A: Transfer,with the aid of water,a portion of the Injectable Suspension,freshly mixed and free from air bubbles,equivalent to about 400,000Penicillin G Units,to a separator,add 50mLof chloroform,and shake by mechanical means for 15minutes.Allow to separate,and filter the lower chloroform layer through about 4g of anhydrous sodium sulfate supported on a pledget of glass wool,collecting the filtrate in a 100-mLvolumetric flask.Repeat the extraction with two 25-mLportions of chloroform,combining the filtrates in the 100-mLvolumetric flask.Dilute with chloroform to volume,and mix.[NOTE—Retain the aqueous phase for Identification test B.]Prepare a Standard solution of USP Penicillin G Procaine RSin chloroform containing about 4.5mg per mL.Apply separately 10µLof each solution to a 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 solvent system consisting of a mixture of butanol,isopropyl alcohol,acetone,and water (4:4:2:2)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,and allow the solvent to evaporate.Expose the plate to iodine vapors in a closed chamber for about 15minutes,and locate the spots:the RFvalues and colors of the two principal spots obtained from the test solution correspond to those obtained from the Standard solution.

B: Dilute the aqueous phase retained from Identificationtest Awith water to obtain a test solution containing about 5mg of dihydrostreptomycin per mL.Prepare a Standard solution of USP Dihydrostreptomycin Sulfate RSin water containing 6.5mg per mL.Apply separately 30µLof each solution to a 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 solvent system consisting of a mixture of n-propyl alcohol,water,pyridine,and glacial acetic acid (15:12:10:2)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,and allow the solvent to evaporate.Spray the plate with a reagent prepared by dissolving 2g of ninhydrin in 100mLof alcohol and adding 20mLof glacial acetic acid,heat the plate at 110for 10minutes,and examine the chromatograms:the RFvalue and color of the principal spot obtained from the test solution correspond to those obtained from the Standard solution.

C: The chromatogram of the Assay preparationobtained as directed in the Assay for chlorpheniramine maleateexhibits a major peak for chlorpheniramine,the retention time of which corresponds to that exhibited in the chromatogram of the Standard preparationsimilarly determined,both relative to the internal standard.

D: The chromatogram of the Assay preparationobtained as directed in the Assay for dexamethasoneexhibits a major peak for dexamethasone,the retention time of which corresponds to that exhibited in the chromatogram of the Standard preparationsimilarly determined,both relative to the internal standard.

Standard preparation— Using USP Penicillin G Potassium RS,prepare as directed for Standard preparationunder Iodometric Assay—Antibiotics á425ñ.

Assay preparation— Dilute an accurately measured volume of Injectable Suspension,freshly mixed and free from air bubbles,quantitatively with Buffer No.1to yield a solution containing about 2000Penicillin G Units per mL.Pipet 2mLof this solution into each of two glass-stoppered,125-mLconical flasks.

Procedure— Proceed as directed for Procedureunder Iodometric Assay—Antibiotics á425ñ,except in the Blank Determinationto add 0.1mLof 1.2Nhydrochloric acid immediately before the 10.0mLof 0.01Niodine VS.Calculate the quantity,in Penicillin G Units,in the portion of Injectable Suspension taken by the formula: in which Lis the labeled quantity,in Penicillin G Units,in the volume of Injectable Suspension taken,and Dis the concentration,in Penicillin G Units per mL,of the Assay preparation,on the basis of the labeled quantity in the portion of Injectable Suspension taken and the extent of dilution,and the other terms are as defined therein.

Internal standard solution— Prepare a solution of brompheniramine maleate in water having a concentration of about 7mg per mL.

Standard preparation— Dissolve an accurately weighed quantity of USP Chlorpheniramine Maleate RSin water to obtain a stock solution having a known concentration of about 6mg per mL.Transfer 5.0mLof this solution to a 50-mLcentrifuge tube.Add 5.0mLof Internal standard solution,and adjust with sodium hydroxide solution (1in 2)to a pHof about 10.Add 25.0mLof hexanes,place the cap on the tube,shake for about 2minutes,and centrifuge.Use the upper hexanes layer as the Standard preparation.

