Mobile phase— Prepare a mixture of n-hexane and dehydrated alcohol (4000:1). Make adjustments, if necessary (see System Suitability under Chromatography 621).

Reference solution 1— Dissolve an accurately weighed quantity of USP Probucol Related Compound A RS in n-hexane, and dilute quantitatively and stepwise with n-hexane to obtain a solution having a known concentration of about 10 µg per mL.

Reference solution 2— Dissolve an accurately weighed quantity of USP Probucol Related Compound B RS in n-hexane, and dilute quantitatively with n-hexane to obtain a solution having a known concentration of about 0.1 mg per mL.

Reference solution 3— Dissolve an accurately weighed quantity of USP Probucol Related Compound C RS in n-hexane, and dilute quantitatively with n-hexane to obtain a solution having a known concentration of about 1 mg per mL.

Standard solution— Transfer about 10 mg of USP Probucol RS, accurately weighed, to a 50-mL volumetric flask. Add 1 mL of Reference solution 1, 4 mL of Reference solution 2, and 10 mL of Reference solution 3, and dilute with n-hexane to volume.

Test solution— Transfer about 1 g of Probucol, accurately weighed, to a 25-mL volumetric flask, dissolve in and dilute with n-hexane to volume, and mix.

System suitability solution— Pipet 1 mL of Reference solution 2 and 1 mL of the Test solution into a 200-mL volumetric flask, dilute with n-hexane to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with 254-nm and 420-nm detectors, connected in series, and a 4.6-mm × 25-cm column that contains packing L3. The flow rate is about 1 mL per minute. Chromatograph the System suitability solution, and record the peak responses detected at 254 nm, for related compound B and probucol. Related compound B elutes first; the resolution, R, of the peaks is not less than 2.5; and the relative standard deviation for replicate injections, determined from the probucol peak, is not more than 2%.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the peak areas. The order of elution is compound C, compound B, compound A, and finally probucol. Compound A is detected at 420 nm, and the others are detected at 254 nm. Calculate the percentage of each related compound in the portion of Probucol taken by the formula: in which C is the concentration, in mg per mL, of the respective USP Probucol Related Compound RS in the Standard solution; W is the weight, in mg, of Probucol taken; and rU and rS are the peak areas obtained from the Test solution; and the Standard solution, respectively: not more than 0.0005% of compound A; not more than 0.02% of compound B; and not more than 0.5% of compound C are found.

Solvent— Use dimethyl sulfoxide.

(Official January 1, 2007)

Mobile phase— Prepare a degassed and filtered mixture of acetonitrile and water (85:15). Make adjustments, if necessary (see System Suitability under Chromatography 621).

System suitability preparation— To about 56 mg of Probucol add 10 mL of n-propyl alcohol, and dissolve the Probucol. Add 1.0 mL of 70% tert-butyl hydroperoxide, and mix. Cover loosely, and heat on a steam bath at about 90 for 30 minutes. Allow to cool to room temperature, dilute with a mixture of n-propyl alcohol and water (17:14) to 200 mL, and mix. Dilute 25 mL of this solution with Mobile phase to 100 mL.

Standard preparation— Dissolve an accurately weighed quantity of USP Probucol RS in Mobile phase, and dilute quantitatively and stepwise with Mobile phase to obtain a solution having a known concentration of about 63 µg per mL.

Assay preparation— Transfer about 63 mg of Probucol, accurately weighed, to a 50-mL volumetric flask. Dissolve in and dilute with Mobile phase to volume, and mix. Pipet 5 mL of this solution into a 100-mL volumetric flask, dilute with Mobile phase to volume, and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 242-nm detector and a 4.6-mm × 25-cm column that contains packing L7. The flow rate is about 2.0 mL per minute. Chromatograph the System suitability preparation, and record the peaks for the degradation product and probucol at relative retention times of approximately 0.8 and 1.0, respectively. The resolution, R, of the peaks is not less than 2.0. Chromatograph the Standard preparation: the relative standard deviation for replicate injections is not more than 1.0%.

Procedure— Separately inject equal volumes (about 50 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 C31H48O2S2 in the portion of Probucol taken by the formula: in which C is the concentration, in µg per mL, of USP Probucol RS in the Standard preparation; and rU and rS are the peak areas obtained from the Assay preparation and the Standard preparation, respectively.