Saturday, February 2, 2013

Assay Method (F)





Assay Method of 
Famotidine Tablet

Sample 
Dissolve powdered sample equivalent to 70 mg Famotidine in previously neutralized 60 ml glacial acetic acid, using crystal violet solution as indicator. Titrate with 0.1 M perchloric acid and determine the end point.Each ml of 0.1M perchloric acid VS is equivalent to 16.87 mg of Famotidine.

% of content =   V x F x 16.87 x 100 / W


Assay Method of 
 Fenofibrate Capsule

Buffer
Take 990 ml distilled water into 1000 ml volumetric flask, adjust the pH 2.5 ± 0.05 with Phosphoric acid  & make volume with distilled water up to 1000  ml.
Ratio   
Buffer ( 40% ) : Acetonitrile ( 60% )    
Standard 
Accurately weigh and transfer about 50 mg of working standard of Fenofibrate into 50 ml volumetric flask. Add 30 ml of Acetonitrile & shake for 30 minutes and dilute with mobile phase up to the mark and mix well. Dilute 5 ml of this solution to 50 ml with  mobile phase. Filter this solution with 0.45 m membrane filter before injection
Sample 
Take the weight of 20 Capsules, then open the capsule & determine the average content  weight. Crush the total content  weight  into fine powder. Take an amount of powder  sample equivalent 50 mg of  Fenofibrate into 50 ml volumetric flask. Add 30 ml of  Acetonitrile & shake for 30 minutes and dilute with mobile phase up to the mark and mix well & Filter.Dilute 5 ml of this solution to 50 ml with mobile phase. Filter this solution with 0.45 m membrane filter before injection.
Condition
Waters Spherisorb C8 5.0 mm ODS2 4.6 x 250 mm / UV at 286 nm / 1.5 ml per minutes / 20 ml / 8.5 minutes


Assay Method of 
 Febuxostat INN Raw Material

Buffer
Take 1 ml 85% Orthophosphoric Acid into 500 ml volumetric flask add 450 ml distilled water, adjust the pH 3.0 ± 0.05 with Triethylamine & make volume 500 ml with distilled water.
Ratio   
Buffer ( 40% ) : ( 60% )    
Solvent
Acetonitrile & Mobile Phase
Standard 
Accurately weigh and transfer about 20 mg of working standard of Febuxostat into 50 ml volumetric flask. Add 30 ml of Acetonitrile & shake for 20 minutes and dilute with mobile phase up to the mark and mix well. Filter this solution with 0.45 m membrane filter before injection.
Sample 
Accurately weigh and transfer about 20 mg of sample of Febuxostat into 50 ml volumetric flask. Add 30 ml of Acetonitrile & shake for 20 minutes and dilute with mobile phase up to the mark and mix well. Filter this solution with 0.45 m membrane filter before injection.
Condition
Waters Spherisorb ODS2 5.0 mm 4.0 x 250 mm / UV at 220 nm / 1ml per minutes / 10 ml / 10 minutes / 45º C


Assay Method of 
 Fexofenadine Hydrochloride Tablet

Buffer

Transfer accurately weighed about 1.36gm of potassium Dihydrogen orthophosphate (KH2PO4) and 1.0 ml of
Triethyl amine in 900 ml purified water, mix well. Adjust PH to 2.5 ± 0.05 with orthophosphoric acid and dilute to 1
liter with distilled water. Filter through a 0.45mm membrane filter.
Ratio   
Buffer : Acetonitrile : Methanol in the proportion of 50 : 30 : 20
Standard 
                                    Weigh accurately about 50mg Fexofenadine Hydrochloride WS and transfer in a 100ml volumetric flask. Add 70ml of Methanol. Shake for 25min. Then add sufficient Methanol to make 100ml. Mix and filter this solution with 0.45mm membrane filter before injection
Sample 
Take 20 tablets in a mortar and finely crushes with passel. Take accurately weight of the Tablet powdered sample
containing about 50mg Fexofenadine Hydrochloride and transfer in a 100 ml volumetric flask. Add 70ml of
Methanol. Shake for 25min and add sufficient Methanol to Make 100ml. Mix and filter with whatman filter paper.
Filter this resultant solution with 0.45 mm membrane filter before injection .
Condition
ODS1  , 5 m 4.6 x 250 mm / UV at 215 nm/12 minutes


Assay Method of 
 Fexofenadine Hydrochloride Suspension

Acid Sol.
Dilute 17 ml Glacial Acetic Acid with water to 1000 ml. Dilute 100 ml of this solution with water to 1 liter.
Buffer

