pH9.2Buffer— Transfer 3.80g of sodium borate to a 1000-mLvolumetric flask,dissolve in and dilute with water to volume,and mix.

Bromothymol blue solution— Transfer 150mg of bromothymol blue and 405mg of sodium carbonate decahydrate to a 100-mLvolumetric flask,dilute with water to volume,and mix.

Standard solution— Dilute 1mLof 60%benzyltrimethylammonium chloride solution,accurately pipeted,quantitatively and stepwise with water to obtain a stock solution having a concentration of 0.2±0.01mg per mL[NOTE—Prepare this solution fresh].Cut a 20-to 25-cm piece of cellulose dialysis tubing*having a molecular weight cut-off of 6,000to 14,000and a dry flat width of 5to 9cm,and place it in water to hydrate until pliable,appropriately sealing one end.Pipet 5mLof the stock solution into the tubing,add 5mLof water,appropriately seal the open end,place the tube in a suitable vessel containing 100mLof water so that it is completely immersed in the water,and stir the fluid for 16hours to effect dialysis.

Test solution— Cut a 20-to 25-cm piece of cellulose dialysis tubing*having a molecular weight cut-off of 6,000to 14,000and a dry flat width of 5to 9cm,and place it in water to hydrate until pliable,appropriately sealing one end.Weigh 2±0.01g of Cholestyramine Resin,and carefully transfer the specimen into the tubing,taking care to ensure that none adheres to the upper walls of the tubing.Add 10mLof water to the contents of the tube,appropriately seal the open end,and place the tube in a suitable vessel containing 100mLof water so that it is completely immersed in the water.Stir the fluid for 16hours to effect dialysis.

Procedure— Pipet the following into each of three separators:separator 1:5mLof Standard solution,5mLof pH9.2Buffer,1mLof Bromothymol blue solution,and 10mLof chloroform;separator 2:5mLof Test solution,5mLof pH9.2Buffer,1mLof Bromothymol blue solution,and 10mLof chloroform;separator 3:5mLof water,5mLof pH9.2Buffer,1mLof Bromothymol blue solution,and 10mLof chloroform.Shake each separator,vigorously,for 1minute,allow the phases to separate until the chloroform phase is clear,and collect the chloroform extracts in separate 25-mLvolumetric flasks.Repeat the extraction process with a second 10-mLportion of chloroform,and combine with the previous extracts.Dilute each solution with chloroform to volume,if necessary,and mix.Concomitantly determine the absorbances of the Test solutionand the Standard solutionat the wavelength of maximum absorbance at about 420nm,with a suitable spectrophotometer,using the solution from separator 3as the blank:the absorbance of the Test solutiondoes not exceed that of the Standard solution(0.05%as benzyltrimethylammonium chloride).

Mobile phase— Prepare a filtered and degassed mixture of 0.08Mmonobasic potassium phosphate and acetonitrile (65:35).Adjust with phosphoric acid to a pHof 3.0.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Potassium phosphate buffer— Transfer about 4g of monobasic potassium phosphate and about 12g of dibasic potassium phosphate to a 1-liter volumetric flask.Dissolve in and dilute with water to volume,and mix.

Sodium glycocholate solution— Transfer about 15g of sodium glycocholate to a 500-mLvolumetric flask,and dissolve in and dilute with Potassium phosphate bufferto volume.

Reference solution— Pipet 4.0mLof Sodium glycocholate solutioninto a 100-mLvolumetric flask,and dilute with water to volume.

Standard solution— Transfer about 100mg of USP Cholestyramine Resin RS,accurately weighed,to a 25-mLconical flask.Pipet 15.0mLof Sodium glycocholate solutioninto the flask,and stir by mechanical means for 2hours.Transfer the contents to a centrifuge tube,and centrifuge for 15minutes.Transfer 5.0mLof the supernatant to a 50-mLvolumetric flask,and dilute with water to volume.

System suitability solution— Prepare a solution in water containing,in each mL,about 0.6mg of sodium glycocholate and about 0.3mg of taurodeoxycholic acid.

Test solution— Transfer about 100mg of anhydrous Cholestyramine Resin,accurately weighed,to a 25-mLconical flask.Pipet 15.0mLof Sodium glycocholate solutioninto the flask,and stir by mechanical means for 2hours.Transfer the contents to a centrifuge tube,and centrifuge for 15minutes.Transfer 5.0mLof the supernatant to a 50-mLvolumetric flask,and dilute with water to volume.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 214-nm detector and a 3.9-mm ×30-cm column that contains packing L1.The flow rate is about 1.5mLper minute.Chromatograph the System suitability solution,and record the peak responses as directed for Procedure:the resolution,R,between sodium glycocholate and taurodeoxycholic acid is not less than 1.5.Chromatograph the Reference solution,and record the peak responses as directed for Procedure:the tailing factor is not more than 2.5;and the relative standard deviation for replicate injections is not more than 1.5%.

Procedure— Separately inject equal volumes (about 50µL)of the Reference solution,the Standard solution,and the Test solutioninto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of sodium glycocholate absorbed on each g of the Resin taken by the formula: in which Mis the stated value,in mg,of sodium glycocholate absorbed per g of USP Cholestyramine Resin RS;rR,rU,and rSare the peak responses obtained from the Reference solution,the Test solution,and the Standard solution,respectively;WUis the weight,in mg,of Cholestyramine Resin,calculated on the dried basis,taken to prepare the Test solution;and WSis the weight,in mg,of USP Cholestyramine Resin RStaken to prepare the Standard solution.