Atovaquone, chemical structure, molecular formula, Reference Standards

Atovaquone

C22H19ClO3 366.84
1,4-Naphthalenedione,2-[4-(4-chlorophenyl)cyclohexyl]-3-hydroxy-,trans-.
2-[trans-4-(p-Chlorophenyl)cyclohexyl]-3-hydroxy-1,4-naphthoquinone [95233-18-4].

»Atovaquone contains not less than 97.5percent and more than 101.5percent of C22H19ClO3,calculated on the anhydrous and organic solvent-free basis.

Packaging and storage— Preserve in tight,light-resistant containers.

USP Reference standards á11ñ— USP Atovaquone RS.USP Atovaquone Related Compound A RS.

Identification—

A: Infrared Absorption á197Mñ.

B: 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.

Water,Method Iá921ñ: not more than 1.0%.

Residue on ignition á281ñ: not more than 0.1%.

Heavy metals—

Test preparation— Mix 1.0g of Atovaquone with 0.5g of magnesium oxide thoroughly in a silica crucible.Ignite to dull redness until a homogeneous white or grayish white mass is obtained.If the mixture remains colored after 30minutes,allow to cool,mix using a fine glass rod,and repeat the ignition.If necessary,repeat the operation.Heat the residue at 800

for about 1hour.Cool,take up the residue in two 5-mLportions of 6Nhydrochloric acid,add 0.1mLof phenolphthalein TS,and then add 13.5Nammonium hydroxide until a pink color is obtained.Cool,add glacial acetic acid until the solution is decolorized,and add 0.5mLin excess.Filter,if necessary,and wash the filter with water.Dilute with water to 20mL.

Standard preparation— Add 1.0mLof Standard Lead Solution(see Special Reagentsunder Heavy Metals á231ñ)to 0.5g of magnesium oxide,and dry between 100

and 105

.Proceed as directed for Test preparation,starting with “Ignite to dull redness”.

Blank preparation— Proceed as directed for Test preparation,omitting the Atovaquone.

Procedure— Transfer 12.0mLof the Test preparationto a 50-mLcolor-comparison tube,10.0mLof the Standard preparationto another,and 10.0mLof the Blank preparationand 2.0mLof the Test preparationto a third.Add 2mLof pH3.5Acetate Buffer(see Heavy Metals á231ñ)to each of the three tubes,mix,add 1.2mLof thioacetamide-glycerin base TS,and mix.Allow to stand for 2minutes,and view downward over a white surface:the solution from the Standard preparationis slightly brown when compared with the solution from the Blank preparation,and the color of the solution from the Test preparationis not darker than that of the solution from the Standard preparation(10µg per g).

Limit of residual organic solvents—

Standard solution— Transfer 1.0mLof methanol and 1.0mLof glacial acetic acid to a 100-mLvolumetric flask,dilute with dimethylformamide to volume,and mix.Transfer 5.0mLof this solution to a second 100-mLvolumetric flask,dilute with dimethylformamide to volume,and mix.

Test solution— Transfer about 100mg of Atovaquone,accurately weighed,to a 2-mLvolumetric flask,dissolve in and dilute with dimethylformamide to volume,and mix.

Chromatographic system (see Chromatography á621ñ)— The gas chromatograph is equipped with a flame-ionization detector and a 4-mm ×2.8-m column that contains 10%liquid phase G16on support S2.The carrier gas is nitrogen,flowing at a rate of about 42.5mLper minute.The column temperature is maintained at about 180

and the detector block temperature is maintained at about 250

.Chromatograph the Standard solution,and record the peak responses as directed for Procedure:the relative retention times are about 0.4for methanol and 1.0for acetic acid;the resolution,R,between methanol and acetic acid is not less than 14;the column efficiency calculated from the acetic acid peak is not less than 700;and the tailing factor for acetic acid is not less than 0.8.

Procedure— Separately inject equal volumes (about 1µL)of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms,and measure the peak areas for methanol and acetic acid.Calculate the percentage,by weight,of methanol and acetic acid in the portion of Atovaquone taken by the formula:

0.1(G/W)(rU/rS),

in which Gis either 0.79,the specific gravity of methanol,or 1.05,the specific gravity of glacial acetic acid,as appropriate;Wis the weight,in mg,of Atovaquone taken to prepare the Test solution;and rUand rSare the peak area responses of methanol or acetic acid,as appropriate,obtained from the Test solutionand the Standard solution,respectively:not more than 0.2%of methanol or of acetic acid is found.

Related compounds— Using the chromatograms of the Assay preparationand the Resolution solutionobtained in the Assay,calculate the percentage of atovaquone related compounds in the portion of Atovaquone taken by the formula:

100(ri/rs),

in which riis the individual peak response of a related compound,if any,in the chromatogram of the Assay preparation;and rsis the sum of the responses of all the peaks in the chromatogram of the Assay preparation,including the atovaquone peak.Not more than 1.0%of any related compound with a retention time corresponding to that of atovaquone related compound A,as determined from the chromatogram of the Resolution solution,is found;not more than 0.5%of any related compound with a retention time of 0.63or 1.8relative to that of atovaquone is found;and not more than 0.3%of any related compound with a retention time of 0.89relative to that of atovaquone is found.Not more than 0.2%of any other individual related compound is found;and the sum of all other such related compounds is not more than 1.0%.The sum of all related compounds is not more than 1.5%.

Assay—

Mobile phase— Prepare a mixture of acetonitrile,water,methanol,and phosphoric acid (525:300:175:5).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Diluent— Prepare a mixture of acetonitrile and water (80:20).

Standard preparation— Dissolve an accurately weighed quantity of USP Atovaquone RSin Diluent,and dilute quantitatively,and stepwise if necessary,with Diluentto obtain a solution having a known concentration of about 0.25mg per mL.

Resolution solution— Prepare a solution in Diluentcontaining about 0.25mg of USP Atovaquone RSand 0.02mg of USP Atovaquone Related Compound A RSper mL.Store in a low-actinic glass container.

Assay preparation— Transfer about 25mg of Atovaquone,accurately weighed,to a low-actinic,100-mLvolumetric flask,dissolve in and dilute with Diluentto volume,and mix.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 220-nm detector and a 4.6-mm ×25-cm column that contains packing L1.The flow rate is about 3mLper minute.Chromatograph the Resolution solution,and record the peak areas as directed for Procedure:the relative retention times are about 0.85for atovaquone related compound Aand 1.0for atovaquone;and the resolution,R,between atovaquone related compound Aand atovaquone is not less than 5.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the column efficiency is not less than 9000theoretical plates;the tailing factor is not more than 1.2;and the relative standard deviation for replicate injections is not more than 2%.

Procedure— Separately inject equal volumes (about 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the areas for the major peaks.Calculate the quantity,in mg,of C22H19ClO3in the portion of Atovaquone taken by the formula:

100C(rU/rS),

in which Cis the concentration,in mg per mL,of USP Atovaquone RSin the Standard preparation;and rUand rSare the atovaquone peak areas obtained from the Assay preparationand the Standard preparation,respectively.

Auxiliary Information— Staff Liaison:Behnam Davani,Ph.D.,MBA,Senior Scientist

Expert Committee:(PA7)Pharmaceutical Analysis 7

USP28–NF23Page 196

Phone Number:1-301-816-8394