Title: Raw Material Test Method

Material Name          : Aceclofenac  BP

Generic Name                        :           Aceclofenac                          

Empirical Formula   :           C16H13Cl2NO4 

Structural Formula   :

Molecular weight      :        354.2

CAS NO.                    :           [89796-99-6]

Table of Content:

Method of Analysis:

01.              Appearance:  

Spread approximately 3.0 g of sample over a white paper and examine visually. Check the appearance for color, nature and any visible foreign particles. Record its appearance, extraneous matter, if any. The sample should be white or almost white, crystalline powder.

02.              Solubility:      

Practically insoluble in water, freely soluble in acetone, soluble in ethanol (96 per cent).

Freely soluble in Acetone:

Take about 0.1 gm of the sample in a clean and dry test tube. To it add 1 ml of acetone and mix. The solution should be clear without any suspended particles.

Soluble in Ethanol (96%):

Take about 0.1 g of sample into a clean and dry test tube. Add 3 ml ethanol (96%) and mix the content. The solution should be clear without any suspended particles

Practically insoluble in Water:

Take about 10 mg of the sample in 200 ml volumetric flask. To it add 100 ml of purified water and shake. The dispersion should not be clear (suspended particles will be observed).

03.              Identification:

A.    Ultraviolet and visible absorption spectrophotometry(2.2.25).

Procedure:

Testsolution Dissolve 50.0 mg in methanol R and dilute to100.0 mL with the same solvent. Dilute 2.0 rnL of the solution to 50.0 rnL with methanolR. Spectra/range 220-370 nm. Absorption maximum 275 nm.

Specific absorbance at the absorption maximum 320 to 350.

B.IR Spectrum

Procedure:

Take about 1 mg of Aceclofenac (sample) and 300 mg of previously dried KBr. Mix uniformly by mortar & pestle. Transfer the mixture into a disc making die and spread the powder uniformly with help of punch. Transfer the die set to the disc making press and compress to form a thin disc. Remove the disc from the die set. Transfer to sample holder and keep the sample holder in the instrument sample compartment.

Scan the sample over the range from 4000 cm-1 to 400 cm-1. Make sure that the transmittance of the standard and sample spectrum at the wavelength of 2000 cm-1 is on or above the 60 % mark.

IR Spectrum of the test sample shall exhibit maximum only at the same wave number as that of Aceclofenac working standard.

Acceptance criteria:

IR Spectrum of the test sample shall exhibit maximum only at the same wavenumbers as that of aceclofenac CRS/Working standard. The correlation should be at least 0.950.

C. Chemical Test

Dissolve about 10 mg in 10 rnL of ethanol (96 per cent) R. To 1 mL of the solution, add 0.2 mL of a mixture, prepared immediately before use, of equal volumes of a 6 gIL solution of potassium ferricyanide R and a 9 gIL solution of ferric chloride R. Allow to stand protected from light for 5 min. Add 3 rnL of a 10.0 gIL solution of hydrochloric acidR. Allow to stand protected from light for 15 min. A blue colour develops and a precipitate is formed.

04.              Related Substances:

Chromatographic Condition:

Column                       : Spherical end-capped C18; 250 mm x 4.6 mm, 5 mm with a pore size of  

   10 nm and a carbon loading of 19 per cent.

Detection, UV             : 275 nm

Flow rate                     : 1.0 ml /min.

Oven Temperature      : 40°C

Inject volume              : 10 ml

Stationary phase:

Spherical end-capped octadecylsilyl silica gel for chromatography R (5 urn) with a pore size of 10 nm and a carbon loading of 19 per cent

Mobile phase:

Mobile phase A: 1.12 g/l solution of phosphoric acid adjusted to pH 7.0 using a 42 g/l solution of sodium hydroxide.

Mobile phase B: purified water, acetonitrile (1:9 V/V). 

Diluent: A mixture of mobile phase A and mobile phase B (30:70)

Test solution:

Dissolve 50.0 mg of the substance to be examined in the solvent mixture and dilute to 25.0 rnL with the solvent mixture.

[Note: prepare test solution freshly just before injection]

Reference solution (a):

Dissolve 21.6 mg of diclofenac sodium CRS (impurity A) in diluent and dilute to 50.0 ml with the same diluents, mix.

Reference solution (b):

Dilute 2.0 ml of the test solution to 10.0 ml with diluent, mix.

Reference solution (c):

Mix 1.0 ml of reference solution (a) and 1.0 ml of reference solution (b) and dilute to 100.0 ml with diluents, mix.