Dilute 15 ml of a solution containing a mixture of Acetonitrile (ACN) and Triethylamine (1 : 1)with acid solution to 1 litre. Adjust pH 5.25 ± 0.05 with Phosphoric Acid (H3PO4)
Ratio   
Buffer: Acetonitrile in the proportion of  64 : 36.
Diluent  
Prepare a mixture of Acetonitril (ACN) and Acid Solution (75:25)
Standard 
                                    Weigh accurately about 30mg Fexofenadine Hydrochloride WS and transfer in a 50 ml volumetric flask. Add 30ml of diluent. Shake for 15 min. Then add sufficient Diluent to make 50 ml. Mix and filter this solution with 0.45mm membrane filter before injection.
Sample 
Take accurately weight 5 ml sample containing about 30mg Fexofenadine HCl and transfer in a 50 ml volumetric
flask. Add 30ml of Diluent. Shake for 15 min and add sufficient diluent to make 50ml.Sonicate for 10 minutes. Mix
and filter with whatman filter paper. Filter this resultant solution with 0.45 mm membrane filter before injection.
Condition
XTerraÒ PR18 5µm. 4.6 × 250mm. / 400 C/ UV at 220 nm/10 minutes


Assay Method of 
 Flavoxate Hydrochloride Tablet

Buffer

Transfer accurately weighed about 6.8 gm of potassium Dihydrogen orthophosphate (KH2PO4) in 950 ml distilled water, mix well. Adjust pH to 3.0 ± 0.05 with orthophosphoric acid and dilute to 1 liter with distilled water. Filter through a 0.45mm membrane filter.
Ratio   
Acetonitrile: Buffer Solution (70:30)
Diluent  
Methanol: Buffer Solution (50:50)
Standard 
Weigh accurately about 25 mg Flavoxate Hydrochloride WS and transfer in to a 50 ml volumetric flask. Add 30 ml of diluents, shake for 10 minutes. Then add sufficient diluents to make 50ml. Take 2 ml of the solution in a 50 ml volumetric flask and add sufficient diluents to make final volume. Mix and filter this solution with 0.45mm membrane filter before injection.
Sample 
Take the weight of 20 tablets and determine the average weight. Then crush the tablets in to fine powder.    Take the powdered sample equivalent to 25 mg Flavoxate Hydrochloride and transfer in to a 50 ml volumetric flask. Add 30 ml of diluents & shake for 10 minutes. Then add sufficient diluents to make 50ml. Mix and Filter with whatman filter paper. Then take 2 ml of the solution in to a 50 ml volumetric flask and add sufficient diluents to make final volume. Filter this resultant solution with 0.45 mm membrane filter before injection.
Condition
ODS1 10µm 4.6 x 250mm / 210 nm / 1 ml per minute / 20 mL / 15 minutes / 8.5 minute approx.


Assay Method of
 Fluconazole Tablet

Standard 
Take 50 mg of Fluconazole WS in a 100 ml Volumetric flask, add 60 ml of methanol. Shake for 15 minutes. Make volume up to 100ml with methanol. Take 10 ml solution in a 25 ml volumetric flask. Make volume up to 25ml with methanol. Measure the absorbenc at 261nm taking methanol as blank. (Concentration =0.02%)
Sample 
Weigh and take the content of 20 capsules. Take powder equivalent to 50 mg of Fluconazole in 100 ml volumetric flask. Add 60 ml of methanol, and shake for 15 minutes. Make volume up to 100ml with methanol. Mix and filter , take 10ml of the filtrate in a 25 ml volumetric flask . Make volume up to 25 ml with methanol. Measure the absorbenc at 261 nm taking methanol as  blank. (Concentration = 0.02%)

                                             Assay Method of 
                        Flucoxacillin  Sodium Tablet, Capsule & PFS

Buffer
Dissolve 173 g of sodium hydroxide & 20.6 g of sodium acetate in sufficient water to produce 1000 ml.
Hydroxylamine Hydrochloride solution
Dissolve 350 g of hydroxylamine hydrochloride in sufficient water to produce 1000 ml.

Neutral Hydroxylamine solution
Mix 1 volume each of Hydroxylamine solution (1) & the buffer solution (2). Check the pH & if necessary adjust the pH 7.0 ± 0.1 by adding an additional amount one of the components. To 1 volume of this neutral solution add 8 volume of water & 2 volume of 95% ethanol. This solution must be freshly prepared.

Ferric Ammonium Sulfate Solution
Dissolve 272 g of Ferric Ammonium Sulphate in a mixture of 26 ml of conc. Sulphuric Acid & sufficient water to produce 1000 ml. Filter if necessary. This reagent should be used for one week.

