Chromatographic purity—
LIMIT OF TRIMETHOPRIM DEGRADATION PRODUCT—
Test solution—
Transfer an accurately measured volume of Oral Suspension,equivalent to about 40mg of trimethoprim,to a separatory funnel.Extract with three 25-mLportions of a mixture of chloroform and methanol (8:2),collecting the extracts in a 125-mLconical flask.Evaporate the combined extracts with the aid of a current of air to dryness on a steam bath.Dissolve the residue in 2.0mLof the mixture of chloroform and methanol (8:2),then centrifuge.
Standard solution A—
Dissolve an accurately weighed quantity of
USP Trimethoprim RSin a mixture of chloroform and methanol (8:2)to obtain a solution having a known concentration of about 20mg per mL.
Standard solution B—
Dilute an accurately measured volume of Standard solution Awith a mixture of chloroform and methanol (8:2)to obtain a solution having a known concentration of about 0.1mg per mL.
Procedure—
Apply 5µLeach of the
Test solution,Standard solution A,and
Standard solution Bto separate points on a thin-layer chromatographic plate (see
Chromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel.Place the plate in a saturated chromatographic chamber,and develop the chromatogram in a solvent system consisting of a mixture of chloroform,methanol,and ammonium hydroxide (80:20:3)until the solvent front has moved at least 15cm.Remove the plate from the chamber,air-dry,and view under short-wavelength UVlight:trimethoprim produces a spot at about
RF0.7,and the trimethoprim degradation product can be seen at
RF0.3to 0.5.Any spot from the
Test solutionat about
RF0.3to 0.5is not greater in size and intensity than the spot produced by
Standard solution Bat about
RF0.7,corresponding to not more than 0.5%.
LIMIT OF SULFANILAMIDE,SULFANILIC ACID,AND SULFAMETHOXAZOLEN4-GLUCOSIDE)—
Alcohol–methanol mixture—
Mix dehydrated alcohol and methanol (95:5).
Modified Ehrlich's reagent—
Dissolve 100mg of p-dimethylaminobenzaldehyde in 1mLof hydrochloric acid,and dilute with alcohol to 100mL.
Test solution—
Using a syringe,transfer an accurately measured volume of Oral Suspension,equivalent to 200mg of sulfamethoxazole,to a 100-mLvolumetric flask containing 10mLof ammonium hydroxide;and add 50mLof methanol.Shake for 3minutes,and dilute with methanol to volume.Centrifuge a portion of the solution for 3minutes.
Standard solution A—
Weigh 20mg of
USP Sulfamethoxazole RSinto a 10-mLvolumetric flask,dissolve in 1mLof ammonium hydroxide,dilute with methanol to volume,and mix.
Standard solution B—
Weigh 10mg of
USP Sulfanilamide RSinto a 50-mLvolumetric flask,dissolve in 5mLof ammonium hydroxide,and dilute with methanol to volume.Pipet 5mLof this solution into a 100-mLvolumetric flask,add 10mLof ammonium hydroxide,and dilute with methanol to volume.
Standard solution C—
Weigh 10mg of
USP Sulfanilic Acid RSinto a 50-mLvolumetric flask,dissolve in 5mLof ammonium hydroxide,and dilute with methanol to volume.Pipet 3mLof this solution into a 100-mLvolumetric flask,add 10mLof ammonium hydroxide,and dilute with methanol to volume.
Standard solution D—
Weigh 3.0mg of USP Sulfamethoxazole N4-Glucoside RSinto a 50-mLvolumetric flask,dissolve in 5mLof ammonium hydroxide,and dilute with methanol to volume.
Procedure—
Apply 50µLeach of the
Test solutionand
Standard solutions A,B,C,and
Dto separate points on a thin-layer chromatographic plate (see
Chromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel.Place the plate in an unsaturated chromatographic chamber,and develop the chromatogram in a solvent system consisting of a mixture of
Alcohol–methanol mixture,heptane,chloroform,and glacial acetic acid (25:25:25:7)until the solvent front has moved at least 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 solutionat about
RF0.5,0.1,and 0.3are not greater in size and intensity than spots produced by
Standard solutions B,C,and
D,respectively,corresponding to not more than 0.5%of sulfanilamide,0.3%of sulfanilic acid,and 3.0%of sulfamethoxazole
N4-glucoside.
Assay—
Mobile phase—
Mix 1400mLof water,400mLof acetonitrile,and 2.0mLof triethylamine in a 2000-mLvolumetric flask.Allow to equilibrate to room temperature,and adjust with 0.2Nsodium hydroxide or dilute glacial acetic acid (1in 100)to a pHof 5.9±0.1.Dilute with water to volume,and filter through a 0.45-µm membrane,making adjustments if necessary (see
System Suitabilityunder
Chromatography á621ñ).
Standard preparation—
Dissolve accurately weighed quantities of
USP Trimethoprim RSand
USP Sulfamethoxazole RSin methanol,and quantitatively dilute with methanol to obtain a solution containing,in each mL,about 0.32mg and 0.32
Jmg,respectively,
Jbeing the ratio of the labeled amount,in mg,of sulfamethoxazole to the labeled amount,in mg,of trimethoprim in the dosage form.Transfer 5.0mLof this solution to a 50-mLvolumetric flask,dilute with
Mobile phaseto volume,and mix to obtain a
Standard preparationhaving known concentrations of about 0.032mg of
USP Trimethoprim RSper mLand 0.032
Jmg of
USP Sulfamethoxazole RSper mL.
Assay preparation—
Transfer an accurately measured volume of Oral Suspension,equivalent to about 80mg of Sulfamethoxazole,to a 50-mLvolumetric flask with the aid of about 30mLof methanol.Sonicate the mixture for about 10minutes with occasional shaking.Allow to equilibrate to room temperature,dilute with methanol to volume,mix,and centrifuge.Transfer 5.0mLof the supernatant to a second 50-mLvolumetric flask,dilute with Mobile phaseto volume,mix,and filter.
Chromatographic system(see Chromatography á621ñ)—
The liquid chromatograph is equipped with a 254-nm detector and a 3.9-mm ×30-cm column that contains packing L1.The flow rate is about 2mLper minute.Chromatograph the
Standard preparation,and record the peak responses as directed for
Procedure:the relative retention times are about 1.0for trimethoprim and 1.8for sulfamethoxazole;the resolution,
R,between sulfamethoxazole and trimethoprim is not less than 5.0;the tailing factor for the trimethoprim and sulfamethoxazole peaks is not more than 2.0;and the relative standard deviation for replicate injections is not more than 2.0%.
Procedure—
Separately inject equal volumes (about 20µL)of the
Standard preparationand the
Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantities,in mg,of trimethoprim (C
14H
18N
4O
3)and sulfamethoxazole (C
10H
11N
3O
3S)in each mLof the Oral Suspension taken by the formula:
(500C/V)(rU/rS),
in which
Cis the concentration,in mg per mL,of the appropriate USP Reference Standard in the
Standard preparation;Vis the volume,in mL,of Oral Suspension taken;and
rUand
rSare the peak responses of the corresponding analyte obtained from the
Assay preparationand the
Standard preparation,respectively.