AKUMS LIFESCIENCES LTD.

ANALYTICAL RESEARCH & DEVELOPMENT

STANDARD TESTING PROCEDURE

Material Name : Faropenem Sodium
Item Code : FRP Customer Name : NA
Revision Number : 00 Category : Finished
Effective Date : NA Supersede No. : NA
Pharmacopoeia Grade : In-House Page Number : 1 of 9

1.0 Description:
1.1 Requirements

Glass Petridish and Spatula

1.2 Procedure

Take about 2-3 g sample in a dry glass petri dish, spread it by spatula and observe its color and
presence of any extraneous matter under suitable light and report the results.
2.0 Solubility
2.1 Requirements:
Analytical Balance and Test tube with stopper
2.2 Procedure:
Weigh accurately a suitable quantity of sample into a glass stoppered test tube and add specified
quantity of specified solvent as per the table given below. Shake vigorously to dissolve, keep aside
for 15 minutes, the solution should be clear.
Volume of solvent Weight of
S. No Solubility term Specified solvents
(mL) sample
1. Freely Soluble 1 to 10 1g Water & Methanol
2. Slightly soluble 10 to 100 10 mg Ethanol (95%)
3. Insoluble more than 100 10 mg Diethyl ether

3. 0 Identification
3.1 (A) Identification By Infrared Absorption Spectrophotometer
3.1.1 Requirements
IR spirit, Isopropyl alcohol
3.1.2 Procedure
Switch on the instrument and clean it with isopropyl alcohol.
Login to the computer using Login Id and Password.
Turn on the FTIR instrument.
Start IR solution software and initialize the instrument.
Load the parameter for the respective sample.
Record the background spectrum from 4000 to 625 cm-1.
Place about 5mg of Reference/Working standard on sample holder and record the spectrum in the
range of 4000-625 cm-1.
Similarly Record the spectrum of the sample in the range 4000-625 cm-1.
Compare it with standard spectrum of working standard.
The Infrared Spectrum of the sample should concordant with the spectrum of the Reference/Working
standard.
3.2 (B) Identification By HPLC
3.2.1 The retention time of principal peak in the chromatogram of test solution should correspond to the
retention time of principal peak in the chromatogram of standard solution during assay.
4.0 Water (By KF)

4.1 Requirements
Karl Fischer Titrator, Analytical Balance, Karl Fischer Reagent, Dried Methanol
4.2 Procedure
Weigh about 0.1 g of sample to be examined and transfer into titration vessel containing previously
neutralized Methanol, wait for dissolve and titrate to end point with Karl Fischer regent, record the
volume of KF reagent.
4.3 Calculations
Calculate the % water content by the formula given under:

Where:
V = Volume in mL of KF reagent
F = Factor of KF reagent (in mg/mL)
W = Weight of sample (in g)

5.0 Specific Optical rotation

5.1 Requirements
Polarimeter, Analytical Balance, HPLC Water

5.2 Procedure
5.2.1 Preparation of Sample Solution
Weigh and transfer accurately about 0.5g (Calculated as on anhydrous basis) of sample into 50 mL
volumetric flask.add about 30 mL of Water, sonicate to dissolve, and further make up the volume up
to the mark with Water and mix well.

5.2.2 Instrument analysis

Stabilize the Polarimeter for 20 min before taking the reading. Rinse the Polarimeter tube with HPLC
water and fill the tube with the water. Make sure that the methanol is free from air bubbles. Record
the readings at 589 nm at 25°C temperature, the scale should show ZERO” at this point, if not, do
zero. Rinse the tube with sample solution and fill the tube with sample solution. Make sure that the
sample solution is free from air bubbles. Record five readings of sample solution at 589 nm at 25°C
temperature.
Average Observed rotation at 25°C x 50
Specific Optical rotation = ---------------------------------------------------------------
Length of the tube in decimeter x Weight of sample (in g)

6.0 Heavy Metals

6.1 Requirements

Analytical Balance. Muffle Furnace, Nessler Tubes, Water bath, Platinum or Porcelain crucible
6.2 Reagents

