Adsorbent: 0.25-mm layer of chromatographic silica gel mixture.

Test solution— Transfer 30 mg of Pamidronate Disodium to a 10-mL volumetric flask, dissolve in and dilute with water to volume, and mix.

Standard solution— Dissolve an accurately weighed quantity of USP Beta Alanine RS in water, and dilute quantitatively, and stepwise if necessary, with water to obtain a solution containing 0.006 mg of beta alanine per mL.

Application volume: 10 µL.

Developing solvent system: a mixture of methanol, diisopropyl ether, and 25% ammonia (9:8:4).

Spray reagent— Dissolve 0.2 g of ninhydrin in 100 mL of a mixture of butyl alcohol and 2 N acetic acid (95:5).

Procedure— Proceed as directed for Thin-Layer Chromatography under Chromatography 621. Dry the plate between 100 and 105 until the ammonia disappears completely. Spray with Spray reagent, and heat between 100 and 105 for about 15 minutes. Examine the plate under white light. The spot obtained from the Test solution having an RF value of about 0.5 is not greater in size or intensity than the corresponding spot obtained from the Standard solution: not more than 0.2% of beta alanine is found. Evaluate any other additional spot in the chromatogram of the Test solution, and determine the percentage of total other impurities (excluding beta alanine).

Mobile phase— Proceed as directed in the Assay.

Impurity stock solution 1— Transfer about 300 mg of ortho-phosphoric acid, accurately weighed, to a 1000-mL volumetric flask, dissolve in and dilute with water to volume, and mix.

Impurity stock solution 2— Transfer about 250 mg of phosphorous acid, accurately weighed, to a 1000-mL volumetric flask, dissolve in and dilute with water to volume, and mix.

Impurity standard solution— Transfer 2.0 mL each of Impurity stock solution 1 and Impurity stock solution 2 to a 50-mL volumetric flask, dilute with water to volume, and mix.

Test solution— Prepare as directed for the Assay preparation.

Chromatographic system (see Chromatography 621)— Proceed as directed in the Assay. Chromatograph the Impurity standard solution, and record the peak responses as directed for Procedure: the elution order is a phosphate peak followed by the phosphite peak; the resolution, R, between the two peaks is not less than 2.5; the relative standard deviation for replicate injections, determined from the phosphate peak, is not more than 10%; and the relative standard deviation for replicate injections, determined from the phosphite peak, is not more than 20%.

Procedure— Separately inject equal volumes (about 100 µL) of the Impurity standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the percentage of phosphates as ortho-phosphoric acid in the portion of Pamidronate Disodium taken by the formula: in which W1 is the weight, in mg, of ortho-phosphoric acid taken to prepare the Impurity stock solution 1; W is the weight, in mg, of Pamidronate Disodium taken to prepare the Test solution; and rU and rS are the phosphate peak responses obtained from the Test solution and the Impurity standard solution, respectively: not more than 0.5% of phosphate, determined as ortho-phosphoric acid, is found.

50C(rU / rS)