Medium: water; 500 mL.

Apparatus 2: 75 rpm.

Time: 60 minutes.

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

Standard solution— Dissolve an accurately weighed quantity of about 25 mg of USP Ubidecarenone RS in 1 mL of ethyl ether, and dilute quantitatively and stepwise with alcohol to obtain a solution having a known concentration of about 2.5 µg per mL. Use a freshly prepared solution only.

Test solution— Dilute quantitatively and stepwise with alcohol an accurately measured volume of the solution under test, previously passed through a suitable 0.45-µm filter, to obtain a solution having a concentration of about 2.5 µg of ubidecarenone per mL.

Procedure— Separately inject equal volumes (about 100 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the percentage of C59H90O4 dissolved by the formula: in which 500 is the volume, in mL, of Medium; C is the concentration, in mg per mL, of USP Ubidecarenone RS in the Standard solution; D is the dilution factor used to prepare the Test solution; rU and rS are the peak areas of ubidecarenone obtained from the Test solution and the Standard solution, respectively; 100 is the conversion factor to percentage; and LC is the label claim, in mg per Capsule.

Tolerances— Not less than 75% of the labeled amount of C59H90O4 is dissolved in 60 minutes.

Solvent— Prepare a mixture of n-hexane and dehydrated alcohol (5:2).

Mobile phase— Prepare a filtered and degassed mixture of acetonitrile, tetrahydrofuran, and water (55:40:5). Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Dissolve an accurately weighed quantity of USP Ubidecarenone RS in Solvent to obtain a solution having a known concentration of 1.0 mg per mL. Dilute a portion of this solution with dehydrated alcohol to obtain a solution having a known concentration of about 40 µg per mL.

Resolution solution— Dissolve an accurately weighed quantity of USP Ubidecarenone Related Compound A RS in Solvent to obtain a solution having a concentration of 1.0 mg per mL. Dilute a portion of this solution with dehydrated alcohol to obtain a solution having a concentration of about 40 µg per mL. Mix equal volumes of this solution and the Standard preparation.

Assay preparation 1 (for soft gelatin Capsules)— Using a suitable cutting instrument, open an accurately counted number of Capsules, equivalent to about 200 mg of ubidecarenone. Quantitatively transfer the shells and contents to a suitable container, add 100 mL of Solvent, and shake by mechanical means for 30 minutes. Using small portions of Solvent, quantitatively transfer this mixture to a 200-mL volumetric flask, dilute with Solvent to volume, and mix. Centrifuge a portion of this solution, transfer 1.0 mL of the supernatant to a 25-mL volumetric flask, add 2.5 mL of a 0.1% solution of anhydrous ferric chloride in alcohol, dilute with alcohol to volume, and mix.

Assay preparation 2 (for hard gelatin Capsules)— Empty and thoroughly mix the contents of not fewer than 20 Capsules. Transfer an accurately weighed portion of the powder, equivalent to about 100 mg of ubidecarenone, to a 100-mL volumetric flask, add 60 mL of Solvent, and shake by mechanical means for 30 minutes. Dilute with Solvent to volume, and mix. Centrifuge a portion of this solution, transfer 1.0 mL of the supernatant to a 25-mL volumetric flask, add 2.5 mL of a 0.1% solution of anhydrous ferric chloride in alcohol, dilute with alcohol to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 280-nm detector and an 8-mm × 10-cm column that contains packing L1. The flow rate is about 2.5 mL per minute. Chromatograph the Resolution solution, and record the peak responses as directed for Procedure: the resolution, R, between ubidecarenone and ubidecarenone related compound A is not less than 2.5. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: 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 15 µL) of the Standard preparation and Assay preparation 1 or Assay preparation 2 into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of ubidecarenone (C59H90O4) in each soft gelatin Capsule taken by the formula: in which C is the concentration, in mg per mL, of USP Ubidecarenone RS in the Standard preparation; N is the number of Capsules taken to prepare Assay preparation 1; and rU and rS are the peak responses obtained from Assay preparation 1 and the Standard preparation, respectively. Calculate the quantity, in mg, of ubidecarenone (C59H90O4) in the portion of hard gelatin Capsules taken by the formula: in which rU is the peak response obtained from Assay preparation 2; and the other terms are as defined herein.