Phenolphthalein ,Ethanol, Ammonia AR Grade (28-30% Content) , Sodium sulfide eneahydrate

,Glycerin, Sodium hydroxide ,Hydrogen sulfide, Iron (III) chloride hexahydrate , Hydrochloric
acid, Lead solution , Magnesium nitrate hexahydrate Sulfuric acid , Acetic acid AR Grade and
Purified water,
6.3 Phenolphthalein TS:

Weigh about 1g of Phenolphthalein in 100mL ethanol (95%)

6.4 Ammonia Solution TS:
Take 400 mL of Ammonia solution (28%) and dilute to 1000 mL with water mix well.
6.5 Sodium sulfide TS:
Dissolve 5 g of Sodium sulfide ennehydrate in a mixture of 10 mL of water add 30 mL of
glycerin. Preserve in well filled, light resistant bottles. Use within 3 months.
6.6 Standard Iron Stock Solution:
Dissolve 4.84 g of Iron (III) chloride hexahydrate in diluted hydrochloric acid to make exactly
100 mL.
6.7 Standard Led Solution:

Measure accurately 10 mL of Standard Led stock solution and add water to make exactly 100 mL
6.8 Procedure:

6.8.1 Test Solution Preparation:

Weigh and transfer accurately about 2.0 g of sample in a Platinum or Porcelain crucible, mix with
10 mL of solution of magnesium nitrate hexahydrate in ethanol 95% (1 in 10 mL),fire the Ethanol
to burn , and carbonized by gradual heating .Cool ,add 1 mL of sulphuric acid ,heat carefully ,and
incinerate by ignition between 500°C and 600°C. If a carbonized substance remains, moisten with
a small amount of sulphuric acid, and incinerate by ignition .Cool, dissolve the residue in 3 mL of
Hydrochloric acid, evaporate on a water bath to dryness, wet the residue with 3 drops of
Hydrochloric acid, add 10 mL of water. And dissolve by warming. Add 1 drop of phenolphthalein
TS, add Ammonia TS drop wise until pale red colour develops , then add 2 mL of dilute acetic
acid, Filter if necessary ,wash with 10 mL of water ,transfer the filtrate and washing to a Nessler
tube, add water to make 50 mL . Use this as a test solution
6.8.2 Standard Solution Preparation:
Take 10 mL solution of magnesium nitrate hexahydrate in Ethanol (95%) (1 in 10 mL) and fire
the ethanol to burn. Cool, add 1 mL of sulphuric acid, heat carefully and ignition between 500°C
and 600°C.Cool add 3 mL of Hydrochloric acid. Hear in after proceed as directed in the test
solution, then add 2.0 mL of Lead solution and water to make 50 mL
Add 1 drop of Sodium sulfied TS to each test solution, mix thoroughly and allow to stand for 5
minutes and view downwards over a white surface, the colour produced with the test solution is not
more intense than that produced with the standard solution.
7.0 Related Substances (By HPLC)
7.1 Requirements
High Performance Liquid chromatograph (HPLC), Analytical Balance
7.2 Reagents
Milli-Q Water
Acetonitrile (HPLC grade) (Make-Rankem)
Potassium dihydrogen phosphate (AR grade) (Make-Rankem),
Disodium hydrogen phosphate dodecahydrate (AR grade) (Make-Sigma),
Tetra n-butyl ammonium bromide (AR grade) (Make-Merck)
7.3 Procedure

7.3.1 Buffer Solution Preparation:

Weigh and transfer accurately about 4.8 gm of Potassium dihydrogen phosphate, 5.4 gm of Disodium
hydrogen phosphate dodecahydrate and 1.0 gm of tetra n-butyl ammonium bromide in 1000 mL of
milli Q water. Sonicate to mix well.

7.3.2 Mobile phase:

Mix Buffer: Acetonitrile in the ratio of (87:13 v/v) sonicate to mix well and filter through 0.45 µm
membrane filter, and degas with sonication.

