SHINIL Pharma Ltd.

B K Bari, Mirzapur, Gazipur.

Standard Test Procedure (Active Ingredient)

Material Name Ciprofloxacin Hydrochloride USP Material Code RA- C01
Revision No. 01 STP No. STP-RA- C01
Effective Date December 06, 2021 Review Date December 05, 2024
1.0 APPERANCE :

Take 1 g of test sample in a watch glass and observe visually with white background.

2.0 IDENTIFICATION :

A. BY INFRARED SPECTROSCOPY

Grind 200 mg Potassium bromide into fine powder. Add 2 mg of test sample mix well and prepare a
disk. Record the Infrared absorption spectrum of the disk from 2000 cm –1 to 400 cm–1. The absorption
maxima in the spectrum obtained with the substance being examined, correspond in position and
relative intensity to those in the spectrum obtained with the Ciprofloxacin Hydrochloride working
standard which is prepared under same operational conditions.

B. The retention time of the major peak of the sample solution corresponds to that of the standard
solution, as obtained in the assy.

C. Dissolve in 2 ml of water a quantity of the substance to be examined equivalent to about 2 mg of

chloride

(Cl–) or use 2 ml of the prescribed solution. Acidify with dilute nitric acid and add 0.4 ml of silver nitrate
solution. Shake and allow standing. A curdled, white precipitate is formed. Centrifuge and wash the
precipitate with three quantities, each of 1 ml, of water. Carry out this operation rapidly in subdued
light, disregarding the fact that the supernatant solution may not become perfectly clear. Suspend the
precipitate in 2 ml of water and add 1.5 ml of ammonia. The precipitate dissolves easily with the
possible exception of a few large particles which dissolve slowly .It gives the reactions of chlorides.

3.0 SOLUBILITY :

Take 100 mg of test sample in nine different test tubes. Add 10 ml of water, 100 ml of acetic acid, 100
ml of methanol, 1000 ml of dehydrated alcohol, 1000 ml of acetone, 1000 ml of acetonitril, 1000 ml of
ethyl acetate, 1000 ml of hexane, 1000 ml of methylene chloride into these test tubes respectively.
Shake properly.

Prepared By Checked By Approved By

Md. Rasheduzzaman Bejoy Karim Md. Mahabub Alam

Officer, Quality Control Senior Officer, PD Manager, Quality Assurance
Page no. 1 of 7
 SHINIL Pharma Ltd.
B K Bari, Mirzapur, Gazipur.

Standard Test Procedure (Active Ingredient)

Material Name Ciprofloxacin Hydrochloride USP Material Code RA- C01
Revision No. 01 STP No. STP-RA- C01
Effective Date December 06, 2021 Review Date December 05, 2024

4.0 WATER :
Weigh about 250 mg of sample and determine its water content by using a suitable Karl Fischer.

5.0 pH ( 2.5% W/V SOLUTION IN WATER) :

Dissolve 1.25 g in carbon dioxide-free water and dilute to 50 ml with the same solvent.
6.0 IMPURITIES :

6.1 INORGANIC IMPURITIES :

A. RESIDUE ON IGNITION :

Ignite a suitable crucible (for example silica, platinum, porcetain or quartz) at 600 ± 500 C for 30 min,
allow to cool in a desiccators over silica gel or other suitable desiccant and weight. Place the 1 g of
test sample in the crucible and weight. Moisten the substance to be examined with a small amount of
sulfuric acid (usually 1 ml) and heat gently at as a low temperature as practicable until the sample is
thoroughly charred. After cooling, moisten the residue with a small amount of sulfuric acid (usually 1
ml), heat gently until white fumes are no longer evolved and ignite at 600 ± 500 C until the residue is
completely incinerated. Ensure that flames are not produced at any time during the procedure. Allow
the crucible to cool in a desiccator over silica gel or other suitable desiccant, weight it again and
calculate the percentage of residue.

Calculation:
W3-W1

Residue on Ignition = ----------------------- X100

W2-W1

Where,W1= Weight of empty crucible (g)

W2=Weight of crucible with sample before ignition(g)

Prepared By Checked By Approved By

Md. Rasheduzzaman Bejoy Karim Md. Mahabub Alam

Officer, Quality Control Senior Officer, PD Manager, Quality Assurance
Page no. 2 of 7
 SHINIL Pharma Ltd.
B K Bari, Mirzapur, Gazipur.

