HPLC Method For Aceclofenac and Paracetamol:

            Determined by liquid chromatography:

Standard solution: Weigh accurately about 162.5 mg of Paracetamol reference standard, and 50 mg of Aceclofenac reference standard in 100ml volumetric flask, dissolve in and dilute to the mark with mobile phase. Shake well and further dilute 2 ml to 25 ml with mobile phase. Shake well and filter through 0.45µm nylon filter.

Test Solution: Weigh accurately about equivalent to 162.5 mg of Paracetamol from crushed powder of  20 tablets in 100 ml volumetric flask, add 50 ml of mobile phase, sonicate for 5 minutes, cool to room temperature and dilute to the mark with mobile phase, filter. Shake well and further dilute 2 ml to 25 ml with mobile phase, shake well and filter through 0.45µm nylon filter.

Procedure: Inject separately standard and test preparation. Inject five replicate injection of working standard and two replicate injection of test preparation. RSD of replicate injection should not more than 2%. Calculate the assay of Paracetamol and Aceclofenac by comparison to peak area of standard preparation.

            Chromatographic system:

  • A stainless steel column (25cm X4.6mm,) C18
  • Mobile phase: Methanol :Water and G.A.A. (80: 20) and (0.01ml); degas
  • Flow rate: 1.2ml per minute,
  • Spectrophotometer set at 280nm,
  • Injection volume: 20µl

Inject the reference solution and test solution.

Calculate the content of Paracetamol and Aceclofenac in tablets.


Area of Test      STD Wt.(mg)      2                 100                     25          Potency

—————–X————–X———-X—————–X———-X———–X   Average weight

Area of STD            100                   25          Test Wt. (mg)         2              100

            Acceptance  criteria: 90.0%-110.0%


For: Serratiopeptidase


Sodium borate hydrochloric acid buffer (pH 9.0): Dissolve 19 g di-sodium tetra borate (Borax) in 900 ml water. Adjust its pH to 9.0 ±0.05 with 1N hydrochloric acid make up the volume to 1000 ml and check the pH. Adjust, if necessary.

Casein substrate: Dissolve 1.2 g (dried) casein in 100 ml of sodium borate-Hydrochloric acid buffer of pH 9.0. Dissolve and keep it on boiling water bath for 1-2 minutes (To make the solution clear). Cool and filter through cotton and make up the volume to 200 ml with buffer.

Trichloro acetic acid (TCA): Dissolve 18 g of TCAand 30 g of sodium acetate (anhydrous) in 500 ml of water. Add 20 ml of G.A.A. Adjust its pH to 4.0 with glacial acetic acid. Make up the volume to 1000 ml with distilled water.

6.0 % Sodium carbonate: Dissolve 6 g of anhydrous sodium carbonate in sufficient water and make up the volume to 100 ml.

0.2 N Hydrochloric acid: Dilute 4.25 ml of hydrochloric acid to 250 ml with water.

Ammonium sulphate: Dissolve 10 g of ammonium sulphate in water and make up the volume to 200 ml.

Folins Cieocaltcu Reagent (FCR): Dilute 10 ml of Folins reagent with 40 ml of water.

Mixed phosphate buffer pH 6.8: Dissolve 28.8 g of disodium hydrogen phosphate and 11.45 g of sodium dihydrogen phosphate in sufficient water to produce 1000 ml.

L-Tyrosine standard preparation: Weigh and transfer accurately about 160 mg of L-Tyrosine to a 100 ml volumetric flask. Dissolve in and make up the volume with 0.2 N Hydrochloric acid. Further, dilute 10 ml the resulting solution to 1000 ml with the same solvent.

Test preparation: Transfer test solution equivalent to 100 mg of Serratiopeptidase to a 250 ml of conical flask. Add 100 ml of phosphate buffer pH 6.8. Gently shake the flask intermittently for 60 minute to release the drug from enteric coating. Dilute 20 ml of the above solution to 100 ml with ammonium sulphate solution. Mix well and keep for 3 to 5 minutes. Dilute 5 ml of the resulting solution to 200 ml with sodium borate hydrochloric acid buffer (pH 9.0). 1 ml shall be taken for analysis.

Proceed for colour development as below:

Reagent Test blank Test Standard Blank Standard
TCA 5 ml Nil Nil Nil
Incubate at 37°±0.5°C for 10 minutes
Casein Nil 5 ml Nil Nil
Enzyme solution 1 ml 1 ml Nil Nil
Incubate at 37°±0.5°C for 20 minutes
TCA Nil 5 ml Nil Nil
Casein 5 ml Nil Nil Nil
Incubate at 37°±0.5°C for 30 minute. Filter the solution using Whattman filter No. 5 Proceed with the clear filtrate.
Filtrate 2 ml 2 ml Nil Nil
0.2 N Hydrochloric acid Nil Nil 2 ml Nil
L-Tyrosine Nil Nil Nil 2 ml
Sodium carbonate 5 ml 5 ml 5 ml 5 ml
Folin reagent 1 ml 1 ml 1 ml 1 ml
Wait for 30 minutes at 37°C, temperature measure absorbance at 660 nm using water as blank.


Calculate the activity of quantity of Serratiopeptidase in the tablet as under.

A1-A2                   1        100       100      200

Serratiopeptidase(mg)=————-X176X——–X——–X——X——-X average weight in mg

A3-A4                   20          Wt       20         5


Total volume of Enzyme reaction mixture (11ml)

176: Conversion factor=——————————————————————–X32 µg

Volume of Filtrate taken for colour development (2 ml)


20: Reaction time in minutes

DF: Dilution factor

A1: Absorbance due to test preparation

A2: Absorbance due to blank preparation


A4: Absorbance due to Tyrosine blank

32 µg: Content of Tyrosine in 2ml Tyrosine standard preparation

Calculate the % of Serratiopeptidase in the tablets as under:


Activity X100

Serratiopeptidase%: ————————–


HPLC Method