Standard 
Dissolve Flucoxacillin  Sodium equivalent to 31.25 mg of Flucloxacillin  in 25 ml of  water .Concentration 1.25mg/ml.

Sample
Weigh and powder the contents of 20 capsules. To a quantity of powder  equivalent to  125 mg of Flucloxacillin, add 60 ml of water . Shake for 20 minutes . Add sufficient  water to make volume  100 ml.  Mix and filter.
Procedure
Take 2 ml solution from each Standard & Sample in two separate test tubes. Add 2 ml of water & 2.5 ml of  Neutral Hydroxylamine solution. Mix and allow to stand for 5 mins. Add 2.5 ml of Ferric Ammonium Sulphate solution & mix.After 3 mins, determine the Absorbance of the resulting solution at 480nm using reagent blank, prepare by treating a volume of water instead of sample solution in the same manner.


Assay Method of 
 Flunarizine

Sample :
Transfer the 1.00 gm powder sample equivalent to 100.0 mg of Flunarizine in a 250 ml Conical flask. Add 100 ml of Ethanol to the Conical flask & shake at least for 20 minutes to dissolve. Then add to 2 ml 0.1 %  Phenolphthalein Solution as indicator and mix well. Titrate with 0.1 M sodium hydroxide VS. Carry out a blank titration. 1 ml of  0.1 M sodium hydroxide VS is equivalent to 20.23 mg of Flunarizine.


Assay Method of 
 Fluoxetine Hydrochloride  Capsule

Standard 
Prepare standard in the same way .as sample preparation . Conc. 0.001 % Fluoxetine Hydrochloride WS in methanol Measure the absorbance at 227 nm , against methanol blank .
Sample 
Weigh and powder 20 capsules. Take powder  equivalent to  50 mg of Fluoxetine Hydrochloride in 100 ml volumetric flask. Add 60 ml of methanol, and shake for 25 minutes. Make volume up to 100 ml with methanol. Mix and filter. Dilute 1 ml of the filtrate to 50 ml with methanol.


Assay Method of 
 Flupentixol dihydrochloride  Tablet

Buffer
Dissolve 8.72 gm of Potassium dihydrogen phosphate, 0.37 gm of anhydrous disodium hydrogen phosphate & 0.77 gm of dodecyltrimethylammonium bromide in 1000 ml distilled water.
Ratio   
Buffer ( 35% ) : Acetonitrile ( 10% ) : Methanol ( 55% )   
Standard 
Weigh  and transfer about 25 mg working standard of Flupentixol di HCl in a 25 ml volumetric flask. Add  15
ml of mobile phase and shake for 20 minutes and  dilute with mobile phase up to the mark and mix well. Dilute 10 ml
of this solution  into  25 ml volumetric flask  & mix well .Filter this solution with 0.45 m membrane filter before 
injection.
Sample 
Take the weight of 30 tablets & determine the average weight. Crush the tablets into fine powder. Take an amount of powder sample equivalent 8.562 mg Flupentixol as 10 mg of Flupentixol dihydrochloride in a 25 ml volumetric flask. Add 15 ml of mobile phase and shake for 20 minutes and  dilute with mobile phase up to the mark and mix well. Filter this solution with Whatman Filter & finally filter this solution with 0.45 m  membrane filter before injection.
Condition
Waters Spherisorb  C18 , 5 m  ODS2  4.6 x 250 mm / UV at 270 nm / 40 minutes
                    

Assay Method of
 Fluvastatin Sodium  Capsule

Buffer
Weigh about 1.95 gm Sodium dihydrogen orthophosphate dihydrate in a cleaned & dry 1000 ml volumetric flask. Add
about 500 ml HPLC grate water & dissolve it & mix well. Add 2 ml Triethylamine using a 2 ml graduated pipette &
make the volume up to mark.Adjust the pH of the resulting buffer solution to 5.0 ±0.05 with controlled addition of
Orthophosphoric acid (85%).
Ratio   
Buffer ( 35% ) : Methanol ( 65% )    
Diluent
Mobile Phase
Standard 
Accurately weigh and transfer about 42.4 mg of working standard of Fluvastatin Sodium  equivalent to 40 mg
Fluvastatin into 50 ml volumetric flask. Add 25 ml of distilled water & shake for 10 minutes and dilute with Methanol
up to the mark and mix well & Filter. Dilute 2.5 ml of this solution to 50 ml with mobile phase. Filter this solution with
0.45 m membrane filter before injection.
Sample 
Take the weight of 20 Capsules, then open the capsule & determine the average content   weight. Crush the total content  weight  into fine powder. Take an amount of powder  sample equivalent 40 mg of  Fluvastatin as Fluvastatin Sodium into 50 ml volumetric flask. Add 25 ml of distilled water & shake for 10 minutes and dilute with Methanol up to the mark and mix well & Filter. Dilute 2.5 ml of this solution to 50 ml with mobile phase. Filter this solution with 0.45 m membrane filter before injection.
Condition
Waters Spherisorb C18 , 10 m ODS1 4.6 x 250 mm / 35°C / UV at 235 nm / 10 minutes
               

