A:Infrared Absorption á197Kñ.

B:UV Absorption á197Uñ—

pH10Buffer— To 33.7g of ammonium chloride add 285mLof ammonium hydroxide,dilute with water to 500mL,and mix.

Procedure— Transfer 20mg of USP Ioxilan Related Compound A RS,accurately weighed,to a 200-mLvolumetric flask,dissolve in a small volume of hot water,cool,dilute with water to volume,and mix.Transfer 2.5mLof this solution to a 50-mLvolumetric flask,add 12mLof water,and mix.Transfer 0.5g of Ioxilan to a second 50-mLvolumetric flask,add 12mLof water,and shake to dissolve.[NOTE—Heat gently,if necessary,to dissolve.Cool to room temperature before proceeding.]To a third 50-mLvolumetric flask,add 12mLof water to serve as the blank,and place the flasks containing the Standard solution,the test solution,and the blank in an ice bath.[NOTE—In conducting the following steps,keep the flasks in the ice bath as much as possible until all of the reagents have been added.]Treat each of the flasks as follows.Add 5mLof sodium nitrite solution (0.5in 100),and shake.[Caution—Considerable pressure is produced with the addition of each reagent. ] Add 10mLof 1Nhydrochloric acid,swirl,and allow to stand for 2minutes.Add 3drops of a 1in 10solution of a-naphthol in alcohol,swirl,and allow to stand for 2minutes.Add 3.5mLof pH10Buffer,swirl,and allow to stand out of the ice bath for 2minutes.Degas all solutions in a water bath at 25for 10minutes,dilute with water to 50mL,and mix.Within 15minutes of this final dilution,concomitantly determine the absorbances of the test solution and the Standard solution at the wavelength of maximum absorbance at about 485nm,with a suitable spectrophotometer,against the blank.The absorbance of the test solution is not greater than that of the Standard solution (0.05%).

Standard stock solution— Transfer 200mg of methanol to a tared 200-mLvolumetric flask containing 20mLof water,dilute with water to volume,and mix.Transfer 1.0mLof this solution to a 100-mLvolumetric flask containing 20mLof water,dilute with water to volume,and mix.

Internal standard solution— Transfer 200mg of dehydrated alcohol to a tared 200-mLvolumetric flask containing 20mLof water,dilute with water to volume,and mix.Transfer 10.0mLof this solution to a 100-mLvolumetric flask,dilute with water to volume,and mix.

Standard solutions A,B,C,and D— Transfer 0.5,1.0,2.0,and 4.0mLof Standard stock solutionto separate 10-mLvolumetric flasks,each containing 2mLof water.Add 1.0mLof Internal standard solutionto each flask,dilute with water to volume,and mix to obtain Standard solutions A,B,C,and Dhaving concentrations of 0.5,1.0,2.0,and 4.0mg of methanol per L,respectively.

Test solution— Transfer about 1g of Ioxilan to a tared 10-mLampul,and add water to the ampul to a final weight of 10.45g,taking care to avoid leaving water in the neck of the ampul.Seal the ampul,and immerse it in a water bath at 90until the Ioxilan is dissolved.Mix,and allow to cool to room temperature.Mix again,open the ampul,and dilute the contents with a volume of water suitable to obtain a methanol concentration within the range of the standard curve (between 0.5and 4mg per L),allowing for the addition of 1.0mLof Internal standard solution.

Chromatographic system(see System Suitabilityunder Chromatography á621ñ)— The gas chromatograph is equipped with a flame-ionization detector and a 10-m ×0.53-mm fused silica capillary column coated with support S2.The column temperature is programmed to increase from 45to 80at a rate of 5per minute.The injection port temperature is maintained at 180,and the detector temperature is maintained at 200.Helium is used as the carrier gas at a flow rate of about 30mLper minute.Chromatograph Standard solution D,and record the peak responses as directed for Procedure:the resolution,R,between the methanol and alcohol peaks is not less than 10;the tailing factor for the methanol peak is not more than 3.0;and the relative standard deviation for replicate injections is not more than 5.0%.

Procedure— Separately inject equal volumes (about 1µL)of each Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the areas of the peak responses.From a linear regression equation calculated from the ratios of the peak responses of methanol to those of alcohol for each of the Standard solutionsversus the methanol concentrations,in mg per L,determine the methanol content in the Test solution:not more than 2.0%is found.

Mobile phase— Prepare a filtered and degassed mixture of acetonitrile and water (91:9).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard solution— Transfer about 60mg of USP Ioxilan RSto a 10-mLvolumetric flask,add 1mLof water,and heat at not more than 80to dissolve.Cool to room temperature,dilute with acetonitrile to volume,and mix.

Test solution— Using about 60mg of Ioxilan,proceed as directed for Standard solution.

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a 245-nm detector and a 25-cm ×4.6-mm stainless steel column that contains 5-µm packing L18.The flow rate is about 2mLper minute,and the column temperature is maintained at 30.Chromatograph the Standard solution,and record the peak responses as directed for Procedure:the retention time of the serinol peak is about 0.9relative to that of the larger of the ioxilan peaks;the resolution,R,between the larger of the ioxilan peaks and the serinol peak is not less than 1.0;and the relative standard deviation of the response of the serinol peak for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10µL)of the Test solutionand the Standard solutioninto the chromatograph,and allow the chromatogram to run for about 35minutes.Measure the areas of the peak responses,and determine the percentage of serinol in the portion of Ioxilan taken:not more than 0.5%is found.

Mobile phase— Prepare a filtered and degassed mixture of acetonitrile and water (87:13).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard solution— Transfer about 60mg of USP Ioxilan RSto a 10-mLvolumetric flask,and add 1mLof water.[NOTE—Heat gently,if necessary,to dissolve.Cool to room temperature before proceeding.]Dilute with acetonitrile to volume,and mix.

Test solution— Using about 60mg of Ioxilan,proceed as directed for Standard solution.

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a 245-nm detector and a 25-cm ×4.6-mm stainless steel column that contains 5-µm packing L8.The column is maintained at 30,and the flow rate is about 1mLper minute.Chromatograph the Standard solution,and record the peak responses as directed for Procedure:the resolution,R,between the two largest impurity peaks eluting immediately after the second ioxilan isomer peak is not less than 0.3.

Procedure— Separately inject equal volumes (about 10µL)of the Test solutionand the Standard solutioninto the chromatograph,record the chromatograms,and measure the areas of the peak responses.Determine the percentage of each impurity (excluding the serinol impurity,which has a retention time of 0.9relative to the larger ioxilan isomer)in the portion of Ioxilan taken:not more than 0.5%of any individual impurity is found;and the total of all impurities is not more than 1.5%.