[Federal Register Volume 59, Number 153 (Wednesday, August 10, 1994)]
[Unknown Section]
[Page 0]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 94-19484]


[[Page Unknown]]

[Federal Register: August 10, 1994]


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DEPARTMENT OF HEALTH AND HUMAN SERVICES
21 CFR Parts 430, 436, and 455

[Docket No. 94N-0184]

 

Antibiotics Drugs; Rifabutin and Rifabutin Capsules

AGENCY: Food and Drug Administration, HHS.

ACTION: Final rule.

-----------------------------------------------------------------------

SUMMARY: The Food and Drug Administration (FDA) is amending the 
antibiotic drug regulations to include accepted standards for a new 
antibiotic drug, rifabutin, and the use of the antibiotic drug in a 
dosage form, rifabutin capsules. The manufacturer has supplied 
sufficient data and information to establish its safety and efficacy.

DATES: Effective September 9, 1994; written comments, notice of 
participation, and requests for a hearing by September 9, 1994; data, 
information, and analyses to justify a hearing by October 11, 1994.

ADDRESSES: Submit written comments to the Dockets Management Branch 
(HFA-305), Food and Drug Administration, rm. 1-23, 12420 Parklawn Dr., 
Rockville, MD 20857.

FOR FURTHER INFORMATION CONTACT: James Timper, Center for Drug 
Evaluation and Research (HFD-520), Food and Drug Administration, 5600 
Fishers Lane, Rockville, MD 20857, 301-443-6714.

SUPPLEMENTARY INFORMATION: FDA has evaluated data submitted in 
accordance with regulations promulgated under section 507 of the 
Federal Food, Drug, and Cosmetic Act (21 U.S.C. 357), as amended, with 
respect to a request for approval of (1) a new antibiotic drug, 
rifabutin, and (2) its use in a dosage form, rifabutin capsules. The 
agency has concluded that the data supplied by the manufacturer 
concerning these antibiotic drugs are adequate to establish their 
safety and efficacy when used as directed in the labeling and that the 
regulations should be amended in 21 CFR parts 430, 436, and 455 to 
include accepted standards for these products.

Environmental Impact

    The agency has determined under 21 CFR 25.24(c)(6) that this action 
is of a type that does not individually or cumulatively have a 
significant effect on the human environment. Therefore, neither an 
environmental assessment nor an environmental impact statement is 
required.

Submitting Comments and Filing Objections

    This final rule announces standards that FDA has accepted in a 
request for approval of an antibiotic drug. Because this final rule is 
not controversial and because when effective it provides notice of 
accepted standards, FDA finds that notice and comment procedure is 
unnecessary and not in the public interest. This final rule, therefore, 
is effective September 9, 1994. However, interested persons may, on or 
before September 9, 1994, submit written comments to the Dockets 
Management Branch (address above). Two copies of any comments are to be 
submitted, except that individuals may submit one copy. Comments are to 
be identified with the docket number found in brackets in the heading 
of this document. Received comments may be seen in the Dockets 
Management Branch between 9 a.m. and 4 p.m., Monday through Friday.
    Any person who will be adversely affected by this final rule may 
file objections to it and request a hearing. Reasonable grounds for the 
hearing must be shown. Any person who decides to seek a hearing must 
file (1) on or before September 9, 1994, a written notice of 
participation and request for a hearing, and (2) on or before October 
11, 1994, the data, information, and analyses on which the person 
relies to justify a hearing, as specified in 21 CFR 314.300. A request 
for a hearing may not rest upon mere allegations or denials, but must 
set forth specific facts showing that there is a genuine and 
substantial issue of fact that requires a hearing. If it conclusively 
appears from the face of the data, information, and factual analyses in 
the request for a hearing that no genuine and substantial issue of fact 
precludes the action taken by this order, or if a request for a hearing 
is not made in the required format or with the required analyses, the 
Commissioner of Food and Drugs will enter summary judgment against the 
person(s) who request(s) the hearing, making findings and conclusions 
and denying a hearing. All submissions must be filed in three copies, 
identified with the docket number appearing in the heading of this 
document and filed with the Dockets Management Branch.
    The procedures and requirements governing this order, a notice of 
participation and request for a hearing, a submission of data, 
information, and analyses to justify a hearing, other comments, and 
grant or denial of a hearing are contained in 21 CFR 314.300.
    All submissions under this order, except for data and information 
prohibited from public disclosure under 21 U.S.C. 331(j) or 18 U.S.C. 
1905, may be seen in the Dockets Management Branch (address above) 
between 9 a.m. and 4 p.m., Monday through Friday.

