A:Infrared Absorption á97Kñ.

B:Ultraviolet Absorption á197Uñ—

C: The retention time of the major peak in the chromatogram of the Assay preparationcorresponds to that in the chromatogram of the Standard preparation,as obtained in the Assay.

Test solution: 5mg per mL,in methanol.

Triethylamine phosphate solution,Mobile phase,System suitability solution,and Chromatographic system— Proceed as directed in the Assay.

Standard solution— Use the Standard preparation,prepared as directed in the Assay.

Test solution— Use the Assay preparation.

Procedure— Separately inject equal volumes (about 20µL)of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the peak responses.Calculate the percentage of each impurity in the portion of Saquinavir Mesylate taken by the formula: in which Fis a response factor and is equal to 2for peaks,if present,at a retention time of 0.32relative to saquinavir,to 0.5for peaks,if present,at retention times of about 0.38and 0.53relative to saquinavir,and to 1for all other peaks;CSis the concentration,in mg per mL,of USP Saquinavir Mesylate RSin the Standard solution;CUis the concentration,in mg per mL,of Saquinavir Mesylate in the Test solution;riis the peak response for each impurity obtained from the Test solution;and rSis the peak response for saquinavir obtained from the Standard solution:not more than 0.1%of any individual impurity is found;and not more than 0.5%of total impurities is found.

Triethylamine phosphate solution— Transfer 10mLof triethylamine to a 1-liter volumetric flask,dilute with water to volume,and mix.Adjust with phosphoric acid to a pHof 2.5,and filter.

Mobile phase— Prepare a filtered and degassed mixture of Triethylamine phosphate solution,tetrahydrofuran,and acetonitrile (14:5:1).[NOTE—Protect from light.]Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

System suitability solution— Dissolve suitable quantities of USP Saquinavir Related Compound A RSand USP Saquinavir Mesylate RSin Mobile phaseto obtain a solution containing about 2µg per mLand 0.25mg per mL,respectively.

Standard preparation— Dissolve an accurately weighed quantity of USP Saquinavir Mesylate RSin Mobile phase,and dilute quantitatively,and stepwise if necessary,with Mobile phaseto obtain a solution having a known concentration of about 0.25mg per mL.

Assay preparation— Transfer about 12.5mg of Saquinavir Mesylate,accurately weighed,to a 50-mLvolumetric flask,dissolve in and dilute with Mobile phaseto volume,and mix for about 20minutes.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 210-nm detector and a 4.6-mm ×25-cm column that contains packing L1.The column temperature is maintained at 20,and the flow rate is about 1mLper minute.Chromatograph the System suitability solution,and record the peak responses as directed for Procedure:the relative retention times are about 0.89for saquinavir related compound Aand 1.0for saquinavir;and the resolution,R,between saquinavir related compound Aand saquinavir is not less than 1.5.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the column efficiency is not less than 500theoretical plates;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of C38H50N6O5·CH4O3Sin the portion of Saquinavir Mesylate taken by the formula: in which Cis the concentration,in mg per mL,of USP Saquinavir Mesylate RSin the Standard preparation;and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.