Medium: water; 500 mL.

Apparatus 2: 50 rpm.

Time: 15 minutes.

Procedure— Place 1 Capsule in each vessel, and allow the Capsule to sink to the bottom of the vessel before starting rotation of the blade. Observe the Capsules, and record the time taken for each Capsule shell to rupture.

Tolerances— The requirements are met if all of the Capsules tested rupture in not more than 15 minutes. If 1 or 2 of the Capsules rupture in more than 15 but not more than 30 minutes, repeat the test on 12 additional Capsules. Not more than 2 of the total of 18 Capsules tested rupture in more than 15 but not more than 30 minutes.

Medium: 0.1 N hydrochloric acid containing 0.5% of sodium lauryl sulfate; 1000 mL.

Apparatus 1: 150 rpm.

Time: 90 minutes.

Mobile phase— Prepare a filtered and degassed mixture of acetonitrile, water, methanol, and phosphoric acid (900:450:50:0.5). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard solution— Quantitatively dissolve an accurately weighed quantity of USP Cyclosporine RS in Dissolution Medium to obtain a solution having a known concentration of about 0.001L mg per mL, L being the labeled quantity, in mg, of cyclosporine in each Capsule. Transfer 25.0 mL of this solution to a 50-mL volumetric flask, dilute with acetonitrile to volume, and mix. This solution contains about 0.0005L mg of USP Cyclosporine RS per mL.

Test solution— Filter a portion of the solution under test. Transfer 5.0 mL of the filtrate to a 10-mL volumetric flask, dilute with acetonitrile to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 210-nm detector and a 4.6-mm × 25-cm column that contains packing L1 and is maintained at a constant temperature of about 80. The flow rate is about 2 mL per minute. Chromatograph the Standard solution, and record the peak areas as directed for Procedure: the column efficiency is not less than 700 theoretical plates; and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10 µL) of the solution estimated to contain 0.1 mg of cyclosporine per mL, or 40 µL of the solution estimated to contain 0.025 mg of cyclosporine per mL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the areas for the major peaks. Calculate the quantity, in mg, of C62H111N11O12 dissolved by the formula: in which C is the concentration, in mg per mL, of USP Cyclosporine RS in the Standard solution; and rU and rS are the cyclosporine peak areas obtained from the Test solution and the Standard solution, respectively.

Tolerances— Not less than 80% (Q) of the labeled amount of C62H111N11O12 is dissolved in 90 minutes.

Mobile phase and Chromatographic system— Proceed as directed in the Assay under Cyclosporine Injection.

Standard preparation— Dissolve an accurately weighed quantity of USP Cyclosporine RS in dehydrated alcohol to obtain a solution having a known concentration of about 1 mg per mL. Use this solution promptly after preparation.

Assay preparation— Using a sharp blade, carefully cut open not fewer than 20 Capsules, and with the aid of dehydrated alcohol transfer the contents of the Capsules to a suitable volumetric flask. Wash the blade with dehydrated alcohol, and transfer the washings to the volumetric flask. Dilute the contents of the volumetric flask with dehydrated alcohol to volume, and mix. Quantitatively dilute an accurately measured volume of this solution with dehydrated alcohol to obtain a solution having a concentration of about 1 mg of cyclosporine per mL.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the areas for the major peaks. Calculate the quantity, in mg, of cyclosporine (C62H111N11O12) in each Capsule taken by the formula: in which L is the labeled quantity, in mg, of cyclosporine in each Capsule taken; D is the concentration, in mg per mL, of the Assay preparation, based on the labeled quantity of cyclosporine in the Capsules taken and the extent of dilution; C is the concentration, in mg per mL, of USP Cyclosporine RS in the Standard preparation; P is the purity, in µg per mg, of USP Cyclosporine RS; and rU and rS are the peak areas obtained from the Assay preparation and the Standard preparation, respectively.

Mobile phase— Prepare a filtered and degassed mixture of acetonitrile, water, methanol, and phosphoric acid (605:400:50:0.5). Make adjustments if necessary (see System Suitability under Chromatography 621).

Diluting solvent— Prepare a mixture of acetonitrile, tetrahydrofuran, and dehydrated alcohol (9:5:4).

Standard preparation— Transfer about 25 mg of USP Cyclosporine RS, accurately weighed, to a 25-mL volumetric flask. Add 2.5 mL of water, and sonicate for 10 minutes. Add about 10 mL of Diluting solvent, sonicate for 5 minutes, dilute with Diluting solvent to volume, and mix.

Assay stock preparation— Transfer the contents of 20 Capsules to a volumetric flask of such capacity, V, in mL, to make a final concentration of 10 mg of cyclosporine per mL. Add 0.1V mL of water to the flask, and sonicate for 10 minutes. Add 0.4V mL of Diluting solvent to the flask, and sonicate for 5 minutes. Dilute with Diluting solvent to volume, and mix.

Assay preparation— Transfer 5.0 mL of Assay stock preparation to a 50-mL volumetric flask, add 5 mL of water, dilute with Diluting solvent to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 210-nm detector and a 4.6-mm × 25-cm column that contains packing L13 and is maintained at a constant temperature of about 70. The flow rate is about 2 mL per minute. Chromatograph the Standard preparation, and record the peak areas as directed for Procedure: the column efficiency is not less than 700 theoretical plates; the tailing factor is not more than 1.5; and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the areas for the major peaks. Calculate the quantity, in mg, of cyclosporine (C62H111N11O12) in each Capsule taken by the formula: in which C is the concentration, in mg per mL, of USP Cyclosporine RS in the Standard preparation; V is the volume, in mL, of the volumetric flask used to prepare the Assay stock preparation; and rU and rS are the cyclosporine peak areas obtained from the Assay preparation and the Standard preparation, respectively.