Theophylline,Ephedrine Hydrochloride,and Phenobarbital Tablets »Theophylline,Ephedrine Hydrochloride,and Phenobarbital Tablets contain not less than 90.0percent and not more than 110.0percent of the labeled amounts of anhydrous theophylline (C7H8N4O2),ephedrine hydrochloride (C10H15NO·HCl),and phenobarbital (C12H12N2O3). Packaging and storage— Preserve in tight containers. USP Reference standards á11ñ USP Theophylline RS.USP Ephedrine Sulfate RS.USP Phenobarbital RS. Identification— Place a quantity of finely powdered Tablets,equivalent to about 24mg of ephedrine hydrochloride,in a 15-mLcentrifuge tube,add 4.0mLof a mixture of chloroform and methanol (4:1),mix by sonication for 10minutes,and filter to obtain the test solution.Prepare separate Standard solutions containing known concentrations of about 36mg of USP Theophylline RSper mL,about 9.6mg of USP Ephedrine Sulfate RSper mL,and about 5mg of USP Phenobarbital RSper mL,respectively,in the mixture of chloroform and methanol (4:1).Apply 2µLof the test solution and of each of the three Standard solutions at equidistant points about 2.5cm above one edge of a suitable thin-layer chromatographic plate (see Chromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel mixture.Allow the spots to dry,and develop the chromatogram in a suitable chamber previously equilibrated with a solvent system consisting of a mixture of chloroform,acetone,methanol,and ammonium hydroxide (50:10:10:1)until the solvent front has moved about three-fourths of the length of the plate.Remove the plate from the developing chamber,mark the solvent front,air-dry,and locate the spots on the plate by viewing under short-wavelength UVlight:the RFvalues of the spots obtained from the test solution correspond to those obtained from the Standard solutions. Dissolution,Procedure for a Pooled Sample á711ñ Medium: water;900mL. Apparatus 1: 100rpm. Time: 30minutes. Determine the amounts of theophylline,ephedrine hydrochloride,and phenobarbital dissolved using the following procedure. Mobile phase— Dissolve accurately weighed quantities of monobasic potassium phosphate and sodium 1-hexanesulfonate in water,and dilute quantitatively with water to obtain a solution having concentrations of 0.953mg per mL(0.007Mmonobasic potassium phosphate)and 0.564mg per mL(0.003Msodium 1-hexanesulfonate),respectively.Adjust,if necessary,with 0.3Mphosphoric acid or 0.2Mmonobasic potassium phosphate to a pHof 3.0±0.05,to obtain a Phosphate buffer.The Mobile phaseis a mixture of Phosphate bufferand methanol (75:25). Standard preparation— Dissolve accurately weighed quantities of USP Theophylline RS,USP Ephedrine Sulfate RS,and USP Phenobarbital RSin water,and dilute quantitatively and stepwise with water to obtain a solution having known concentrations of about 145µg of anhydrous theophylline per mL,28µg of ephedrine sulfate per mL,and 9µg of phenobarbital per mL. Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 215-nm detector and a 3.9-mm ×30-cm column that contains packing L1.The flow rate is about 3.0mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the resolution,R,between the theophylline and phenobarbital peaks is not less than 4.0,the tailing factors for the ephedrine and phenobarbital peaks are not more than 3.0and 2.0,respectively,and the relative standard deviation for replicate injections is not more than 2.0%. Procedure— Inject an accurately measured volume (about 75µL)of a filtered portion of the solution under test into the chromatograph,record the chromatogram,and measure the responses for the major peaks.The elution order is theophylline,ephedrine,and phenobarbital (last).Calculate the quantity,in mg,of C7H8N4O2dissolved by the formula: (0.9C)(rU/rS), in which Cis the concentration,in µg per mL,of USP Theophylline RSin the Standard preparation,and rUand rSare the peak responses for theophylline obtained from the solution under test and the similarly chromatographed Standard preparation,respectively.Calculate the quantity,in mg,of C10H15NO·HCl dissolved by the formula: (201.69/214.27)(0.9C¢)(r¢U/r¢S), in which 201.69is the molecular weight of ephedrine hydrochloride,214.27is one-half the molecular weight of ephedrine sulfate,C¢is the concentration,in µg per mL,of USP Ephedrine Sulfate RSin the Standard preparation,and r¢Uand r¢Sare the peak responses for ephedrine obtained from the solution under test and the similarly chromatographed Standard preparation,respectively.Calculate the quantity,in mg,of C12H12N2O3dissolved by the formula: (0.9C¢¢)(r¢¢U/r¢¢S), in which