Adsorbent , Developing solvent system, and Spray reagent—Proceed as directed for Related compounds test.

Application volume: 25 µL.

Standard solution— Prepare a solution of USP Ursodiol RS in methanol containing about 1 mg per mL.

Test solution— Transfer a quantity of finely powdered Tablets, equivalent to about 25 mg of ursodiol, to a conical flask. Add 25.0 mL of methanol, and mix for 20 minutes. Centrifuge this solution for 10 minutes at 4000 rpm, and use the clear supernatant.

Procedure— Proceed as directed for Related compounds test. The principal indigo-colored spot observed in the chromatogram of the Test solution corresponds in color and in RF value to that in the chromatogram of the Standard solution.

Medium: simulated intestinal fluid TS, prepared without pancreatin and adjusted with 0.1 N sodium hydroxide or 0.1 N hydrochloric acid to a pH of 8.0; 900 mL.

Apparatus 2: 75 rpm.

Time: 45 minutes.

Mobile phase and Chromatographic system—Prepare as directed in the Assay.

Procedure— Inject a volume (about 25 µL) of a filtered portion of the solution under test into the chromatograph, record the chromatogram, and measure the heights of responses for the major peaks. Calculate the quantity of C24H40O4 dissolved in comparison with a Standard solution having a known concentration of USP Ursodiol RS in the same Medium and similarly chromatographed.

Tolerances— Not less than 80% (Q) of the labeled amount of C24H40O4 is dissolved in 45 minutes.

Adsorbent: 0.25-mm layer of chromatographic silica gel mixture (see Chromatography 621 ), activated for at least 4 hours at 105.

Developing solvent system: a mixture of chloroform, acetone, and acetic acid (7:2:1).

Standard solution 1— Prepare a solution of USP Ursodiol RS in methanol containing 20 µg per mL.

Standard solution 2— Prepare a solution of lithocholic acid in methanol containing 10 µg per mL.

Standard solution 3— Prepare a solution of chenodeoxycholic acid in methanol containing 300 µg per mL.

Test solution— Transfer a quantity of finely powdered Tablets, equivalent to about 250 mg of ursodiol, to a conical flask. Add 25.0 mL of methanol, and mix for 20 minutes. Centrifuge this solution for 20 minutes at 4000 rpm, and use the clear supernatant.

Application volume: 25 µL each of Standard solution 1, Standard solution 2, and Standard solution 3, and 50 µL of the Test solution.

Spray reagent— Dissolve about 2.5 g of phosphomolybdic acid in 50 mL of glacial acetic acid, add 2.5 mL of concentrated sulfuric acid, and mix well.

Procedure— Proceed as directed for Thin-Layer Chromatography under Chromatography 621. Spray the plate lightly with Spray reagent. Dry the plate by heating at 105 for about 7 minutes. The spot due to lithocholic acid observed in the chromatogram of the Test solution, if present, is not greater in size and intensity than that obtained from Standard solution 2 (0.05%). The spot due to chenodeoxycholic acid observed in the chromatogram of the Test solution, if present, is not greater in size and intensity than that obtained from Standard solution 3 (1.5%). No other unidentified spot observed in the chromatogram of the Test solution is greater in size and intensity than the spot obtained from Standard solution 1 (0.1%).

(Official January 1, 2007)

Mobile phase— Prepare a filtered and degassed mixture of methanol, water, and phosphoric acid (77:23:0.6). Make adjustments if necessary (see System Suitability under Chromatography 621).

Internal standard solution— Dissolve an accurately weighed quantity of propylparaben in Mobile phase to obtain a solution having a known concentration of about 3.75 mg per mL.

Standard preparation— Dissolve an accurately weighed quantity of USP Ursodiol RS in Internal standard solution to obtain a solution having a known concentration of about 3.75 mg per mL.

Assay preparation— Weigh and finely powder 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 37.5 mg of ursodeoxycholic acid, to a glass-stoppered conical flask. Add 10.0 mL of Internal standard solution, and shake by mechanical means for 15 minutes. Sonicate at 40 for an additional 15 minutes, and filter.

Chromatographic system (see Chromatography 621 )—The liquid chromatograph is equipped with a differential refractive index detector and a 4.6-mm × 25-cm column that contains packing L7. The flow rate is about 1.0 mL per minute. The detector temperature is maintained at 40. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative retention times are about 0.73 for propylparaben and 1.0 for ursodiol; the resolution, R , between ursodiol and propylparaben is not less than 3.0; the column efficiency is not less than 1600 theoretical plates; the tailing factor is not more than 2.0; and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of ursodiol (C24H40O4) in the portion of Tablets taken by the formula: in which C is the concentration, in mg per mL, of USP Ursodiol RS in the Standard preparation; and R U and R S are the peak response ratios obtained from the Assay preparation and the Standard preparation, respectively.