7.3.3 Diluent
Use Water as diluent.

7.3.4 Chromatographic conditions

Instrument A suitable High performance Liquid chromatograph (HPLC)
equipped with a UV Detector and suitable software for
integration
Column Hypersil ODS-C18 (250x4.6)mm, 5µm
Detector UV at 240 nm
Column Temperature 40°C
Sample Temperature 10°C
Injection volume 20µl
Run Time 75 min
Flow rate 1.8 mL/min.
Needle wash solution Water : Acetonitrile (90:10 v/v)
Retention time About 11 minutes

Note: Adjust the flow rate to attain RT of Faropenem sodium at about 11 min
7.3.5 Test Solution Preparation
Note: Freshly prepared solution.
Weigh accurately and transfer about 25 mg of sample into 50 mL volumetric flask, add about 10 mL
of diluent, sonicate to dissolve and further make up the volume up to the mark with diluent and mix
well.

7.3.6 Standard Solution Preparation:

Transfer accurately 1 mL of the above test solution to 100 mL with the diluent and mix well.

7.3.7 Test for required Detectability Solution Preparation:

Transfer accurately 2.0 mL of Standard solution into 20 mL volumetric flask and dilute upto the
mark with diluent and mix well.
7.3.8 Test Solution Preparation
Note: Freshly prepared solution.
Weigh accurately and transfer about 25 mg of sample into 50 mL volumetric flask, add about 10 mL
of diluent, sonicate to dissolve and further make up the volume up to the mark with diluent and mix
well.

7.3.9 Injection sequence (for information only)

Sr. No. Sample Name No. of Injection
1. Blank 1

2. Test Detectability 1

3. Standard Solution 6

4. Test Solution 1

5. Standard Solution BKT 1

7.3.10 Evaluation of system suitability

%RSD for Faropenem peak area from six replicate injections of standard solution should not be
more than 2.0.
Peak area of Faropenem obtained with test required detectability solution should obtained with
standard solution is equivalent to 7 to 13%.
7.4 Calculations
For Epimer (at RRT about 1.1):

Where,

AT = Area of Faropenem epimer (RRT about 1.1) in test solution.

AS= Average area of Faropenem peak in six replicate injections of
standard solution.
WS =Weight of Faropenem sodium Reference/Working standard in mg.
WT = Weight of test sample in mg
For Total impurities

Where,

AT2 = Total Area of all peak other than Faropenem in test solution.
AS=Average area of Faropenem peak in six replicate injections of standard solution.
WS =Weight of Faropenem sodium Reference/Working standard in mg.
Wt = weight of test sample in mg
8.0 Assay (By HPLC)
8.1 Requirements
High Performance Liquid chromatograph (HPLC), Analytical Balance

8.2 Reagents

Milli-Q Water

Acetonitrile (HPLC grade) (Make-Rankem),

Potassium dihydrogen phosphate (AR grade) (Make-Rankem),
Disodium hydrogen phosphate dodecahydrate (AR grade) (Make-Sigma),
m-Hydroxyacetophenone (AR grade),
Tetra n-butyl ammonium bromide (AR grade) (Make-Rankem),

8.3 Procedure

8.3.1 Buffer Solution Preparation:

Weigh and transfer accurately about 4.8 gm of Potassium dihydrogen phosphate, 5.4 gm of Disodium
hydrogen phosphate dodecahydrate and 1.0 gm of tetra n-butyl ammonium bromide in 1000 mL of
milli Q water, sonicate to dissolve and mix well.
8.3.2 Mobile phase:
Mix Buffer: Acetonitrile in the ratio of (87:13 v/v) sonicate to mix well and filter through 0.45 µm
membrane filter, and degas with sonication.

