Method of Analysis Ceftriaxone and Sulbactam For Injection

Description

White to yellowish crystalline powder, slightly hygroscopic.

Identification

A

Liquid

Chromatography

1. Refer to the assay.

B

Sodium Test

Procedure:

1. Prepare a solution to contain 0.1 g of the sodium compound in 2 ml of water.

2. Add 2 ml of a 15% w/v solution of potassium carbonate and heat to boiling.

3. No precipitate is formed.

4. Add 4 ml of potassium pyroantimonate solution and heat to boiling.

5. Allow to cool in ice and if necessary rub the inside of the test-tube with a glass rod.

6. A dense, white precipitate is formed.

7. Sodium compounds impart an intense yellow colour to a non-luminous flame.

Average Net Filled Weight

1. Remove any adherent labels from a container and wash and dry the outside.

1. Open the container and immediately weigh the container

and its contents empty the container as completely as

possible by gentle tapping.

2. Rinse if necessary with water and then with ethanol (95%) and dry at 100°C to 105°C for 1 hour.

3. If the nature of the container precludes such treatment, dry at a lower temperature to constant weight.

4. Allow to cool in a desiccators and weigh the difference between the weights represents the weight of the contents.

5. Repeat the procedure with a further 9 containers.

6. Average net filled weight = S W/10

Completeness and Clarity of Solution

Standard preparation:

1. Dissolve 1.0 g of hydrazine sulphate in sufficient water to produce 100 ml.

2- Set aside for 6 hours.

3- To 25 ml of this solution add 25 ml of a 10% w/v solution of

hexamine.

4. Mix well and allow to stand for 24 hours.

5. Keep in a glass container with smooth surfaces so that suspensions does not adhere to the glass.

(Note: Prepare the standard suspension by diluting 15 ml of the well mixed suspension to 1000 ml with water. The standard solution should be used within 24 hours of preparation)

Opalescence standard:

1. From standard solution mix 5 ml in 95 ml of water this is final Opalescence standard.

Test preparation:

1. Dissolve 12 g of the sample in 50 ml carbon dioxide free water.

2. Shake well and finally make up the volume 100 ml with same water.

Procedure:

1. Transfer to a flat-bottom test tube of neutral glass 15 to 25 mm in diameter a suitable volume of test solution such that test tube is filled to a depth of 40 mm.

2. Into another matched test tube add the same volume of opalescence standard.

3. After 5 minutes compare the contents of the test tube against the black background.

Constituted Solution

1. The solid dissolves completely.

1. Leaving no visible residue as undissolved matter.

2. The constituted solution is clear.

Crystallinity

1. Mount a few particles of the specimen in mineral oil on a clean glass slide.

2. Examine the mixture using a polarizing microscope: the particles show birefringence (interference colors) and extinction positions when the microscope stage is revolved.

pH

1. Dissolve 5 g of substance to be examined in 50 ml volumetric flask

2. Dissolve and makeup the volume 50 ml with water.

Procedure:

1. Wash the electrode of the pH meter with distilled water and clean with tissue paper.

2. Take 40 ml of the sample in 50 ml glass beaker.

3. Dip the electrode in a beaker.

4. Wait for 5 minutes to stable the pH.

5. Note down the pH.

6. Wash the electrode twice with distilled water.

7. Keep it in distilled water.

Water

1. Start Karl Fischer Titrator apparatus.

2. Neutralize the glass bottle with Karl Fischer reagent, which must be completely free from moisture.

Determination of water factor:

1. First take 50 ml of dried methanol in titration beaker.

2. Neutralize with Karl Fischer.

3. Now add accurately about 50.0 mg purified water.

4. Start titration and find out the water moisture factor of Karl Fischer solution.

Calculation:

Wt. of water in mg

= —————————————————— =…………mg/ml

Volume Fischer consumed of Karl in ml

Determination of water in sample:

1. Weigh accurately about 0.5 g of substance.

2. Transfer it into glass bottle containing magnetic stirrer.

3. Start the instrument.

4. Find out the water content of substance being examined.

Volume consumed of Karl Fischer x Factor of water x 100 Calculation: = ——————————————————————-

Wt. of substance in mg

=…………..%

Particulate Matter

Sample preparation:

1. Invert the container of the preparation under examination 20 times successively in order to mix the contents.

2. Wash the outer surface of the container with a jet of particle-free water and remove the closure carefully, avoiding contamination of the contents.

Test procedure :

1. Reconstitute the sample with particle free water.

2. Combine the content of 10 units or more in a clean container and test it through liquid particle counter.

3. During this procedure sample 4 portions each not less than 5 ml.

4. Ignore the result obtained from the first portion and calculate the average number of particle equal to or greater than 10 µm and 25 µm for rest of three portions.

Assay

Content of Ceftriaxone and Sulbactam:

Determine by liquid chromatography:

Reagent:

1. Dibasic potassium phosphate (AR Grade)

2. Potassium hydroxide (AR Grade)

3. Phosphoric acid (LR Grade)

4. Acetonitrile (HPLC Grade)

5. Water (HPLC Grade)

Chromatographic system:

1. Column : A stainless steel column C18 250

x 4.6 mm, packed with

octadecylsilane bonded to porous

silica (5 µm)

2. Flow Rate : 1 ml/minute

3. Wavelength : 220 nm

4. Injection Volume : 20 µl

Buffer solution:

1. Dissolve 4.25 g of dibasic potassium phosphate in 1000 ml of water.

2. Adjust to a pH-4.0 with dilute orthophosphoric acid or dilute potassium hydroxide.

Composition of mobile phase:

Buffer solution : Acetonitrile

75 : 25

Standard solution:

1. Weigh accurately equivalent to 50.0 mg of Ceftriaxone

sodium and 50.0 mg of Sulbactam sodium working

standard in 100 ml of volumetric flask.