Standard Test Procedure (Active Ingredient)

Material Name Ciprofloxacin Hydrochloride USP Material Code RA- C01
Revision No. 01 STP No. STP-RA- C01
Effective Date December 06, 2021 Review Date December 05, 2024
W3= Weight of crucible with sample after ignition

B. SULFATE: Dissolve 375 mg of the substance under test in 30 ml to 40 ml of water or where the
substance is already in solution, add water to make a total volume of 30 to 40 ml, and if necessary
neutralize the solution with hydrochloric acid to litmus. Add 1 ml of 3 N hydrochloric acid, 3 ml of
barium chloride TS, and sufficient water to make 50 ml. Mix and allow to stand for 10 minutes.
Compare the turbidity, if any with that produced in a solution containing the volume of 0.020N sulfuric
acid specified in the monograph corresponds to 0.15 ml of 0.020 N sulfuric acid (0.04%).

C. HEAVY METALS :
pH 3.5 Acetate Buffer: Dissolve 25.0 g of ammonium acetate in 25 ml of water, and add 38.0 ml of 6
N hydrochloric acid. Adjust, if necessary, with 6 N ammonium hydroxide or 6 N hydrochloric acid to a
pH of 3.5 dilute with water to 100 ml, and mix.
Standard Preparation: Into a 50-mL color-comparison tube pipet 2 ml of Standard Lead Solution (20
µg of Pb), and dilute with water to 25 ml. Using a pH meter or short-range pH indicator paper as
external indicator, adjust with 1 N acetic acid or 6 N ammonium hydroxide to a pH between 3.0 and
4.0, dilute with water to 40 ml, and mix.

Test Preparation— Into a 50-mL color-comparison tube place 25 ml of the solution prepared for the
test as directed in the individual monograph; or, using the designated volume of acid where specified
in the individual monograph, dissolve in and dilute with water to 25 ml the quantity, in g, of the
substance to be tested, as calculated by the formula: 2.0/(1000L).In which L is the Heavy metals limit,
in percentage. Transfer the weighed quantity of the substance to a suitable crucible, add sufficient
sulfuric acid to wet the substance, and carefully ignite at a low temperature until thoroughly charred.
(The crucible may be loosely covered with a suitable lid during the charring.) Add to the carbonized
mass 2 ml of nitric acid and 5 drops of sulfuric acid, and heat cautiously until white fumes no longer
are evolved. Ignite, preferably in a muffle furnace, at 500°to 600° until the carbon is completely burned
off. Cool, add 4 ml of 6 N hydrochloric acid, cover, digest on a steam bath for 15 minutes, uncover,
and slowly evaporate on a steam bath to dryness. Moisten the residue with 1 drop of hydrochloric
acid, add 10 ml of hot water, and digest for 2 minutes. Add 6 N ammonium hydroxide dropwise until
the solution is just alkaline to litmus paper, dilute with water to 25 ml, and adjust with 1 N acetic acid to
a pH between 3.0 and 4.0, using short-range pH indicator paper as an external indicator. Filter if

Prepared By Checked By Approved By

Md. Rasheduzzaman Bejoy Karim Md. Mahabub Alam

Officer, Quality Control Senior Officer, PD Manager, Quality Assurance
Page no. 3 of 7
 SHINIL Pharma Ltd.
B K Bari, Mirzapur, Gazipur.

Standard Test Procedure (Active Ingredient)

Material Name Ciprofloxacin Hydrochloride USP Material Code RA- C01
Revision No. 01 STP No. STP-RA- C01
Effective Date December 06, 2021 Review Date December 05, 2024
necessary, rinse the crucible and the filter with 10 ml of water, combine the filtrate and rinsing in a 50-
mL color-comparison tube, dilute with water to 40 ml, and mix.

Procedure— To each of the tubes containing the Standard Preparation and the test Preparation, add 2
ml of pH 3.5 Acetate Buffer, then add 1.2 ml of thioacetamide–glycerin base TS, dilute with water to 50
ml, mix, allow to stand for 2 minutes, and view downward over a white surface the color of the solution
from the test Preparation is not darker than that of the solution from the Standard Preparation.

6.2 ORGANIC IMPURITIES :

A. Procedure 1: Limit of Fluoroquinolonic acid:

Standard Solution: Transfer 5.0 mg of USP Fluoroquinolonic acid RS to a 50 ml volumetric flask

containing 0.05 ml of 6N ammonium hydroxide, add water to volume. And mix. Transfer 2.0 ml of this
solution to a 10 ml volumetric flask. And dilute with water to volume.

Sample Solution: 10 mg/ml of ciprofloxacin hydrochloride in water.

Chromatographic system: Thin Layer Chromatography:

Mode : TLC

Adsorbent: 0.25 mm layer of silica gel mixture.

Application volume: 5 µl.