Reference solution (d):

Dissolve 4.0 mg of aceclofenac impurity F CRS and 2.0 mg of aceclofenac impurity H CRS in diluents then dilute to 10.0 ml with the same diluent.

Reference solution (e) :

Dissolve 2.0 mg of aceclofenac  impurity H CRS in the solvent mixture and dilute to 10.0 rnL with the solvent mixture.

Reference solution (f):

Mix 1.0 mL of reference solution (b), 1.0 mL of reference solution (d) and 1.0 mL of reference solution (e) and dilute to 100.0 mL with the solvent mixture.

Referencesolution (g)

Dissolve 5.0 mg of aceclofenac impurity I CRS in the solvent mixture and dilute to 50.0 mL with solvent mixture. Dilute 1.0 mL of the. solution to 50.0 mL with the solvent mixture.

Reference solution (h)

Dissolve 4 mg of aceclofenac for peak identification CRS (containing impurities B, C, D, E and G) in 2 rnL of the solvent mixture

.Procedure:

Equilibrate the column at least 30 minutes with the initial composition of mobile phase at a flow rate of 1 ml/ min and carry out the gradient elution as follows.

Inject 10 ml of reference solution (c) into the chromatograph and record the chromatogram.

Injection:

Inject separately equal volume (10 ml) of blank (diluent), reference solutions (e), (f), (g) (h) and test solution into the chromatograph and record the chromatograms.

Identification of impurities:

Use the chromatogram obtained with reference solution (c) to identify the peak due to impurity A; use the chromatogram supplied with aceclofenac for peak identification CRS and the chromatogram obtained with reference solution (h) to identify the peaks due to impurities B, C, D, E and G; use the chromatogram obtained with reference solution (d) to identify the peak due to impurity F; use the chromatogram obtained with reference solution (e) to identify the peak due to impurity H; use the chromatogram obtained with reference solution (g) to identify the peak due to impurity I.

Relative retention:

With reference to aceclofenac (retention time = about 11 min):

impurity A = about 0.8;impurity G = about 1.3; impurity H = about 1.5; impurity I = about 2.3;

impurity D = about 3.1;impurity B = about 3.2; impurity E = about3.3;

impurity C = about 3.5; impurity F = about 3.7.

System suitability: Reference solution (c):

Resolution: minimum 5.0 between the peaks due to impurity A and aceclofenac.

Acceptance criteria:

Impurity A: not more than the area of the corresponding peak in the chromatogram obtained with reference solution (c) (0.2 per cent);

Impurities B, C, D, E, G: for each impurity, not more than the area of the peak due to aceclofenac in the chromatogram obtained with reference solution (e) (0.2 per cent);

Impurity F: not more than the area of the corresponding peak in the chromatogram obtained with reference solution (e) (0.2 per cent);

Impurity H: not more than the area of the corresponding peak in the chromatogram obtained with reference solution (e) (0.15 per cent);

Impurity I: not more than the area of the corresponding peak in the chromatogram obtained with reference solution (f) (0.15 per cent);

Unspecified impurities: not more than 0.5 times the area of the peak due to aceclofenac in the chromatogram obtained with reference solution (e) (0.10 per cent);

Total impurities: not more than 0.7 per cent.

Disregard limit: 0.25 times the area of the peak due toaceclofenac in the. chromatogram obtained with referencesolution (f) (0.05 per cent )

05.              Loss on Drying:

Procedure:

Dry the weighing bottle at 105°C until constant weight and weigh (W1) in gm. Weigh accurate 1.0 g of sample (W2) in the weighing bottle. Dry the sample at 105°C for 3 hours. Cool the sample in desiccator and weigh (W3) in g. Calculate the loss on drying (LOD) in percentage by following formula.

LOD (% w/w) =

Acceptance criteria:  Maximum 0.5%.

06.              Sulfated Ash:

Procedure:

Ignite a silica crucible 600°C ± 50° for 30 minutes; allow to cool in a desiccator over silica gel and weigh (W1) in gm. Weigh 1 gm of sample (W2) and transfer into the crucible. Moisten the test sample with a small amount of sulphuric acid (usually 1 ml) and heat gently at as low a temperature as practicable until the sample is thoroughly charred.

After cooling, moisten the residue with a small amount of sulphuric acid, heat gently until white fumes are no longer evolved and ignite at 600°C ± 50° 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, weigh it again and weigh (W3) of the residue.

If the weight of residue so obtained exceeds the prescribed limit, repeat the moistening with sulphuric acid and ignition, as previously, for 30 min periods until 2 consecutive weighings do not differ by more than 0.5 mg or until the percentage of residue complies with the prescribed limit.

Calculate the residue in percentage by following formula:

Ash (% w/w) = 

Where,

W1      =          Weight of empty ignited crucible in gm.