Assay Method of 
 Folic Acid + Pyridoxine HCl + Cyanocobalamin Tablet

For Folic Acid + Pyridoxine HCl

M. Phase
Methanol: Water = 65 : 35
Standard 
Accurately weigh and transfer about 25 mg Pyridoxine Hydrochloride WS and 2.5 mg Folic Acid WS into a 100 ml
volumetric flask.  Add 60 ml water and 2 % of Ammonium hydroxide solution and  shake for 20 minutes and make the
volume with water up to the mark & mix well. Filter this solution with 0.45m membrane filter before injection.
Concentration of Pyridoxine Hydrochloride 0.025% and Concentration of Folic Acid 0.0025%.
Sample 
Weigh and transfer about 0.2gm powder equivalent to 25 mg of Pyridoxine Hydrochloride  and 2.5 mg of Folic Acid in
a 100 ml volumetric flask. Add 60 ml of water and shake for 20 minutes & than make the volume 100 ml with water
and mix well. Filter this solution with whatman filter paper. Dilute 20 ml to 100 ml with same water & mix well. Filter
this solution with 0.45 m membrane filter before injection. Concentration of sample 0.001%.
Condition
XTerraÒ RP18 5mm 4.6 ´ 250mm./ 40º C / UV at 280 nm / 20 minutes.
7.5 minutes for Pyridoxine HCl & 13.5 minutes for Folic Acid (approx.)

For Cyanocobalamin

M. Phase
Methanol: Water = 65 : 35
Standard 
Accurately weigh and transfer about 10 mg Cyanocobalamin (100%) in WS in a 100 ml volumetric flask.  Add 60 ml
water and shake for 20 minutes and make the volume up to mark & mix well. Dilute 10 ml to 100 ml with water & mix
well. Filter this solution with 0.45m membrane filter before injection. Concentration of standard 0.001%.
Sample 
Weigh and transfer about 1gm powder equivalent to 5 mg of Cyanocobalamin (100%) in a 100 ml volumetric flask.
Add 60 ml of water and shake for 20 minutes & than make the volume 100 ml with water and mix well. Filter this
solution with whatman filter paper. Dilute 20 ml to 100 ml with same water & mix well. Filter this solution with 0.45 m
membrane filter before injection. Concentration of sample 0.001%.
Condition
XTerraÒ RP18 5mm 4.6 ´ 250mm./ 40º C / UV at  550 nm / 5 minutes.
3 minutes for Cyanocobalamin (approx.)

 
Assay Method of 
 Folic Acid + Zinc Tablet For Folic Acid

Condition
Standard solution preparation: Accurately weigh and transfer about 15 mg Working Standard of Folic Acid into 50 ml volumetric flask. Add 30 ml of 0.1 M NaOH and shake for 10 minutes. Adjust the volume up to the mark with the same solvent. Take 1 ml of this solution in a 50 ml volumetric flask.  Make the volume up to the mark with 0.1 M NaOH & mix well.
Sample solution preparation: Take 20 Tablets & determine the average weight. Crush the tablets into fine powder & transfer a quantity of powdered sample equivalent to 15 mg of Folic Acid into a 50 ml volumetric flask. Add 30 ml of 0.1 M NaOH and shake for 10 minutes. Adjust the volume up to the mark with the same solvent. Filter the solution. Take 1 ml of the filtrate into a 50 ml volumetric flask. Make the volume up to the mark with 0.1 M NaOH & mix well.
Measurement: Measure at a wavelength of 256 nm using 0.1 M NaOH as blank solution.