List of Subjects

21 CFR Part 430

    Administrative practice and procedure, Antibiotics.

21 CFR Parts 436 and 455

    Antibiotics.
    Therefore, under the Federal Food, Drug, and Cosmetic Act and under 
authority delegated to the Commissioner of Food and Drugs, 21 CFR parts 
430, 436, and 455 are amended as follows:

PART 430--ANTIBIOTIC DRUGS; GENERAL

    1. The authority citation for 21 CFR part 430 continues to read as 
follows:

    Authority: Secs. 201, 501, 502, 503, 505, 507, 701 of the 
Federal Food, Drug, and Cosmetic Act (21 U.S.C. 321, 351, 352, 353, 
355, 357, 371); secs. 215, 301, 351 of the Public Health Service Act 
(42 U.S.C. 216, 241, 262).

    2. Section 430.4 is amended by adding new paragraph (a)(69) to read 
as follows:


Sec. 430.4  Definitions of antibiotic substances.

    (a) * * *
    (69) Rifabutin. Rifabutin is an antibiotic substance having the 
chemical structure described by the following name: 
(9S,12E,14S,15R,16S,17R,18R,19R, 20S,21S,22E, 24Z)-6,16, 18,20-
tetrahydroxy-1'-isobutyl-14-methoxy-7,9,15,17,19,21,25-
heptamethylspiro[9,4-(epoxypentadeca[1,11,13]trienimino)-2H-
furo[2',3':7,8]naphth[1,2-d]imidazole-2,4'-piperidine]-5,10,26-(3H,9H)-
trione-16-acetate.
* * * * *
    3. Section 430.5 is amended by adding new paragraphs (a)(104) and 
(b)(106) to read as follows:


Sec. 430.5  Definitions of master and working standards.

    (a) * * *
    (104) Rifabutin. The term ``rifabutin master standard'' means a 
specific lot of rifabutin that is designated by the Commissioner as the 
standard of comparison in determining the potency of the rifabutin 
working standard.
    (b) * * *
    (106) Rifabutin. The term ``rifabutin working standard'' means a 
specific lot of a homogeneous preparation of rifabutin.
    4. Section 430.6 is amended by adding new paragraph (b)(106) to 
read as follows:


Sec. 430.6  Definitions of the terms ``unit'' and ``microgram'' as 
applied to antibiotic substances.

* * * * *
    (b) * * *
    (106) Rifabutin. The term ``microgram'' applied to rifabutin means 
the rifabutin (potency) contained in 1.022 micrograms of the rifabutin 
master standard.

PART 436--TESTS AND METHODS OF ASSAY OF ANTIBIOTIC AND ANTIBIOTIC-
CONTAINING DRUGS

    5. The authority citation for 21 CFR part 436 continues to read as 
follows:

    Authority: Sec. 507 of the Federal Food, Drug, and Cosmetic Act 
(21 U.S.C. 357).

    6. Section 436.215 is amended by alphabetically adding a new entry 
to the table in paragraph (b) and by adding new paragraph (c)(18) to 
read as follows:


Sec. 436.215  Dissolution test.