2- Add 50 ml of mobile phase.

3- Sonicate it to dissolve.

4- Make up the volume with mobile phase.

5- Dilute 10 ml of this solution to 50 ml with mobile phase.

Test solution:

1. Weigh accurately equivalent to 50.0 mg of sample in100

ml of volumetric flask.

1. Add 50 ml of mobile phase.

2. Sonicate it to dissolve.

3. Make up the volume with mobile phase.

4. Dilute 10 ml of this solution to 50 ml with mobile phase.

Procedure:

Inject standard solution. The test is not valid unless the tailing factor is maximum 2 and the relative standard deviation for replicate injections is maximum 2.0%.

Inject standard solution and the test solution.

Name	Number of Injections
Blank	01
Standard solution	05
Test solution	02
Bracketing Standard solution	01

Calculation:

Test Area Std. Wt. Test Dil.

= ————–X ———–X ———- X Avg. filled wt. X Potency of W.S.

Std. Area Std. Dil. Test Wt.

= …….. mg/vial

= ……%.

Bacterial Endotoxin

Reconstitution of LAL reagent:

Reconstitute the LAL Reagent with LRW as indicated on the vial of LAL Reagent. Homogenize it by revolving it smoothly. Do not vortex. After reconstitution store LAL Reagent at 0ºC.

Reconstitution of CSE and its dilution:

Reconstitute the CSE with LRW as indicated on the certificate of analysis. Vortex it for 3 minutes. After reconstitution store it at 2ºC to 8ºC.

Now prepare 4 λ, 2 λ, λ and λ/2.

Dilutions of CSE :

Preparation of maximum valid dilution of the product:

For preparation of Maximum Valid Dilution (MVD) of the product, use the following formula and calculate the MVD as follows:

Endotoxin Limit x Potency of product

Maximum Valid Dilution = ————————————————–

Sensitivity of LAL Reagent

Test procedure:

After preparation of CSE dilution and MVD, the test may be carried out as follows:

Checking the sensitivity of LAL reagent:

For testing the sensitivity of LAL Reagent 2 λ, λ and λ/2 CSE dilutions are used:

(i) Take 100 µl of CSE ( 2 λ) into sample tube put in Heating Block and add 100 µl of LAL Reagent into it and incubate for one hour at 37ºC ±1ºC.

(ii) Take 100 µl of CSE (λ) into sample tube put in Heating Block and add 100 µl of LAL Reagent into it and incubate for one hour at 37ºC ±1ºC.

(iii) Take 100 µl of CSE (λ/2) into sample tube put in Heating block and add 100 µl of LAL Reagent into it and incubate for one hour at 37ºC ±1ºC.

Negative product control (NPC):

Take 100 µl of sample (MVD) into sample tube put in Heating Block and add 100 µl of LAL Reagent into it and incubate for one hour at 37ºC ±1ºC.

Positive product control (PPC):

Take 50 µl of sample (MVD/2) into sample tube and add to it 50 µl of 4 λ CSE and vortex it for one minute and put this tube in Heating Block and add 100 µl of LAL Reagent into it and incubate for one hour at 37ºC (±1ºC).

Negative water control (NWC):

Take 100 µl of LRW into sample tube put in Heating Block and add 100 µl of LAL Reagent into it and incubate for one hour at 37ºC ±1ºC.

Positive water control (PWC):

Take 50 µl of LRW into sample tube and add to it 50 µl of 4 λ CSE and vortex it for one minute and put this tube in Heating Block and add 100 µl of LAL Reagent into it and incubate for one hour at 37ºC ±1ºC.

Sterility

Method used:

Membrane filtration:

Procedure:

Prepare the fluid thioglycollate medium and soyabean casein digest medium as indicated on the container of the medium. Dispense100 ml of the medium in the test tubes (38 x 200 mm diameter) and plug them. Also prepare peptone water and dispense 100-100 ml of it in conical flasks. Sterilize medium tubes and peptone water for 121º C at 15 psi for 20 minutes in a M.H.S. Add the filter paper (0.45 æ pore size, without grid and 47 mm diameter) to the filtration manifold and wrap it using butter paper. Also wrap the silicon tubings, manifold holder (suction flask), cutter, forceps and surgical blades.

Now sanitize the samples by dipping these into a beaker containing 70% IPA solution. Leave it for at least half an hour prior to sterility test.

The materials which are to be taken to sterile area of Microbiology lab should be previously sanitized using 70% IPA solution.

Clear the stage of Laminar air flow with 70%IPA solution using lint free cloth. Light the burner. Assemble the filtration apparatus (filtration manifold containing filter paper, manifold holder, silicone tubing and vacuum pump) aseptically. Take the samples out of the beaker and allow to dry on the stage of Laminar air flow. Now cut the ampoule of sterile water for injection using sterile scissor and filter 2 ml of the water sample aseptically using filtration apparatus. Repeat the process for 20 ampoules. After filtration cut the filter paper into two halves using sterile surgical blade and aseptically transfer one part of the filter paper in the test tube containing 100 ml of FTGM and another part in the test tube containing 100 ml of SCDM.

Leave one tube of FTGM and one tube of SCDM as negative control. Incubate the tubes of FTGM at 32.5ºC ±2.5ºC and tubes of SCDM at 22.5ºC ±2.5ºC. Apply positive control. Negative control of the test should be performed simultaneously.