Identification—
A: Ultraviolet Absorption 
197U
—
Medium:
a mixture of methanol and water (1:1).
Solution—
Transfer 5.0 mL of the Assay preparation and 5.0 mL of the Standard preparation, prepared in the Assay, to separate 50-mL volumetric flasks, dilute with Medium to volume, and mix.
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.
Sedimentation—
FOR ORAL SUSPENSION PACKAGED IN MULTIPLE-UNIT CONTAINERS—
Transfer 50 mL of well-mixed Oral Suspension to a glass-stoppered graduated cylinder, and allow to stand for 16 hours. Measure the volume, if any, of clear liquid observed in the cylinder: not more than 1 mL of clear liquid is found.
Related compounds—
Using the chromatograms of the
Resolution solution, the
Standard preparation, and the
Assay preparation obtained in the
Assay, calculate the percentage of atovaquone-related compounds, based on the labeled strength of atovaquone, by the formula:
in which
C is the concentration, in mg per mL, of
USP Atovaquone RS in the
Standard preparation; D is the density of Oral Suspension, in g per mL (1.04 g per mL at 20
to 25
);
S is the weight, in g, of Oral Suspension taken to prepare the
Assay preparation; L is the labeled amount, in mg per mL, of atovaquone in the Oral Suspension;
Fi is the response factor of an individual atovaquone related compound relative to the response of atovaquone, specifically, 1.08 for any peak observed at a relative retention time of about 0.65, 0.85 for any peak observed at a retention time corresponding to that of atovaquone related compound A, as determined from the chromatogram of the
Resolution solution, and 1.0 for any other related compound peak;
ri is the individual peak response of an atovaquone related compound, if any, in the chromatogram of the
Assay preparation; and
rS is the peak response of atovaquone in the chromatogram of the
Standard preparation. Disregard any peak having a relative retention time of about 0.3, which is due to photodegradation during preparation of the
Assay preparation. Not more than 0.5% of an atovaquone related compound with a relative retention time of about 0.65 is found; not more than 1.0% of atovaquone related compound A is found; not more than 0.3% of an atovaquone related compound with a relative retention time of about 0.88 is found; not more than 0.2% of any other atovaquone related compound is found; and the sum of all related compounds is not more than 2.0%.
Assay—
Mobile phase—
Prepare a mixture of acetonitrile, water, methanol, and phosphoric acid (480:360:160:5). Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Standard preparation—
Transfer about 30 mg of
USP Atovaquone RS, accurately weighed, to a low-actinic 10-mL volumetric flask, and add 2 mL of water and 6 mL of 0.1 M methanolic sodium hydroxide. Sonicate for about 5 minutes or until the material has dissolved. Allow to cool, dilute with 0.1 M methanolic sodium hydroxide to volume, and mix. Transfer 3.0 mL of this solution to a low-actinic 100-mL volumetric flask, dilute with a mixture of methanol and water (1:1), and mix.
[NOTE—Minimize exposure of this solution to light.
]
Assay preparation—
Transfer approximately 5.2 g of the well-mixed Oral Suspension, accurately weighed, to a low-actinic 250-mL volumetric flask. Add 50 mL of water, swirl for about 5 minutes, add 150 mL of 0.1 M methanolic sodium hydroxide, and sonicate for about 15 minutes. Allow to cool, dilute with 0.1 M methanolic sodium hydroxide to volume, and mix. Immediately filter a 20-mL portion, discarding the first 5 mL of the filtrate. Transfer 3.0 mL of the clear filtrate to a low-actinic 100-mL volumetric flask, dilute with a mixture of methanol and water (1:1) to volume, and mix. [NOTE—Minimize exposure of this solution to light.]
Chromatographic system (see Chromatography 621)—
The liquid chromatograph is equipped with a 220-nm detector and a 4.6-mm × 12.5-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 0.86 for atovaquone related compound A and 1.0 for atovaquone. Chromatograph the
Standard preparation, and record the peak areas as directed for
Procedure: the tailing factor is not more than 1.5; and the relative standard deviation for replicate injections is not more than 2.0%.
Procedure—
Separately inject equal volumes (about 20 µL) of the
Standard preparation, the
Resolution solution, and the
Assay preparation into the chromatograph, record the chromatograms, and measure the areas for the major peaks. Calculate the quantity, in mg, of atovaquone (C
22H
19ClO
3) in each mL of the Oral Suspension taken by the formula:
(25,000/3)(C/V)(rU / rS),
in which
C is the concentration, in mg per mL, of
USP Atovaquone RS in the
Standard preparation; V is the volume, in mL, of Oral Suspension taken to prepare the
Assay preparation; and
rU and
rS are the atovaquone peak areas obtained from the
Assay preparation and the
Standard preparation, respectively.
;)