* * * * *
    (b) * * *

--------------------------------------------------------------------------------------------------------------------------------------------------------
                   Dosage form                               Dissolution medium              Rotation rate\1\     Sampling time(s)        Apparatus     
--------------------------------------------------------------------------------------------------------------------------------------------------------
            *                    *                    *                    *                    *                    *                    *             
Rifabutin capsules                                         900 mL 0.01N hydrochloric acid                  100               45 min                    1
            *                    *                    *                    *                    *                    *                    *             
--------------------------------------------------------------------------------------------------------------------------------------------------------
\1\Rotation rate of basket or paddle stirring element (revolutions per minute).                                                                         

    (c) * * *
    (18) Rifabutin--(i) Preparation of the working standard solution. 
Accurately weigh approximately 45 milligrams of the rifabutin working 
standard into a suitable-sized volumetric flask. Dissolve and dilute to 
volume with 0.01N hydrochloric acid (prepared by diluting 5.0 
milliliters of hydrochloric acid (37 percent) to 6 liters with 
distilled water) to obtain a concentration of approximately 13 
micrograms rifabutin activity per milliliter.
    (ii) Preparation of sample solutions. Forty-five minutes after the 
beginning of the rotation, withdraw a 10-milliliter aliquot from the 
vessel. Dilute a 2-milliliter portion of the sample to 25 milliliters 
with 0.01N hydrochloric acid.
    (iii) Procedure. Using a suitable spectrophotometer and 0.01N 
hydrochloric acid as the blank, determine the absorbance of each 
standard and sample solution at the absorbance maximum at approximately 
280 nanometers. Determine the exact position of the absorbance maximum 
for the particular instrument used.
    (iv) Calculations. Determine the total amount of rifabutin 
dissolved as follows:

                                                                        
                                                                        
                                                    AU X c X d X 900    
           T                       =           -------------------------
                                                        AS X 1,000      
                                                                        

where:
T = Total milligrams of rifabutin activity dissolved;
AU = Absorbance of sample;
AS = Absorbance of the standard;
c = Rifabutin activity of the working standard solution in 
micrograms per milliliter; and
d = Dilution factor of the sample filtrate.
* * * * *
    7. New Secs. 436.369 and 436.370 are added to subpart F to read as 
follows:


Sec. 436.369  Thin layer chromatography test for free N-
isobutylpiperidone content in rifabutin.

    (a) Equipment--(1) Chromatography tank. A rectangular tank, 
approximately 23 X 23 X 9 centimeters, with a glass solvent trough on 
the bottom and a tight-fitting cover.
    (2) Iodine vapor chamber. A rectangular tank, approximately 23 X 23 
X 9 centimeters, with a suitable cover, containing iodine crystals.
    (3) Plates. Use 20 X 20 centimeter thin layer chromatography plates 
coated with silica gel 60F 254 or equivalent to a thickness of 250 
microns.
    (b) Reagents--(1) Developing solvent. Mix petroleum ether (b.p. 60 
to 80  deg.C) and acetone in volumetric proportions of 100:30, 
respectively.
    (2) Spray solution. Prepare a 1 percent solution of soluble starch 
in water (containing 0.01 percent mercuric iodide).
    (c) Preparation of spotting solutions--(1) Sample solution. Prepare 
a solution of the rifabutin sample in 1:1 chloroform/methanol to 
contain 10 milligrams per milliliter.
    (2) Standard solution. Prepare a solution of N-isobutylpiperidone 
standard in 1:1 chloroform/methanol to contain 1 milligram per 
milliliter. Transfer aliquots of 0.5, 1.0, 2.0, 5.0, and 10.0 
milliliters into separate 100-milliliter volumetric flasks and dilute 
to volume with 1:1 chloroform/methanol. These solutions contain, 
respectively, the equivalent of 0.05, 0.1, 0.2, 0.5, and 1.0 percent of 
N-isobutylpiperidone.
    (d) Procedure. Pour 100 milliliters of developing solvent into the 
glass trough on the bottom of the unlined chromatography tank. Cover 
and seal the tank. Allow it to equilibrate while the plate is being 
prepared. Prepare a plate as follows: on a line 2.0 centimeters from 
the base of the thin layer chromatography plate, and at intervals of 
2.0 centimeters, apply 10 microliters of each of the standard solutions 
and the sample solution prepared as directed above. After the spots are 
thoroughly dry, place the plate into the trough in the bottom of the 
tank. Cover and tightly seal the tank, allow the solvent front to 
travel about 15 centimeters from the starting line and then remove the 
plate from the tank. Air dry the plate. Warm the iodine vapor chamber 
to vaporize the iodine crystals and place the dry plate in the iodine 
vapor chamber until the spots are visible (usually about 5 minutes). 
Remove the plate from the iodine vapor chamber and spray with 1 percent 
starch solution.
    (e) Evaluation. Measure the distance the solvent front traveled 
from the starting line and the distance the spots are from the starting 
line. Calculate the Rf value by dividing the latter by the former. 
N-isobutylpiperidone has an Rf value of about 0.3. Rifabutin has 
an Rf value of about 0.1. Compare the size and intensity of any N-
isobutylpiperidone spots in the sample lane with the N-
isobutylpiperidone spots in the standard lanes, and report the 
percentage of N-isobutylpiperidone in the sample.


