

Interpretation:
If colonies grow on MacKonkey Agar plates these indicate the possible presence of
Escherichia coli. This is confirmed by identification tests.
The product complies with the test if no colonies are present OR if the identification
tests are negative.

Test for Staphylococcus aureus:

Sample Preparation and Pre-incubation:

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Prepare a sample using a 1 in 10 dilution of not less than 10 g of Rifaxa 200 mg
Tablets in 100 mL Tryptic Soy Broth, mix well and incubate at 30 35 C for 24
hours.

Subculturing:
After completion of 24 hours of incubation time, shake the bottle of sample containing
Tryptic Soy Broth, transfer 1 mL of Tryptic Soy Broth to Mannitol Salt Agar plates in
duplicate. Incubate at 30 35 C for 72 hours.

Interpretation:
The possible presence of Staphylococcus aureus is indicated by the growth of yellow
OR white colonies surrounded by a yellow zone. This is confirmed by identification
tests. The product complies with the test if colonies of the types described are not
present OR if the confirmatory identification tests are negative.

Test for Pseudomonas aeruginosa:

Sample Preparation and Pre-incubation:


Prepare a sample using a 1 in 10 dilution of not less than 10 g of Rifaxa 200 mg
Tablets in 100 mL Tryptic Soy Broth, mix well and incubate at 30 35 C for 24
hours.

Subculturing:

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After completion of 24 hours incubation, subculture a small aliquot on plates of
Cetrimide Agar and incubate at 30 35 C for 24 hours.

Interpretation:
Growth of colonies on Cetrimide Agar plates with yellow-green to blue-green OR
yellow-green to green coloration these indicate the presence of Pseudomonas
aeruginosa. This is confirmed by identification tests.
The product complies with the test if no colonies are present OR if the identification
tests are negative.

Test for Salmonella:

Sample Preparation and Pre-incubation:


Prepare a sample using a 1 in 10 dilution of not less than 10 g of Rifaxa 200 mg
Tablets in 100 mL Tryptic Soy Broth, mix well and incubate at 30 35 C for 24
hours.

Subculturing:
After completion of 24 hours of incubation time, transfer 0.1 mL of Tryptic Soy Broth to
10 mL of Rappaport Vassiliadis Salmonella Enrichment Broth and incubate at 30 35
C for 24 hours.
After completion of 24 hours incubation, subculture a small aliquot on plates of Xylose
Lysine Deoxycholate Agar and incubate at 30 35 C for 48 hours.

Interpretation:

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The possible presence of Salmonella is indicated by the growth of well-developed, red
colonies with OR without black centers. This is confirmed by identification tests.
The product complies with the test if no colonies are present OR if the identification
tests are negative.
Test for Clostridia:

Sample Preparation and Pre-incubation:


Prepare a sample using a 1 in 10 dilution of not less than 2 g of the product to be
examined in Tryptic Soy Broth.
Take two equal portions corresponding to not less than 1 g of the product to be
examined. Heat 1 portion at 80 C for 10 minutes and cool rapidly. Dont heat the other
portion.
Transfer 10 mL OR the quantity corresponding to 1 g of each portion to 2 containers
containing 100 mL / suitable amount of Reinforced Clostridial Medium. Incubate under
anaerobic conditions at 30 35 C for 48 hours.

Subculturing:
After completion of 48 hours incubation, subculture a small aliquot from each bottle of
Reinforced Clostridial Medium on plates of Columbia agar and incubate at 30 35 C
for 48 hours under anaerobic conditions.

Interpretation:
The occurrence of anaerobic growth of rods (with OR without endospores) giving a
negative catalase reaction indicates the presence of Clostridia.

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The product complies with the test if colonies of the types described are not present
OR if the confirmatory identification tests are negative.
Test for Candida albicans:

Sample Preparation and Pre-incubation:


Prepare a sample using a 1 in 10 dilution of not less than 10 g in 100 mL Tryptic Soy
Broth, and mix well.
Transfer 10 mL OR the quantity corresponding to not less than 1 g to Sabouraud
Dextrose Broth, mix well and incubate at 20 25 C for 72 hours.

Subculturing:
After completion of 72 hours incubation, subculture a small aliquot from Sabouraud
Dextrose Broth on plates of Sabouraud Dextrose Agar and incubate at 30 35 C for
48 hours.

Interpretation:
Growth of white colonies may indicate the presence of Candida albicans. This is
confirmed by identification tests.
The product complies with the test if colonies of the types described are not present
OR if the confirmatory identification tests are negative.
Negative Controls:
To verify testing conditions, a negative control is performed using the chosen diluent in
place of the test preparation. There must be no growth of microorganisms observed /
detected. A negative control is also performed when testing the products as described
under the Testing of Products.

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If product sample shows no growth but negative control shows any growth then retest
the product sample. If product sample shows growth but negative control also shows
any growth then again retest the sample. A failed negative control requires an
investigation.

TRAINING:
Training of the concerned SOP is given to the personnel of the Quality control analyst who
will be exercising the SOP. And training record is to be maintained on Annexure I.

DISTRIBUTION:
Distribution
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Title
Signature

Date

Copy No.

Signature

Date

HISTORY:
Date
June 27, 2016

Reason of Change
To replace fQ number with new PAM number and addition of
dissolution test.

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