Limit of N-isobutylpiperidone—
Prepare a test solution of Rifabutin in a mixture of chloroform and methanol (1:1) containing 10 mg per mL. Prepare a series of Standard solutions of
N-isobutylpiperidone in a mixture of chloroform and methanol (1:1) containing 0.005, 0.01, 0.02, 0.05, and 0.1 mg per mL, respectively. Separately apply 10-µL spots of the test solution and the Standard solutions to the starting line of a thin-layer chromatographic plate (see
Chromatography 
621
) coated with a 0.25-mm layer of chromatographic silica gel mixture, and allow to dry. Develop the chromatograms in a solvent system consisting of a mixture of hexanes and acetone (100:30) in an equilibrated unlined chromatographic chamber until the solvent front has moved about three-fourths of the length of the plate. Remove the plate from the chromatographic chamber, allow the plate to air-dry, and place it in an iodine vapor chamber until the spots are visible (about 5 minutes). Remove the plate from the chamber, spray the plate with
starch TS, and examine the plate: no spot in the chromatogram of the test solution at an
RF value corresponding to that of
N-isobutylpiperidone is more intense than that of the principal spot observed in the chromatogram obtained from the Standard solution containing 0.05 mg of
N-isobutylpiperidone per mL (0.5%).
Chromatographic purity—
Using the chromatogram of the
Assay preparation obtained as directed in the
Assay, calculate the percentage of impurities by the formula:
100(ri / rS),
in which
ri is the response of an individual impurity and
rS is the sum of the responses of all peaks: any impurity peak detected at a retention time of about 0.5, 0.6, 0.8, or 1.4 relative to the retention time of the rifabutin peak does not exceed 1.0%, not more than 0.5% of any other impurity is detected, and the total of all impurity peaks is not more than 3.0%.
Assay—
0.1 M Monobasic potassium phosphate—
Prepare a solution containing 13.6 g of monobasic potassium phosphate per L.
Mobile phase—
Prepare a mixture of acetonitrile and
0.1 M Monobasic potassium phosphate (50:50). Adjust by dropwise addition of 2 N sodium hydroxide to a pH of 6.5 ± 0.1. Filter through a 0.5-µm or finer porosity filter, and degas. Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Standard preparation—
Transfer about 25 mg of
USP Rifabutin RS, accurately weighed, to a 50-mL volumetric flask. Add 5 mL of acetonitrile, dilute with
Mobile phase to volume, and mix.
Assay preparation—
Transfer about 25 mg of Rifabutin, accurately weighed, to a 50-mL volumetric flask. Add 5 mL of acetonitrile, dilute with Mobile phase to volume, and mix.
Resolution solution—
Dissolve about 10 mg of Rifabutin and 2 mL of methanol, add 1 mL of 2 N sodium hydroxide, and allow to stand for about 4 minutes. Add 1 mL of 2 N hydrochloric acid, and dilute with Mobile phase to 50 mL. [NOTE—Portions of this solution may be stored in the frozen state for future use.]
Chromatographic system
(see
Chromatography 621)—The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm × 12.5-cm column that contains 5-µm diameter packing L7. The flow rate is about 1 mL per minute. Chromatograph the
Resolution solution, and record the peak responses as directed for
Procedure: the chromatogram exhibits a major peak for a degradant, two minor peaks for degradants, and a major peak for rifabutin at relative retention times of about 0.5, 0.6, 0.8, and 1.0, respectively. The resolution,
R, between the rifabutin peak and the degradant peak eluting at a relative retention time of about 0.8 is not less than 1.3. Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the column efficiency is not less than 2000 theoretical plates, and the relative standard deviation for replicate injections is not more than 2.0%.
Procedure—
Separately inject equal volumes (about 10 µL) of the
Standard preparation and the
Assay preparation into the chromatograph, record the chromatograms for a period of time that is twice the retention time of the major rifabutin peak, and measure the area responses for the major peaks. Calculate the quantity, in µg, of C
46H
62N
4O
11 in each mg of Rifabutin taken by the formula:
50(CP / W)(rU / rS),
in which
C is the concentration, in mg per mL, of
USP Rifabutin RS in the
Standard preparation,
P is the designated potency, in µg per mg, of
USP Rifabutin RS,
W is the weight, in mg, of Rifabutin taken to prepare the
Assay preparation, and
rU and
rS are the rifabutin peak area responses obtained from the
Assay preparation and the
Standard preparation, respectively.
;)