W2      =          Weight of test sample before ignition in gm.

W3      =          Weight of crucible after ignition with the residue sample in gm

Acceptance criteria: Maximum 0.1%.

07.              Assay:

Reagents:

i)   Methanol

ii)  0.1 M sodium hydroxide VS

Procedure:

Dissolve 0.300 g in 40 ml of methanol. Titrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically.

1 ml of 0.1 M sodium hydroxide is equivalent to 35.42 mg of aceclofenac.

Calculate the content of Aceclofenac in percentage by the equation given below.

Content of Aceclofenac(A) =  %w/w (as it is)

CO1     =                     Required titre for sample

CO2       =                   Factor of 0.1 M sodium hydroxide VS

CO3     =                     35.42

CO4     =                     100

COO    =                     Weight of sample in mg

Carry out two assays and take average of the two assays.

Then calculate the content of Aceclofenac in  percentage on dried basis by the equation given below.

OR Alternative UV Method:

Standard Solution

Weight accurately about 50 mg of Aceclofenac WS and transfer into a 100 ml volumetric flask and dissolve in 30 ml of ethanol and volume upto 100 ml with ethanol. Dilute 2 ml of the solution into a 100 ml volumetric flask and volume upto 100 ml with ethanol.

Sample Solution

Weight accurately about 50 mg of test sample and transfer into a 100 ml volumetric flask and dissolve in 30 ml of ethanol and volume upto 100 ml with ethanol. Dilute 2 ml of the solution into a 100 ml volumetric flask and volume upto 100 ml with ethanol.

Measurement:

Determine the absorbance of standard and sample solution in 1cm cell at 275 nm using ethanol as blank.

Calcukation:

Content of Aceclofenac (% w/w asis):     

Asam  X Wstd X   2    X 100   X 100 X Pstd

Astd    X 100   X 100 X Wsam  X  2  

Where,

Astd       :               Absorbance from standard solution

Wstd      :               Weight of standard     

Asam      :               Absorbance from sample solution     

Wsam    :               Weight of sample

Pstd        :               Potency of standard

Content of Aceclofenac =  %w/w (on dried basis)

A= assay (as it is)

Acceptance criteria: 99.0 % to 101.0 % of Aceclofenac on dried basis.

OR Alternative liquid chromatography Method:

NOTE — Use freshly prepared solutions.

Solvent mixture: 40 volumes of acetonitrile and 60 volumes of water.

Test solution: Weigh and powder 20 tablets. Disperse a quantity of the powdered tablets containing 325 mg of Paracetamol in 40 ml of acetonitrile, with the aid of ultrasound with intermittent shaking. Add 100 ml of the solvent mixture and sonicate for 15 minutes with intermittent shaking, dilute to 200.0 ml with the solvent mixture. Dilute 5.0 ml of the solution to 50.0 ml with the solvent mixture.

Reference solution (a): Dissolve 32.5 mg of paracetamol IPRS in 5 ml of acetonitrile, with the aid of ultrasound for 5 minutes and dilute to 20.0 ml with the solvent mixture.

Reference solution (b): Dissolve 25 mg of aceclofenac IPRS in 10 ml of acetonitrile, with the aid of ultrasound for 5 minutes and dilute to 50.0 ml with the solvent mixture.

Reference solution (c): Transfer 5.0 ml, each of, reference solution (a) and reference solution (b) to a 50- ml volumetric flask. Add 10 ml of acetonitrile and dilute to 50.0 ml with the solvent mixture.

Chromatographic System:

– a stainless steel column 15 cm x 4.6 mm, packed with octadecylsilane bonded to porous silica (5 µm), ( Such as X-Terra RP 18),

– mobile phase: A. a mixture of 90 volumes of 0.005 M disodium hydrogen phosphate, adjusted to pH 8.0 with dilute orthophosphoric acid, 9 volumes of acetonitrile and 1 volume of methanol, B. a mixture of 90 volumes of acetonitrile and 10 volumes of methanol.

− flow rate: 1 ml per minute

− a gradient programme using the conditions given below,

− spectrophotometer set at 280 nm,

− injection volume: 10 µl.

Inject reference solution (c). The test is not valid unless the column efficiency is not less than 2000 theoretical plates, the tailing factor is not more than 2.0 and the relative standard deviation for replicate injections is not more than 5.0 per cent, for both the peaks. Inject reference solution (c) and test solution.

Calculate the content of C16H13C12NO4 and C8H9NO2 in the tablets.

Storage. Store protected from moisture, at a temperature not exceeding 30°.

08.  Revision History: