TEST A(FOR TRIMETHOPRIM DEGRADATION PRODUCT)—
Hydrochloric acid solution—
Dilute 2mLof 3Nhydrochloric acid with water to 100mL.
Test preparation—
Transfer an accurately measured volume of Injection,equivalent to about 48mg of trimethoprim and 240mg of sulfamethoxazole,to a glass-stoppered,50-mLcentrifuge tube.Add 15mLof Hydrochloric acid solution,and mix.Add 15mLof chloroform,shake for 30seconds,and centrifuge at high speed for 3minutes.Transfer the supernatant layer to a 125-mLseparator.Extract the chloroform layer in the centrifuge tube with 15mLof Hydrochloric acid solution,centrifuge at high speed,and add the extract to the separator.Add 2mLof sodium hydroxide solution (1in 10)to the solution in the separator,and extract with three 20-mLportions of chloroform,collecting the organic layer in a 125-mLconical flask.Evaporate the chloroform under a stream of nitrogen to dryness.Dissolve the residue in 1mLof a mixture of chloroform and methanol (1:1).
Standard preparation A—
Using an accurately weighed quantity of
USP Trimethoprim RS,prepare a solution in chloroform and methanol (1:1)having a known concentration of 48mg per mL.
Standard preparation B—
Dilute an accurately measured volume of Standard preparation Awith a mixture of chloroform and methanol (1:1)to obtain a solution having a known concentration of 240µg per mL.
Procedure—
Apply 10µLeach of the
Test preparation,Standard preparation A,and
Standard preparation Bto separate points on a thin-layer chromatographic plate (see
Chromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel.Develop the chromatogram in a solvent system consisting of a mixture of chloroform,methanol,and ammonium hydroxide (97:7.5:1)until the solvent front has moved at least 12cm.Remove the plate from the developing chamber,and air-dry.Locate the bands by viewing under short-wavelength UVlight and by spraying with a freshly prepared mixture of ferric chloride solution (1in 10)and potassium ferricyanide solution (1in 20)(1:1).Trimethoprim produces a spot at about
RF0.5,and the trimethoprim degradation product produces a spot at about
RF0.6to 0.7.Any spot from the
Test preparationat about
RF0.6to 0.7is not greater in size and intensity than the spot produced by
Standard preparation Bat about
RF0.5,corresponding to not more than 0.5%.
[NOTE—There may be spots due to concentrate excipients at about
RF0.1.
]
TEST B(FOR SULFANILAMIDE AND SULFANILIC ACID)—
Alcohol–methanol mixture—
Mix dehydrated alcohol and methanol (95:5).
Ammonium hydroxide solution—
Dilute 1mLof ammonium hydroxide with Alcohol-methanol mixtureto 100mL.
Modified Ehrlich's reagent—
Dissolve 100mg of p-dimethylaminobenzaldehyde in 1mLof hydrochloric acid,dilute with alcohol to 100mL,and mix.
Test preparation—
Transfer an accurately measured volume of Injection,equivalent to about 32mg of trimethoprim and 160mg of sulfamethoxazole,to a 25-mLgraduated cylinder,dilute with Ammonium hydroxide solutionto 16mL,and mix.
Standard preparation A—
Transfer about 50mg of
USP Sulfamethoxazole RS,accurately weighed,to a 5-mLvolumetric flask,dissolve in and dilute with
Ammonium hydroxide solutionto volume,and mix.
Standard preparation B—
Transfer about 25mg of
USP Sulfanilamide RS,accurately weighed,to a 250-mLvolumetric flask,dissolve in and dilute with
Ammonium hydroxide solutionto volume,and mix.Pipet 5mLof this solution into a 10-mLvolumetric flask,dilute with
Ammonium hydroxide solutionto volume,and mix.
Standard preparation C—
Transfer about 25mg of
USP Sulfanilic Acid RS,accurately weighed,to a 250-mLvolumetric flask,dissolve in and dilute with
Ammonium hydroxide solution,and mix.Pipet 3mLof this solution into a 10-mLvolumetric flask,dilute with
Ammonium hydroxide solutionto volume,and mix.
Procedure—
Apply 10µLeach of the
Test preparationand
Standard preparations A,B,and
Cto separate points on a thin-layer chromatographic plate (see
Chromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel.Develop the chromatogram in a solvent system consisting of a mixture of
Alcohol-methanol mixture,heptane,chloroform and glacial acetic acid (30:30:30:10)until the solvent front has moved not less than 12cm.Remove the plate from the developing chamber,air-dry,spray with
Modified Ehrlich's reagent,and allow the plate to stand for 15minutes:sulfamethoxazole produces a spot at about
RF0.7.Any spots from the
Test preparationat about
RF0.5or 0.1are not greater in size or intensity than spots produced by
Standard preparations Band
C,respectively,corresponding to not more than 0.5%of sulfanilamide and 0.3%of sulfanilic acid.