Assay preparation— Transfer an accurately measured volume of Injectable Suspension,freshly mixed and free from air bubbles,equivalent to about 30mg of chlorpheniramine maleate,to a 50-mLcentrifuge tube.Proceed as directed under Standard preparation,beginning with “Add 5.0mLof Internal standard solution.”Use the upper hexanes layer as the Assay preparation.

Chromatographic system(see Chromatography á621ñ)— The gas chromatograph is equipped with a flame-ionization detector,and contains a 4-mm ×1.8-m glass column packed with 1.2%liquid phase G16and 0.5%potassium hydroxide on 100-to 120-mesh support S1A.The column is maintained isothermally at about 180,and the injection port and the detector block are maintained at about 200.Dry nitrogen is used as the carrier gas at a flow rate of about 50mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the resolution,R,between the analyte and internal standard peaks is not less than 2.0,and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 1.5µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.The relative retention times are about 0.75for chlorpheniramine and 1.0for brompheniramine.Calculate the quantity,in mg,of chlorpheniramine maleate (C16H19ClN2·C4H4O4)in each mLof the Injectable Suspension taken by the formula: in which Cis the concentration of USP Chlorpheniramine Maleate RSin the stock solution used to prepare the Standard preparation,Vis the volume,in mL,of Injectable Suspension taken,and RUand RSare the peak response ratios of the chlorpheniramine maleate peak to the internal standard peak obtained from the Assay preparationand the Standard preparation,respectively.

Mobile phase— Prepare a suitable filtered mixture of water and acetonitrile (2:1).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Internal standard solution— Dissolve about 30mg of beclomethasone in 2mLof methanol in a 50-mLvolumetric flask,dilute with methylene chloride to volume,and mix.

Standard preparation— Transfer about 25mg of USP Dexamethasone RS,accurately weighed,to a 50-mLvolumetric flask.Add about 1mLof methanol,swirl to dissolve,dilute with methylene chloride to volume,and mix.Transfer 5.0mLof this solution to a suitable flask,and add 5.0mLof Internal standard solution.Heat the flask on a steam bath,and evaporate under a stream of nitrogen just to dryness.Add 10.0mLof methanol to the flask,and swirl to dissolve the residue.This Standard preparationcontains about 0.25mg of USP Dexamethasone RSand 0.3mg of beclomethasone per mL.

Assay preparation— Transfer an accurately measured volume of Injectable Suspension,freshly mixed and free from air bubbles,equivalent to about 2.5mg of dexamethasone,to a separator containing 50mLof 0.1Nhydrochloric acid,add 5.0mLof Internal standard solution,and extract with four 25-mLportions of methylene chloride,combining the extracts in a second separator.Wash the combined extracts with 50mLof sodium bicarbonate solution (1in 20),filtering the lower methylene chloride layer through about 4g of anhydrous sodium sulfate supported on a cotton pledget previously washed with methylene chloride,and collecting the filtrate in a suitable flask.Wash the aqueous layer with 25mLof methylene chloride,and filter the lower methylene chloride layer through the same filter,collecting the filtrate in the same flask.Heat the flask on a steam bath,and evaporate under a stream of nitrogen just to dryness.Add 10.0mLof methanol,and swirl to dissolve the residue.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector and a 4-mm ×30-cm column that contains packing L1.The flow rate is about 1.2mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the resolution,R,of the analyte and the internal standard peaks is not less than 2.0,and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.The relative retention times are about 0.8for dexamethasone and 1.0for beclomethasone.Calculate the quantity,in mg,of dexamethasone (C22H29FO5)in each mLof the Injectable Suspension taken by the formula: in which Cis the concentration,in mg per mL,of USP Dexamethasone RSin the Standard preparation,Vis the volume,in mL,of Injectable Suspension taken,and RUand RSare the peak response ratios of the dexamethasone peak to the internal standard peak obtained from the Assay preparationand the Standard preparation,respectively.