A:Infrared Absorption á197Kñ.

B: Asolution containing 0.5g in 10mLof water meets the requirements of the test forSodium á191ñ.

Test solution: 30mg per mL.

Delete the following:

Test Preparation— Provide separate 10-g specimens for each of the tests called for below.Dissolve Chondroitin Sulfate Sodium in pH7.2Phosphate buffer.[NOTE—On the basis of results for Preparatory Testing,modify the Test Preparationas appropriate.]

Preparatory Testing— Incubate Clostridium sporogenes(ATCC No.11437)for 18to 24hours,and then dilute with pH7.2Phosphate Buffer.Inoculate the Test Preparation,to obtain a final concentration of less than 100cfu per mL.Controls containing the inoculum but without the material under test are prepared at the same time.Proceed as directed under Procedure,making sure to evaluate the growth after each time a medium is added.

Results— Proceed as directed for Preparatory Testingunder Microbial Enumeration Tests—Nutritional and Dietary Supplements á2021ñ.

Procedure— Take two equal portions of the Test Preparation,heat one to 80for 10minutes,and cool rapidly.Transfer 10mLof each portion to separate containers,each containing each 100mLof Reinforced Medium for Clostridia,and incubate under anaerobic conditions at 35to 37for 48hours.After incubation subculture each specimen on Columbia Agar Mediumto which gentamicin has been added,and incubate under anaerobic conditions at 35to 37for 48hours.Examine the plates,and interpret as follows:if no growth of Gram-positive rods is detected,the test specimen meets the requirement for the absence of Clostridium species.

Change to read:

0.1MUSP28Barium acetate buffer,pH5.0— Dissolve about 25.24gUSP28of barium acetate in water,and dilute with water to 900mL.Adjust with acetic acid to a pHof 5.0,dilute with water to 1L,and mix.

USP28

Staining reagent: 0.1%toluidine blue in acetic acid;dissolve 1g of toluidine blue in 1000mLof 0.1Macetic acid.USP28

Standard solution 1— Prepare a solution of USP Chondroitin Sulfate Sodium RSin water having a known concentration of about 30mg per mL.USP28

Standard solution 2— Dilute 1mLofStandard solution 1with water to 50mL,USP28and mix.

Test solution— Transfer 150mg of Chondroitin Sulfate Sodium,accurately weighed,to a 5.0-mLUSP28volumetric flask,dissolve in and dilute with water to volume,and mix.

Procedure— Fill the chambers of an electrophoresis apparatus suitable for separations on cellulose acetate membranes1(a small submarine gel chamber or one dedicated to membrane media)with 0.1M Barium acetate buffer,pH5.0.Soak a cellulose acetate membrane about 5to 6cm ×12to 14cm in 0.1M Barium acetate buffer,pH5.0for 10minutes,or until evenly wetted,then blot dry between two sheets of absorbent paper.Using an applicator2suitable for electrophoresis,apply equal volumes (about 0.5µL)of the Test solution,Standard solution 1,and Standard solution 2to the brighter side of the membrane held in position in an appropriate applicator stand or on a separating bridge in the chamber.Ensure that both ends of the membrane are dipped at least 0.5-to 1.0-cm deep into the buffer chambers.Apply a constant 60volts (about 6mAat the start)for 2hours.[NOTE—Perform the application of solutions and voltage within 5minutes because further drying of the blotted paper reduces sensitivity.]Place the membrane in a plastic staining tray,and with the application side down,float or gently immerse in Staining reagentfor 5minutes.Then stir the solution gently for 1minute.Remove the membrane,and destain in 5%acetic acid until the background clears.Compare the bands.The electropherogram obtained from the Test solutionexhibits a major band that is identical in position to the band obtained from Standard solution 1.The band obtained from Standard solution 2is clearly visible at a mobility similar to the band obtained from Standard solution 1.Any secondary band in the electropherogram of the Test solutionis not more intense than the band obtained from Standard solution 2.Not more than 2%of any individual impurity is found.Document the results by taking a picture within 15minutes of completion of destaining.USP28

Change to read:

Alkaline cupric tartaric reagent— Dissolve 200mg of sodium tartrate dihydrate in 10mLof water,and mark as Solution A.Dissolve 100mg of cupric sulfate in 10mLof water,and mark as Solution B.Dissolve 2.0g of anhydrous sodium carbonate in 0.1Msodium hydroxide,dilute with 0.1Msodium hydroxide to 100mL,and mark as Solution C.Mix well 1mLof Solution Aand 1mLof Solution B,and to the mixture slowly add 100mLof Solution Cwith stirring.Use within 24hours,and discard afterwards.

Standard solution— Transfer an accurately measured volume of 7percent bovine serum albumin certified standard to a suitable container,and dilute quantitatively and stepwise with water to obtain a solution having a known concentration of about 35µg per mL.

Test solution— Transfer an accurately weighed quantity of Chondroitin Sulfate Sodium,equivalent to 60mg of the dried substance,to a 100-mLvolumetric flask,dissolve in and dilute with water to volume,and mix.

Procedure— Add 2.0mLof freshly prepared Alkaline cupric tartaric reagentto test tubes containing 2.0mLof water,2.0mLof theTest solution,or 2.0mLof theStandard solution,and mix.After about 10minutes,add 1.0mLof Folin-Ciocalteu phenol TS,diluted with water (1:5)andUSP28prepared immediately before use,to each test tube,and mix immediately and vigorously.USP28After 30minutes,measure the absorbance of each solution at 750nm against the blank.The absorbance of theTest solution is not greater than the absorbance of theStandard solution:not more than 6.0%of proteins is found,calculated on the dried basis.

Change to read:

Cetylpyridinium chloride solution— Prepare a solution of cetylpyridinium chloride in water having a concentration of about 1mg per mL.Degas before use.

Diluent— Weigh about 297mg of monobasic potassium phosphate,492mg of dibasic potassium phosphate,and 250mg of polysorbate 80,and transfer into a 1-Lbeaker.Dissolve in 1000mLof water,and adjust with potassium hydroxide or phosphoric acid to a pHof 7.0±0.2.USP28

Standard solutions— Prepare a solution having a known concentration of USP Chondroitin Sulfate Sodium RSin water,USP28and dilute with water,quantitatively and stepwise if necessary,to obtain threeStandard solutionshaving known concentrations of about 1.5mg per mL,1.0mg per mL,and 0.5mg per mL,respectively.USP28

Test solution— Transfer about 100mg of dried Chondroitin Sulfate Sodium,accurately weighed,to a 100-mLvolumetric flask,dissolve in 30mLof water,USP28dilute with water to volume,and mix.

Procedure— Transfer 5.0mLof eachStandard solution and theTest solution to four separate titration vessels,and add about 25mLof Diluent.Use a phototrode to determine the endpoint turbidimetrically,either at 420nm,550nm,or 660nm.Stir until a steady reading is obtained.Set the instrument to zero in absorbance mode.Titrate withCetylpyridinium chloride solution.From a linear regression equation derived from the volumes ofCetylpyridinium chloride solution consumed,and the masses,in mg,of USP Chondroitin Sulfate Sodium RSin the aliquots of the respective Standard solutions,USP28determine the mass of chondroitin sulfate sodium in the aliquot of theTest solution taken.Calculate the percentage of chondroitin sulfate sodium in the portion taken by the formula: in which Mis the mass of Chondroitin Sulfate Sodium foundUSP28in the aliquot of theTest solution;and Wis the weight,in mg,of Chondroitin Sulfate Sodium taken to prepare the Test solution.