Heavy metals—
Test preparation—
Mix 1.0 g of Atovaquone with 0.5 g 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 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.
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”.
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 and 2.0 mL of the
Test preparation to a third. 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).
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.
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).
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.
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 5. 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 1.2; 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 C
22H
19ClO
3 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.
;)