For Zinc

Condition
Procedure: Take powdered sample equivalent to 160 mg of Zinc sulfate Monohydrate into a 250 ml conical flask, add 10 ml 2.0 M acetic acid to dissolve and dilute to 100 ml with water. Add 15 ml ammonia-ammonium chloride buffer solution. Titrate with 0.1 M Sodium edetate and determine the end point using Solochrome black as an indicator.
1 ml 0.1M Sodium edetate VS ≡ 17.95 mg of Zinc sulfate Monohydrate
Calculation: % of Zinc sulfate Monohydrate per Tablet = V x f x C x 100 / W
Where, V = Consume volume of Sodium edetate, f = Factor of Sodium edentate & C = 17.95, W = Weight of powder sample as Zinc sulfate Monohydrate.
Note: Sample taken weight = 636.3 mg of powder = 62.69 mg of Zinc
Required volume of 0.1 M Disodium EDTA (VS) = 9.5 ml, Factor of 0.1 M Disodium EDTA (VS) = 1.00968, Each ml of 0.1 M Disodium EDTA (VS) = 6.538 of Zinc                         
% of content = 9.5 x 1.00968 x 6.538 x 100 / 62.69 = 100.035
               
                 
    Assay Method of 
  Furosemide  Syrup
                             .
Sample 
Take 5 ml of Frusin syrup equivalent to 40 mg of Furosemide in a 200 ml volumetric flask. Add 150 ml of Distilled water and mix well by 10 minutes shaking. Adjust the volume up to the mark (200 ml) with the same solvent. Filter the solution by Whatman filter paper then take 5 ml of this filtrate in 100 ml volumetric flask. Make the volume up to the mark (100 ml) with Distilled water  & mix well. Measure the absorbance of the sample solution at wavelength 271 nm using Distilled water as blank.
Calculation
 % of Furosemide   =  Absorbance of Sample ´ 100 / Conc. in gm per 100 ml ´ 580                                              

                           
 Assay Method of  
 Furosemide Injection
                             .
Sample 
To a volume containing the equivalent of 20 mg of  Furosemide add sufficient water to produce 100 ml. Dilute 5 ml to 100 ml with 0.1M Sodium  Hydroxide and measure the measure the absorbance of the resulting solution at the maximum at 271 nm .
Calculate the content of Furosemide taking 580 as the value of A (1%, 1cm) at the maximum at 271 nm . 
Calculation
 % of Furosemide   =  Absorbance of Sample ´ 100 / Conc. in gm per 100 ml ´ 580                                              


Assay Method of 
 Furosemide + Spironolactone  Tablet

Buffer
Dissolve 2.0 gm of  Potassium dihydrogen orthophosphate in 1000 ml distilled water , adjust pH 7.0± 0.05 with 6M ammonia solution.
Ratio   
Buffer ( 40 % ) : Acetonitrile ( 60 % )
Standard 
Accurately weigh and transfer about 20 mg of working standard of Frusemide & about 50 mg of working standard of  Spironolactone into a 50 ml volumetric flask. Add 30 ml of mobile phase & shake for 15 minutes and dilute with mobile phase up to the mark and mix well. Dilute 2.0ml of this solution to 50 ml with the same solvent. Filter this solution with 0.45 m membrane filter before injection
Sample 
Weigh and powder the contents of 20 Tablets. Take powder-containing equivalent to 20 mg of Frusemide & about 50 mg of  Spironolactone into a 50 ml volumetric flask. Add 30 ml of mobile phase & shake for 15 minutes and dilute with mobile phase up to the mark and mix well. Dilute 2.0ml of this solution to 50 ml with the same solvent. Filter this solution with 0.45 m membrane filter before injection.
Condition
Waters Spherisorb C8 5.0 mm  4.6 x 250 mm / UV at 238 nm/25 minutes
 Frusemide  :  2.0  minutes  &  Spironolactone                :  5.4  min. approx.


Assay Method of 
 Fusidic Acid + Hydrocortisone  Cream

Mobile Phase
A mixture of 10 volumes of Methanol, 20 volumes of a 10 g/l solution of  Phosporic Acid ,20 volumes of water and 50 volumes of Acetonitrile.
Diluents
Mobile Phase
Standard 
Dissolve about 10 mg of Hydrocortisone (1 mg Hydrocortisone ≡ 1.116 mg Hydrocortisone Acetate) WS & 20 mg of Fusidic Acid WS in a 50 ml volumetric flask add 35 ml diluent and shake for 15 minutes and dilute with diluent up to mark 50 ml and mix well finally filter this solution with 0.20 micron membrane filter before injection.
Sample 
Take about 1 gm of sample cream equivalent to 10 mg of Hydrocortisone & 20 mg of Fusidic Acid in a 50 ml volumetric flask then add 35 ml diluent and shake for 25 minutes and dilute with diluent up to mark 50 ml and mix well, filter this solution with whattman filter paper & finally filter this solution with 0.20 micron membrane filter before injection.
Condition
Waters Spherisorb ODS1 10 mm 4.6 ´ 250mm./ 40°C  / UV at 235 nm / 10 ml / 1.5 ml per minute / 15 minutes /
3 minutes for Hydrocortisone & 10.8 minutes for Fusidic Acid approximately.

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