Sec. 436.370  Spectrophotometric identity test for rifabutin capsules.

    (a) Equipment. A suitable spectrophotometer capable of recording 
the ultraviolet spectrum in the 200 to 400 nanometer range, using 
suitable quartz cells of 1 centimeter pathlength.
    (b) Preparation of working standard and sample solution--(1) 
Working standard solution. Suspend approximately 200 milligrams of 
rifabutin working standard in 20 milliliters of methanol and sonicate 
for approximately 5 minutes. Filter the resulting solution through a 
suitable 0.5 micrometer filter. Transfer a 2-milliliter aliquot of the 
filtered solution to a 100-milliliter volumetric flask and fill to 
volume with methanol. Further dilute with methanol to obtain a solution 
containing 20 micrograms of rifabutin activity per milliliter.
    (2) Sample solution. Empty and combine the contents of five 
capsules. Suspend a quantity of the capsule contents equivalent to 200 
milligrams of rifabutin in 20 milliliters of methanol. Sonicate for 
about 5 minutes and then filter through an appropriate 0.5 micrometer 
filter. Transfer a 2-milliliter aliquot to a 100-milliliter volumetric 
flask and dilute to volume with methanol. Further dilute with methanol 
to obtain a solution containing 20 micrograms of rifabutin activity per 
milliliter (estimated).
    (c) Procedure. Using a suitable spectrophotometer equipped with 1.0 
centimeter cells and methanol as the blank, determine the absorbance 
spectra of the working standard and sample solutions over the 
ultraviolet range of 250 to 300 nanometers.
    (d) Evaluation. Compare the spectrum of the sample to that of the 
working standard. The identity of the rifabutin capsules is confirmed 
by quantitative comparison of the two spectra with an absorbance 
maximum being observed at about 275 nanometers.

PART 455--CERTAIN OTHER ANTIBIOTIC DRUGS

    8. The authority citation for 21 CFR part 455 continues to read as 
follows:

    Authority: Sec. 507 of the Federal Food, Drug, and Cosmetic Act 
(21 U.S.C. 357).

    9. New Sec. 455.88 is added to subpart A to read as follows:


Sec. 455.88  Rifabutin.