Developing Solvent System: methylene chloride , methanol, acetonitril and ammonium hydroxide
(4:4:1:2)

Procedure: standard solution and sample solution prepared as directed in the above. Place a beaker
containing 50 ml of ammonium hydroxide in a suitable chamber, and then place the plate in the
chamber. After 15 min, transfer the plate to a suitable chromatographic chamber, and develop the
chromatogram in the developing solvent system until the solvent front has moved about three-fourth of
the length of the plate. Remove the pate from the chamber, mark the solvent front, and allow the plate

Prepared By Checked By Approved By

Md. Rasheduzzaman Bejoy Karim Md. Mahabub Alam

Officer, Quality Control Senior Officer, PD Manager, Quality Assurance
Page no. 4 of 7
 SHINIL Pharma Ltd.
B K Bari, Mirzapur, Gazipur.

Standard Test Procedure (Active Ingredient)

Material Name Ciprofloxacin Hydrochloride USP Material Code RA- C01
Revision No. 01 STP No. STP-RA- C01
Effective Date December 06, 2021 Review Date December 05, 2024
to dry for about 15 min. Examine the plate under short wavelength UV light.

Any spot from the sample solution, at an R F value corresponding to the principal spot from the
standard solution, is not greater in size or intensity than the principal spot from the standard solution
(0.2%)

B. Procedure 2: Mobile phase, standard solution, system suitability solution, sample solution,
chromatographic system and system suitability: Proceed as directed in the assay.

Analysis:

Sample: Sample solution

Inject the sample solution and run for 30 minutes and calculate the percentage of each impurity in the
portion of Ciprofloxacin hydrochloride taken:

Result= (rU /rT )x100

rU = Peak response from each impurity

rT = sum of the responses of all the peaks

7.0 Assay :

Diluent: Water

Mobile Phase: Acetonitrile and solution A at the ratio of (13:87).

Standard Solution: Take 6.0 mg /ml of Gonadorelin Acetate BP RS in 100 ml and volume upto with
diluent

System Suitability Solution: Take 6.0 mg /ml of Gonadorelin Acetate BP RS in 100 ml and volume
upto with diluent.

Prepared By Checked By Approved By

Md. Rasheduzzaman Bejoy Karim Md. Mahabub Alam

Officer, Quality Control Senior Officer, PD Manager, Quality Assurance
Page no. 5 of 7
 SHINIL Pharma Ltd.
B K Bari, Mirzapur, Gazipur.

Standard Test Procedure (Active Ingredient)

Material Name Ciprofloxacin Hydrochloride USP Material Code RA- C01
Revision No. 01 STP No. STP-RA- C01
Effective Date December 06, 2021 Review Date December 05, 2024
Sample solution: Take about 3 ml sample solution into 25 ml volumetric flask. and volume upto with
diluent.

Chromatographic System:

Mode: LC

Detector: UV 215 nm

Inject size: 50 µl.

Column temperature: ambient

Flow rate : 1.5 m/min

System suitability

Samples: Standard solution Run at least 5 times.

[Note: The relative retention times for Gonadorelin is 0.7 and 1.0, respectively.]

Suitability Requirements:

Resolution: NLT 6 between the Ciprofloxacin ethylenediamine analog peak and the Ciprofloxacin
peak, system suitability solution.

Column Efficiency: NLT 2500 theoretical plates from the ciprofloxacin peak, Standard solution.

Tailing factor: NMT 2.5 for the Gonadorelin. Standard solution

Relative Standard Deviation: NMT 1.5 % Standard solution.

Calculation:

Prepared By Checked By Approved By

Md. Rasheduzzaman Bejoy Karim Md. Mahabub Alam

Officer, Quality Control Senior Officer, PD Manager, Quality Assurance
Page no. 6 of 7
 SHINIL Pharma Ltd.
B K Bari, Mirzapur, Gazipur.

Standard Test Procedure (Active Ingredient)

Material Name Ciprofloxacin Hydrochloride USP Material Code RA- C01
Revision No. 01 STP No. STP-RA- C01
Effective Date December 06, 2021 Review Date December 05, 2024
Calculate the percentage of Ciprofloxacin by the following way:

Pu x Ws x P
Result = ---------------------- = ----------------- % (As Gonadorelin as it is)
Ps x Wu
Here,
Pu = Peak area of sample preparation
Ps = Peak area of working standard preparation
Ws = Standard weight taken
Wu = Sample weight taken
P = Standard potency (As Ciprofloxacin as it is)

Calculate the potency of Gonadorelin by using following equation:

367.60 100
Result = Potency of Ciprofloxacin x ------------ x ----------------- =------ % (On anhydrous basis)
331.40 100 - W

List of Distribution

1) Quality Assurance
2) Product Development
3) Quality Control
4) Commercial

Prepared By Checked By Approved By

Md. Rasheduzzaman Bejoy Karim Md. Mahabub Alam

Officer, Quality Control Senior Officer, PD Manager, Quality Assurance
Page no. 7 of 7