Medium: water;900mL.

Apparatus 2: 50rpm.

Time: 15minutes.

Buffer solution and Mobile phase— Prepare as directed in the Assay.

0.05%9-Fluorenylmethyl chloroformate solution— Transfer 100mg of 9-fluorenylmethyl chloroformate to a 200-mLvolumetric flask,dilute with acetonitrile to volume,and mix.Prepare this solution fresh.

Borate buffer— Dissolve 6.2g of boric acid in approximately 950mLof water,adjust with 1Nsodium hydroxide to a pHof 9.0,and dilute with water to 1L.

Diluent— Transfer 176.4g of sodium citrate dihydrate to a 1000-mLvolumetric flask,dissolve in and dilute with Dissolution Mediumto volume,and mix.

Standard stock solution— Dissolve an accurately weighed quantity of USP Alendronate Sodium RSin Dissolution Medium,and dilute quantitatively and stepwise with the same solvent to obtain a solution having a known concentration corresponding to the concentration that would be obtained by dissolving 1Tablet in 900mLof the same Medium.Calculate the concentration,C,in mg per mL,of anhydrous alendronate sodium in this solution.

Standard solution— Transfer 5.0mLof the Standard stock solutionto a 50-mLpolypropylene screw-cap centrifuge tube containing 1.0mLof Diluentand 5.0mLof Borate buffer,and mix for about 3minutes.Add 4.0mLof 0.05%9-Fluorenylmethyl chloroformate solution,and agitate for about 30seconds.Allow the solution to stand at room temperature for 25minutes.Add 25mLof methylene chloride,and agitate for about 40seconds.Centrifuge the mixture for 5minutes.Use a portion of the clear upper aqueous layer.

Reagent blank— Using 5mLof water,proceed as directed for Standard solution,beginning with “to a 50-mLpolypropylene screw-cap centrifuge tube.”

Test solution— After 15minutes,withdraw a portion of the solution under test,and centrifuge immediately.Using 5.0mLof the clear supernatant,proceed as directed for Standard solution,beginning with “to a 50-mLpolypropylene screw-cap centrifuge tube.”

Chromatographic system (see Chromatography á621ñ)— Proceed as directed in the Assay.Chromatograph the Standard solution,and record the peak responses as directed for Procedure:the capacity factor,k¢,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 50µL)of the Standard solution,the Test solution,and the Reagent blankinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of alendronic acid (C4H13NO7P2)dissolved by the formula: in which Cis defined under the Standard stock solution;and rUand rSare the peak areas obtained from the Test solutionand the Standard solution,respectively.[NOTE—827.1is the molecular weight conversion factor (C4H13NO7P2/C4H12NNaO7P2)multiplied by the volume of the Medium(900mL).]

Tolerances— Not less than 80%(Q)of the labeled amount of alendronic acid (C4H13NO7P2)is dissolved in 15minutes.Tablets labeled for weekly dosing:not less than 75%(Q)of the labeled amount of alendronic acid (C4H13NO7P2)is dissolved in 15minutes.

Diluent— Transfer 29.4g of sodium citrate dihydrate to a 1000-mLvolumetric flask,dissolve in and dilute with water to volume,and mix.

Buffer solution— Transfer 14.7g of sodium citrate dihydrate and 7.05g of anhydrous dibasic sodium phosphate to a 1000-mLvolumetric flask,dissolve in about 900mLof water,adjust with phosphoric acid to a pHof 8.0,dilute with water to volume,and mix.

0.1%9-Fluorenylmethyl chloroformate solution— Transfer 250mg of 9-fluorenylmethyl chloroformate to a 250-mLvolumetric flask,dilute with acetonitrile to volume,and mix.Prepare this solution fresh just prior to use.

Borate solution— Transfer 38.1g of sodium borate to a 1000-mLvolumetric flask,dissolve in and dilute with water to volume,and mix.

Mobile phase— Prepare a filtered and degassed mixture of Buffer solution,acetonitrile,and methanol (75:20:5).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard stock preparation— Prepare a solution of USP Alendronate Sodium RSin diluent containing 0.03mg of anhydrous alendronate sodium per mL.

Standard preparation— Transfer 5.0mLof the Standard stock preparationto a 50-mLpolypropylene screw-cap centrifuge tube containing 5mLof Borate solution,and mix for about 3minutes.Add 4mLof 0.1%9-Fluorenylmethyl chloroformate solution,and agitate for about 30seconds.Allow the solution to stand at room temperature for 25minutes.Add 25mLof methylene chloride,and agitate for about 40seconds.Centrifuge the mixture for 10minutes.Use the clear upper aqueous layer.

Assay stock preparation— Transfer not fewer than 10Tablets to a 1000-mLvolumetric flask.Add 500mLof Diluent,shake by mechanical means for 30minutes,and sonicate for 5minutes.Dilute with Diluentto volume,mix,and centrifuge a portion of this solution.Quantitatively dilute a portion of the clear supernatant to a concentration in a range of 0.02to 0.03mg per mL.

Assay preparation— Using 5.0mLof the Assay stock preparation,proceed as directed for Standard preparation,beginning with “to a 50-mLpolypropylene screw-cap centrifuge tube.”

Reagent blank— Using 5mLof Diluent,proceed as directed for Standard preparation,beginning with “to a 50-mLpolypropylene screw-cap centrifuge tube.”

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 266-nm detector and a 4.1-mm ×25-cm column that contains packing L21.The column is maintained at a constant temperature of about 35.The flow rate is about 1mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the capacity factor,k¢,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 50µL)of the Standard preparation,the Assay preparation,and the Reagent blankinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of alendronic acid (C4H13NO7P2)in the portion of Tablets taken by the formula: in which Dis the dilution factor for the Assay stock preparation;Cis the concentration,in mg per mL,of anhydrous USP Alendronate Sodium RSin the Standard stock preparation;and rUand rSare the peak areas obtained from the Assay preparationand the Standard preparation,respectively.[NOTE—0.919is the molecular weight conversion factor (C4H13NO7P2/C4H12NNaO7P2).]USP28