Atovaquone
(a toe' va kwone).
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366.841,4-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 97.5 percent and not more than 101.5 percent of C22H19ClO3, calculated on the anhydrous and organic solvent-free basis.
Packaging and storage—
Preserve in tight, light-resistant containers.
USP Reference standards 
11
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11—
USP Atovaquone RS 
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USP Atovaquone Related Compound A RS
cis-2[4-(4-Chlorophenyl)cyclohexyl]-3-hydroxy-1,4-naphthoquinone.
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cis-2[4-(4-Chlorophenyl)cyclohexyl]-3-hydroxy-1,4-naphthoquinone.
Identification—
B:
The retention time of the major peak in the chromatogram of the Assay preparation corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay.
Water, Method I 921:
not more than 1.0%.
921:
not more than 1.0%.
Residue on ignition 281:
not more than 0.1%.
281:
not more than 0.1%.
Heavy metals—
Test preparation—
Thoroughly mix 1.0 g of Atovaquone with 0.5 g of magnesium oxide in a silica crucible. Ignite to dull redness until a homogeneous white or grayish-white mass is obtained. If the mixture remains colored after 30 minutes, 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 1 hour. Cool, take up the residue in two 5-mL portions of 6 N hydrochloric acid, add 0.1 mL of phenolphthalein TS, and then add 13.5 N ammonium hydroxide until a pink color is obtained. Cool, add glacial acetic acid until the solution is decolorized, and add 0.5 mL in excess. Filter, if necessary, and wash the filter with water. Dilute with water to 20 mL.
for about 1 hour. Cool, take up the residue in two 5-mL portions of 6 N hydrochloric acid, add 0.1 mL of phenolphthalein TS, and then add 13.5 N ammonium hydroxide until a pink color is obtained. Cool, add glacial acetic acid until the solution is decolorized, and add 0.5 mL in excess. Filter, if necessary, and wash the filter with water. Dilute with water to 20 mL.
Standard preparation—
Add 1.0 mL of Standard Lead Solution (see Special Reagents under Heavy Metals 231) to 0.5 g of magnesium oxide, and dry between 100 and 105. Proceed as directed for Test preparation, starting with “Ignite to dull redness”.
231) to 0.5 g 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 12.0 mL of the Test preparation to a 50-mL color-comparison tube, 10.0 mL of the Standard preparation to another, and 10.0 mL of the Blank preparation to a third. Then add 2.0 mL of the Test preparation to the Standard preparation as well as to the Blank preparation. Add 2 mL of pH 3.5 Acetate Buffer (see Heavy Metals 231) to each of the three tubes, mix, add 1.2 mL of thioacetamide–glycerin base TS, and mix. Allow to stand for 2 minutes, and view downward over a white surface: the solution from the Standard preparation is slightly brown when compared with the solution from the Blank preparation, and the color of the solution from the Test preparation is not darker than that of the solution from the Standard preparation (10 µg per g).
231) to each of the three tubes, mix, add 1.2 mL of thioacetamide–glycerin base TS, and mix. Allow to stand for 2 minutes, and view downward over a white surface: the solution from the Standard preparation is slightly brown when compared with the solution from the Blank preparation, and the color of the solution from the Test preparation is not darker than that of the solution from the Standard preparation (10 µg per g).
Limit of residual organic solvents—
Standard solution—
Transfer 1.0 mL of methanol and 1.0 mL of glacial acetic acid to a 100-mL volumetric flask, dilute with dimethylformamide to volume, and mix. Transfer 5.0 mL of this solution to a second 100-mL volumetric flask, dilute with dimethylformamide to volume, and mix.
Test solution—
Transfer about 100 mg of Atovaquone, accurately weighed, to a 2-mL volumetric flask, dissolve in and dilute with dimethylformamide to volume, and mix.
Chromatographic system (see Chromatography 621)—
The gas chromatograph is equipped with a flame-ionization detector and a 4-mm × 2.8-m column that contains 10% liquid phase G16 on support S2. The carrier gas is nitrogen, flowing at a rate of about 42.5 mL per 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 0.4 for methanol and 1.0 for 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.
621)—
The gas chromatograph is equipped with a flame-ionization detector and a 4-mm × 2.8-m column that contains 10% liquid phase G16 on support S2. The carrier gas is nitrogen, flowing at a rate of about 42.5 mL per 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 0.4 for methanol and 1.0 for 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 1 µL) of the Standard solution and the Test solution into 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 G is either 0.79, the specific gravity of methanol, or 1.05, the specific gravity of glacial acetic acid, as appropriate; W is the weight, in mg, of Atovaquone taken to prepare the Test solution; and rU and rS are the peak area responses of methanol or acetic acid, as appropriate, obtained from the Test solution and 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 preparation and the Resolution solution obtained in the Assay, calculate the percentage of atovaquone related compounds in the portion of Atovaquone taken by the formula:
100(ri / rs)
in which ri is the individual peak response of a related compound, if any, in the chromatogram of the Assay preparation; and rs is 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 0.63 or 1.8 relative to that of atovaquone is found; and not more than 0.3% of any related compound with a retention time of 0.89 relative 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 Suitability under Chromatography 621).
621).
Diluent—
Prepare a mixture of acetonitrile and water (80:20).
Standard preparation—
Dissolve an accurately weighed quantity of USP Atovaquone RS in Diluent, and dilute quantitatively, and stepwise if necessary, with Diluent to obtain a solution having a known concentration of about 0.25 mg per mL.
Resolution solution—
Prepare a solution in Diluent containing about 0.25 mg of USP Atovaquone RS and 0.02 mg of USP Atovaquone Related Compound A RS per mL. Store in a low-actinic glass container.
Assay preparation—
Transfer about 25 mg of Atovaquone, accurately weighed, to a low-actinic, 100-mL volumetric flask, dissolve in and dilute with Diluent to volume, and mix.
Chromatographic system (see Chromatography 621)—
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 3 mL per minute. Chromatograph the Resolution solution, and record the peak areas as directed for Procedure: the relative retention times are about 0.85 for atovaquone related compound A and 1.0 for atovaquone; and the resolution, R, between atovaquone related compound A and atovaquone is not less than 4. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the column efficiency is not less than 9000 theoretical plates; the tailing factor is not more than 1.5; and the relative standard deviation for replicate injections is not more than 2%.
621)—
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 3 mL per minute. Chromatograph the Resolution solution, and record the peak areas as directed for Procedure: the relative retention times are about 0.85 for atovaquone related compound A and 1.0 for atovaquone; and the resolution, R, between atovaquone related compound A and atovaquone is not less than 4. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the column efficiency is not less than 9000 theoretical plates; the tailing factor is not more than 1.5; and the relative standard deviation for replicate injections is not more than 2%.
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 C22H19ClO3 in the portion of Atovaquone taken by the formula:
100C(rU / rS)
in which C is the concentration, in mg per mL, of USP Atovaquone RS in the Standard preparation; and rU and rS are the atovaquone peak areas obtained from the Assay preparation and the Standard preparation, respectively.
Auxiliary Information—
Please check for your question in the FAQs before contacting USP.
| Topic/Question | Contact | Expert Committee |
|---|---|---|
| Monograph | Behnam Davani, Ph.D., M.B.A.
Senior Scientific Liaison 1-301-816-8394 |
(SM12010) Monographs - Small Molecules 1 |
| Reference Standards | RS Technical Services 1-301-816-8129 rstech@usp.org |
USP35–NF30 Page 2265
Pharmacopeial Forum: Volume No. 34(2) Page 247