    (a) Requirements for certification--(1) Standards of identity, 
strength, quality, and purity. Rifabutin is an amorphous red-violet 
powder. It is (9S,12E,14S,15R,16S,17R,18R,19R,20S,21S,
22E,24Z)-
6,16,18,20-tetrahydroxy-1'-isobutyl-14-methoxy-
7,9,15,17,19,21,25-heptamethylspiro[9,4-(epoxypentadeca
[1,11,13]trienimino)-2H-furo[2',3':7,8] naphth[1,2-d]imidazole-2,4'-
piperidine]-5,10,26-(3H,9H)-trione-16-acetate. It is very slightly 
soluble in water, sparingly soluble in ethanol, and soluble in 
chloroform and methanol. It is so purified and dried that:
    (i) Its potency is not less than 950 micrograms and not more than 
1,020 micrograms of rifabutin activity per milligram on an anhydrous 
basis.
    (ii) Its content for the four major related substances detected by 
high-performance liquid chromatography (HPLC) is not more than 1.0 
percent each. All other unknown related substances are not more than 
0.5 percent. The total of all related substances is not more than 3.0 
percent.
    (iii) Its moisture content is not more than 2.5 percent.
    (iv) Its N-isobutylpiperidone content is not more than 0.5 percent.
    (v) It gives a positive identity test.
    (2) Labeling. It shall be labeled in accordance with the 
requirements of Sec. 432.5 of this chapter.
    (3) Requests for certification; samples. In addition to complying 
with the requirements of Sec. 431.1 of this chapter, each such request 
shall contain:
    (i) Results of tests and assays on the batch for rifabutin potency, 
related substances, moisture, N-isobutylpiperidone, and identity.
    (ii) Samples, if required by the Director, Center for Drug 
Evaluation and Research: 10 packages each containing approximately 300 
milligrams.
    (b) Tests and methods of assay--(1) Potency. Proceed as directed in 
Sec. 436.216 of this chapter, using ambient temperature, an ultraviolet 
detection system operating at a wavelength of 254 plus-minuss> 1 
nanometers, an 11 centimeters X 4.7 millimeters (i.d.) column packed 
with microparticulate (5 to 7 micrometers in diameter) packing material 
such as octylsilane chemically bonded to porous silica (U.S. 
Pharmacopeia designation L7), a flow rate of about 1.0 milliliter per 
minute, and a manual or automatic injector capable of injecting 10 
microliters. The retention time for rifabutin is between 9 and 11 
minutes. Reagents; working standard, sample, and resolution solutions; 
system suitability requirements; and calculations are as follows:
    (i) Reagents--(A) Hydrochloric acid, 2N. Dilute 85 milliliters of 
hydrochloric acid (37 percent) with distilled water to 500 milliliters.
    (B) Potassium dihydrogen phosphate, 0.1M. Prepare a solution 
containing 15.4 grams of potassium dihydrogen phosphate monohydrate 
(potassium phosphate monobasic) per liter of distilled water.
    (C) Sodium hydroxide, 2N. Dissolve 8 grams of sodium hydroxide 
pellets in 100 milliliters of distilled water.
    (D) Mobile phase. Acetonitrile:phosphate buffer, pH 6.5, 50:50. Mix 
equal quantities of acetonitrile and 0.1M potassium dihydrogen 
phosphate and adjust to an apparent pH of 6.5 plus-minuss> 0.1 by 
dropwise addition of 2N sodium hydroxide. Filter through a suitable 
filter capable of removing particulate matter 0.5 micron in diameter 
and degas it just prior to its introduction into the chromatograph. 
Slight adjustments of the mobile phase components ratio may be made in 
order to meet the system suitability requirements described in the 
system suitability tests in paragraph (b)(1)(iii) of this section.
    (ii) Preparation of working standard, sample, and resolution test 
solution--(A) Working standard solution. Accurately weigh approximately 
25 milligrams of the rifabutin working reference standard into a 50-
milliliter volumetric flask. Add 5 milliliters of acetonitrile. 
Dissolve and dilute to volume with mobile phase and mix to obtain a 
solution having a known concentration of about 0.5 milligram of 
rifabutin per milliliter.
    (B) Sample solution. Accurately weigh approximately 25 milligrams 
of sample into a 50-milliliter volumetric flask. Add 5 milliliters of 
acetonitrile. Dissolve and dilute to volume with mobile phase and mix 
to obtain a solution containing 0.5 milligram of rifabutin per 
milliliter (estimated).
    (C) Resolution test solution. Dissolve approximately 10 milligrams 
of rifabutin in 2 milliliters of methanol and add 1 milliliter of 2N 
sodium hydroxide. Allow to stand for 3 to 4 minutes and then add 1 
milliliter of 2N hydrochloric acid. Mix and dilute to 50 milliliters 
with mobile phase. Store aliquots of this solution in the frozen state 
for future use.
    (iii) System suitability requirements. Using the apparatus and 
conditions described in this section, test the chromatographic system 
by injecting the resolution test solution. The chromatogram shows one 
major degradation peak and two minor degradation peaks eluting at 
relative retention times (RRT) of 0.5-0.6, 0.65-0.75, and 0.8-0.9, 
respectively, followed by the rifabutin peak.
    (A) Asymmetry factor. The asymmetry factor (AS) is 
satisfactory if it is not less than 1.0 and not more than 4.0 for the 
rifabutin peak.
    (B) Efficiency of the column. The absolute efficiency (hr) is 
satisfactory if it is not more than 11 for the rifabutin peak, 
equivalent to 2,000 theoretical plates for a 11-centimeter column of 5-
micrometer particles.
    (C) Resolution factor. The resolution factor (R) between the peak 
for rifabutin and its closest eluting degradation product (generated in 
situ as described in paragraph (b)(1)(iii) of this section and eluting 
at RRT of 0.8-0.9) is satisfactory if it is not less than 1.3.
    (D) Coefficient of variation (relative standard deviation). The 
coefficient of variation (SR in percent of 5 replicate injections 
of the rifabutin working standard solution) is satisfactory if it is 
not more than 2.0 percent. If the system suitability parameters have 
been met, then proceed as described in Sec. 436.216(b) of this chapter.
    (iv) Calculations. Calculate the micrograms of rifabutin per 
milligram of sample on an anhydrous basis as follows:

                                                                        
                                                       AU X PS X 100    
Micrograms of rifabutin             =            -----------------------
     per milligram                                   AS X CU X (100-m)  
                                                                        

where:
AU = Area of the rifabutin peak in the chromatogram of the 
sample (at a retention time equal to that observed for the 
standard);
AS = Area of the rifabutin peak in the chromatogram of the 
rifabutin working standard;
PS = Rifabutin activity in the rifabutin working standard 
solution in micrograms per milliliter;
CU = Milligrams of sample per milliliter of sample solution; 
and
m = Percent moisture content of the sample.
    (2) Related substances. Proceed as directed in paragraph (b)(1) of 
this section for potency using the sample prepared as described in 
paragraph (b)(1)(ii)(B) of this section and calculating the amounts of 
related substances as follows.
    (i) Calculations. Calculate the percentage of related substances as 
follows:

                                                                        
                                                         Ai x 100       
Percent individual HPLC-            =            -----------------------
   related substance                                        At          
                                                                        


                                                                        
                                                          A x 100       
  Percent total HPLC-               =            -----------------------
   related substances                                       At          
                                                                        

where:
Ai = Area of the individual related substance peak;
A = The sum of areas of all peaks minus the area due to the 
rifabutin peak and solvent front peak; and
At = The sum of areas of all peaks in the chromatogram 
excluding the solvent peak.
    (ii) [Reserved]
    (3) Moisture. Proceed as directed in Sec. 436.201 of this chapter.
    (4) N-Isobutylpiperidone. Proceed as directed in Sec. 436.369 of 
this chapter.
    (5) Identity. (i) Proceed as directed in Sec. 436.211 of this 
chapter, using the sample preparation method described in paragraph 
(b)(1) of that section using a 1 to 2 percent mixture in potassium 
bromide.
    (ii) The identity of rifabutin is confirmed by the qualitative 
comparison of the HPLC of the sample to the rifabutin working standard 
as directed in paragraph (b)(1) of this section.
    10. New Sec. 455.188 is added to subpart B to read as follows:


Sec. 455.188  Rifabutin capsules.