8.3.3 Diluent:
Use Water as diluent.

8.3.4 Chromatographic conditions:

Instrument A suitable High performance Liquid chromatograph (HPLC)
equipped with a UV Detector and suitable software for integration
Column Hypersil ODS-C18 (250×4.6)mm
Detector UV at 305 nm
Column Temperature 40°C
Sample Temperature 10°C
Injection volume 20µl
Run Time 30 minutes.
Flow rate 1.8 mL/minute
Needle wash solution Water : Acetonitrile (85:15)
Retention Time About 11 minutes

Note: Adjust the flow rate to attain RT of Faropenem sodium hydrate at about 11 min

8.3.1 Internal Standard Solution Preparation:

Weigh accurately and transfer about 500.0 mg of m-Hydroxyacetophenone Reference/Working

standard into 200 mL of volumetric flask, add about 20 mL of Acetonitrile and sonicate to dissolve,
and further make up the volume up to the mark with diluent, and mix well.

8.3.2 Standard Solution Preparation:

Weigh accurately and transfer about 25 mg of Faropenem sodium Reference/Working standard into
50 mL volumetric flask, add 10 mL of internal standard sonicate to dissolve then make up the
volume with diluent.

8.3.3 Test Solution Preparation:

Note: Freshly prepared solution.

Weigh accurately and transfer about 25 mg of sample into 50 mL volumetric flask, add 10 mL of
internal standard solution, sonicate to dissolve and make up the volume up to the mark with diluent.

8.3.4 Injection sequence (for information only)

Sr. No. Sample Name No. of Injection

1. Blank 1
2. Standard solution 6
3. Test solution 2
4. Standard solution BKT 1

8.3.5 Evaluation of system suitability

%RSD for Faropenem peak area from six replicate injections of standard solution should not be
more than 2.0.
Resolution should not be less than 1.5, between Internal standard and Faropenem sodium hydrate
peak.

8.4 Calculations:
Calculate the Assay in µg/mg by area ratio, dividing area of Faropenem peak and Internal standard
area.

AT WS 50 Potency
%Assay of Faropenem = ------ x ------ x-------x-------------------- x 100
(on anhydrous basis) AS 50 WT (100 - % water)

Where,

AT = Area ratio of Internal standard and Faropenem peak in test solution.

AS = Average area ratio of Internal standard and Faropenem peak in six replicate injections of
standard solution.
WS = Weight of Faropenem sodium Reference/Working standard in mg.
WT = Weight of sample in mg

Additional tests:
9.0 Particle Size by Malvern Mastersizer (as per customer requirement)

9.1 Requirements

The Malvern Mastersizer 2000 equipped with Scirocco 2000, flow cell, Fourier lenses and a multi
element detector.

Data Handling System, Malvern Mastersizer 2000 Version 5.54 or equivalent

9.2 Procedure

9.2.1 Instrument Parameters

Optical Parameters Particle RI 1.45

Absorption 0.01

Analysis Model Polydisperse

Lens 300 mm

Technique Used Dry Method

Note: On examination of data, fit curve and histogram shape, the absorption values may vary in
multiples of 10 from 0 to 1.0 to obtain the best fit curve.

9.2.2 Instrumental Analysis

Carefully assemble the flow and the lens into the main unit of the instrument.
Connect the dry powder feeder unit (connected with the air cylinder) to the main unit. Connect the
out let of the cell to a vacuum calendar.
Switch on the instrument, dry powder feeder unit and the data handling system.
Switch on the cylinder and maintain air pressure of about 120 psi in the inlet of the cylinder
regulator.
Switch on the vacuum cleaner.
Align the instrument after feeding the requirements.
Place 5 - 10 g of sample in the hopper tray of the dry powder feeder unit close it.
Measure the background.
Start the particle size measurement by feeding into the main unit by turning the knob to 'Feed'
position of the dry powder unit.
Adjust the feed rate at 70% to get an obscuration of 0.5 - 5.0% start the particle size measurement
after getting a proper obscuration value.
Analyses the sample in triplicate at air pressure 1.5 bar with one measurement for each sample and
report the value closest to the mean value.
After sample measurement, click at Air 1 Vacuum position at the software window feeder unit.
9.2.3 Precautions
Flow cell and Scirocco 2000 unit must be absolutely clean.
Weighted residual preferably should be less than 2.0%.
Sample should be put in middle part of hopper tray of the dry powder feeder.
For sharp histogram set distance of slit.
9.3 Calculations: NA