A: Dissolve 0.25g in 3mLof 1Nsodium hydroxide,transfer to a 500-mLvolumetric flask,dilute with water to volume,and mix.Transfer a 5-mLaliquot to a 250-mLvolumetric flask containing 12.5mLof pH7phosphate buffer (see Buffer Solutionsin the section Reagents,Indicators,and Solutions),dilute with water to volume,and mix.This solution,when compared in a suitable spectrophotometer against a blank of the same buffer in the same concentration,exhibits absorbance maxima at 265±2and 299±2nm,and the ratio A265/A299is between 1.50and 1.56.

B: Place about 1g in a small,round-bottom flask,and add 10mLof acetic anhydride.Heat the flask on a steam bath for 30minutes,add 40mLof water,mix,filter,cool,and allow to stand until the diacetyl derivative has crystallized.Collect the precipitate on a filter,wash well with water,and dry at 105for 1hour:the diacetyl derivative so obtained melts between 191and 197.

C: Shake 0.1g with 10mLof water,and filter.To 5mLof the filtrate add 1drop of ferric chloride TS:a violet color is produced.

Mobile phase— Prepare as directed in the Assay.

Internal standard solution— Prepare a solution of sulfanilamide in Mobile phasehaving a concentration of about 5µg per mL.

Standard solution— Dissolve an accurately weighed quantity of USPm-Aminophenol RSin Mobile phaseto obtain a solution having a known concentration of about 12µg per mL.Transfer 10.0mLof this solution and 10.0mLof Internal standard solutionto a 100-mLlow-actinic volumetric flask,dilute with Mobile phaseto volume,and mix.

Test solution— Transfer about 50mg of Aminosalicylic Acid,accurately weighed,to a 100-mLlow-actinic volumetric flask,add 50mLof Mobile phase,and swirl to dissolve.Add 10.0mLof Internal standard solution,dilute with Mobile phaseto volume,and mix.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 280-nm detector and a 4.6-mm ×25-cm column that contains 10-µm packing L1.The flow rate is about 1.5mLper minute.Chromatograph the Standard solution,and record the peak responses as directed for Procedure:the relative retention times are about 0.66for sulfanilamide and 1.0for m-aminophenol;the resolution,R,between m-aminophenol and sulfanilamide is not less than 2.5;and the relative standard deviation for replicate injections is not more than 7%.

Procedure— [NOTE—After use,wash the column for 30minutes with a filtered and degassed mixture of methanol,water,and phosphoric acid (77:23:0.6),and then wash for 30minutes with a filtered and degassed mixture of methanol and water (50:50).]Separately inject equal volumes (about 20µL)of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the percentage of m-aminophenol,in relation to the quantity of aminosalicylic acid in the portion of Aminosalicylic Acid taken by the formula: in which Cis the concentration,in µg per mL,of USPm-Aminophenol RSin the Standard solution,Wis the quantity of aminosalicylic acid,in mg,in the portion of Aminosalicylic Acid taken,as determined in the Assay;and RUand RSare the ratios of the response of the m-aminophenol peak to the response of the sulfanilamide peak obtained from the Test solutionand the Standard solution;respectively:not more than 0.25%of m-aminophenol is found.

Mobile phase— Prepare a mixture of 425mLof 0.05Mdibasic sodium phosphate,425mLof 0.05Mmonobasic sodium phosphate,and 150mLof methanol containing 1.9g of tetrabutylammonium hydroxide.Filter,and degas.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Internal standard solution— Prepare a solution of acetaminophen in Mobile phasehaving a concentration of about 5mg per mL.

Standard preparation— Transfer about 12.5mg of USP Aminosalicylic Acid RS,accurately weighed,to a 25-mLlow-actinic volumetric flask,add 15mLof Mobile phase,and swirl to dissolve.Add 2.5mLof Internal standard solution,dilute with Mobile phaseto volume,and mix.

Assay preparation— Prepare as directed for Standard preparation,except to use Aminosalicylic Acid instead of USP Aminosalicylic Acid RS.

Chromatographic system (see Chromatography á621ñ)—The chromatograph is equipped with a 254-nm detector and a 4.6-mm ×25-cm column that contains packing L1.The flow rate is about 1.5mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed under Procedure:the relative retention times are about 0.83for acetaminophen and 1.0for aminosalicylic acid;the resolution,R,between aminosalicylic acid and acetaminophen is not less than 1.7;and the relative standard deviation of the ratios of the response of the aminosalicylic acid peak to the response of the acetaminophen peak is not more than 1.0%.

Procedure— [NOTE—After use,wash the column for 30minutes with a filtered and degassed mixture of methanol,water,and phosphoric acid (77:23:0.6),and then wash for 30minutes with a filtered and degassed mixture of methanol and water (50:50).]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 quantity,in mg,of C7H7NO3in the Aminosalicylic Acid taken by the formula: in which Cis the concentration,in mg per mL,of USP Aminosalicylic Acid RSin the Standard preparation;and RUand RSare the ratios of the response of the aminosalicylic acid peak to the response of the acetaminophen peak obtained from the Assay preparationand the Standard preparation,respectively.