    (a) Requirements for certification--(1) Standards of identity, 
strength, quality, and purity. Rifabutin capsules are gelatin capsules 
containing rifabutin with a suitable and harmless filler and with or 
without binders, lubricants, and stabilizers. Each capsule contains 
rifabutin equivalent to 150 milligrams of rifabutin. Its rifabutin 
content is satisfactory if it is not less than 90 percent and not more 
than 110 percent of the number of milligrams of rifabutin that it is 
represented to contain. Its content of the four major related 
substances detected by high-performance liquid chromatography (HPLC) is 
not more than 1.0 percent each. All other unknown related substances 
are not more than 0.5 percent. The total of all related substances is 
not more than 4.5 percent. It passes the dissolution test if the 
quantity (Q) dissolved is 75 percent at 45 minutes. It passes the 
identity test. The rifabutin used conforms to the standards prescribed 
by Sec. 455.88(a)(1).
    (2) Labeling. It shall be labeled in accordance with the 
requirements of Sec. 432.5 of this chapter.
    (3) Requests for certification; samples. In addition to complying 
with the requirements of Sec. 431.1 of this chapter, each such request 
shall contain:
    (i) Results of tests and assays on:
    (A) The rifabutin used in making the batch for potency, related 
substances, moisture, N-isobutylpiperidone, and identity.
    (B) The batch for content, related substances, dissolution, and 
identity.
    (ii) Samples, if required by the Director, Center for Drug 
Evaluation and Research:
    (A) The rifabutin used in making the batch: 10 packages, each 
containing approximately 300 milligrams.
    (B) The batch: A minimum of 30 capsules.
    (b) Tests and methods of assay--(1) Rifabutin content. Proceed as 
directed in Sec. 455.88(b)(1), preparing the sample solution and 
calculating the rifabutin content as follows:
    (i) Preparation of sample solution. Empty 20 capsules, collecting 
the contents quantitatively. Weigh the powder and determine the average 
capsule fill weight. Mix the powder and accurately weigh a portion 
containing the equivalent of about 25 milligrams of rifabutin into a 
50-milliliter volumetric flask. Add 5 milliliters of acetonitrile. 
Dilute to volume with mobile phase and mix to yield a solution 
containing 0.5 milligram of rifabutin per milliliter (estimated). 
Filter through a suitable filter capable of removing particulate matter 
0.5 micron in diameter prior to injection into the chromatographic 
system.
    (ii) Calculations. Calculate the rifabutin content as follows:

                                                                        
                                                     AU X CS X PS X Wa  
Milligrams of rifabutin             =            -----------------------
      per capsule                                     AS X CU X 1,000   
                                                                        

where:
AU = Area of the rifabutin peak in the chromatogram of the 
sample (at a retention time equal to that observed for the 
standard);
AS = Area of the rifabutin peak in the chromatogram of the 
rifabutin working standard;
CS = Milligrams of rifabutin working standard per milliliter of 
standard solution;
CU = Milligrams of sample per milliliter of sample solution;
PS = Rifabutin activity in the rifabutin working standard 
solution in micrograms per milliliter; and
Wa = Average capsule fill weight in milligrams.
    (2) Related substances. Proceed as directed in paragraph (b)(1) of 
this section for rifabutin content using the sample prepared as 
described in paragraph (b)(1)(i) of this section and calculating the 
amounts of related substances as follows.
    (i) Calculations. Calculate the percentage of related substances as 
follows:

                                                                        
                                                         Ai x 100       
Percent individual HPLC-            =            -----------------------
   related substance                                        At          
                                                                        


                                                                        
                                                          A x 100       
  Percent total HPLC-               =            -----------------------
   related substances                                       At          
                                                                        

where:
Ai = Area of the individual related substance peak;
A = The sum of areas of all peaks minus the area due to the 
rifabutin peak and solvent front peak; and
At = The sum of areas of all peaks in the chromatogram 
excluding the solvent peak.
    (ii) [Reserved]
    (3) Dissolution test. Proceed as directed in Sec. 436.215 of this 
chapter. The quantity (Q) (the amount of rifabutin activity dissolved) 
is 75 percent within 45 minutes.
    (4) Identity. (i) The retention time of the rifabutin response in 
the HPLC procedure described in paragraph (b)(1) of this section as 
applied to the sample solution compares qualitatively to that of the 
rifabutin reference standard.
    (ii) The identity of rifabutin capsules is also confirmed by the 
spectrophotometric identity test described in Sec. 436.370 of this 
chapter.

    Dated: August 1, 1994.
 Stephanie R. Gray,
 Acting Director, Office of Compliance, Center for Drug Evaluation and 
Research.
[FR Doc. 94-19484 Filed 8-9-94; 8:45 am]
BILLING CODE 4160-01-F