C¢¢is the concentration,in µg per mL,of USP Phenobarbital RSin the Standard preparation,and r¢¢Uand r¢¢Sare the peak responses for phenobarbital obtained from the solution under test and the similarly chromatographed Standard preparation,respectively. Tolerances— Not less than 75%(Q)of the labeled amounts of C7H8N4O2,C10H15NO·HCl,and C12H12N2O3are dissolved in 30minutes. Uniformity of dosage units á905ñ: meet the requirements. Assay— Mobile phase— Mix 240mLof acetonitrile with 760mLof 0.01M,pH7.8phosphate buffer (see Buffer Solutionsunder Solutionsin the section Reagents,Indicators,and Solutions). Internal standard solution— Dissolve 25mg of butabarbital sodium in 50mLof dibasic potassium phosphate solution (17in 1000).Mix 40mLof this solution with 10mLof sodium metaperiodate solution (1in 100). Standard preparation— Transfer about 120mg of USP Theophylline RS,about 8mg of USP Phenobarbital RS,and about 25mg of USP Ephedrine Sulfate RS,each accurately weighed,to a 200-mLvolumetric flask.Add 10mLof methanol and 100mLof chloroform,mix to dissolve,add chloroform to volume,and mix.Pipet 15mLof this solution into a 50-mLvolumetric flask,add chloroform to volume,and mix.The concentrations of anhydrous theophylline,phenobarbital,and ephedrine sulfate in the Standard preparationare about 180µg per mL,12µg per mL,and 37.5µg per mL,respectively. Assay preparation— Weigh and finely powder not less than 20Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 120mg of anhydrous theophylline,to a 200-mLvolumetric flask,and shake by mechanical means for 20minutes with a mixture of 10mLof methanol and 100mLof chloroform.Add chloroform to volume,mix,and filter.Pipet 15mLof the clear filtrate into a 50-mLvolumetric flask,dilute with chloroform to volume,and mix. Standard solution and Assay solution—Pipet 10-mLportions of the Standard preparationand the Assay preparationinto separate glass-stoppered,25-mLconical flasks,and treat each as follows:Evaporate in a warm-water bath with the aid of a current of air to dryness.Add by pipet 4mLof Internal standard solution,insert the stopper in the flask,mix to dissolve the residue,and allow to stand at room temperature for 30minutes.Pipet 1mLof propylene glycol solution (1in 100)into the flask,insert the stopper,and mix. Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 241-nm detector and a 30-cm ×4-mm stainless steel column that contains packing L1.Set a flow rate of 1.0mLper minute for the Mobile phase,and allow the system to equilibrate until a stable baseline is obtained on the recorder.Chromatograph a 10-µLportion of the Standard solution,and record the peak responses as directed for Procedure:the peaks are completely resolved,and the resolution factors between each two neighboring peaks are not less than 1.5.Five replicate injections of the Standard preparationshow a relative standard deviation of not more than 2.0%. Procedure— Separately inject equal volumes (about 10µL)of the Standard solutionand the Assay solutioninto the chromatograph,record the chromatograms,and measure the responses for the major peaks.In the order of increasing elution times,the five peaks correspond to the reagent (iodate),theophylline,phenobarbital,butabarbital,and benzaldehyde (from ephedrine).Designate the peak response ratio of each component to the internal standard in the Standard solutionas RS,and that of each component to the internal standard in the Assay solutionas RU. Calculation for theophylline— Calculate the quantity,in mg,of anhydrous theophylline (C7H8N4O2)in the portion of Tablets taken by the formula: 0.667C(RU/RS), in which Cis the concentration,in µg per mL,of USP Theophylline RSin the Standard preparation. Calculation for ephedrine hydrochloride— Calculate the quantity,in mg,of ephedrine hydrochloride (C10H15NO·HCl)in the portion of Tablets taken by the formula: (201.69/214.27)(0.667C)(RU/RS), in which 201.69is the molecular weight of ephedrine hydrochloride,214.27is one-half the molecular weight of ephedrine sulfate,and Cis the concentration,in µg per mL,of USP Ephedrine Sulfate RSin the Standard preparation. Calculation for phenobarbital— Calculate the quantity,in mg,of phenobarbital (C12H12N2O3)in the portion of Tablets taken by the formula: 0.667C(RU/RS), in which Cis the concentration,in mg per mL,of USP Phenobarbital RSin the Standard preparation. Auxiliary Information— Staff Liaison:Karen A Russo,Ph.D.,Scientist Expert Committee:(PA1)Pharmaceutical Analysis 1 USP28–NF23Page 1900 Pharmacopeial Forum:Volume No.30(1)Page 189 Phone Number:1-301-816-8379