Rifabutin

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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1 DEFINITION

Rifabutin contains NLT 950 µg/mg and NMT 1020 µg/mg of rifabutin (C₄₆H₆₂N₄O₁₁), calculated on the anhydrous basis.

2 IDENTIFICATION

A. Spectroscopic Identification Tests 〈197〉, Infrared Spectroscopy: 197K

B. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.

3 ASSAY

Procedure

Solution A: 13.6 g/L of monobasic potassium phosphate

Mobile phase: Acetonitrile and Solution A (50:50).

Adjust by dropwise addition of 2 N sodium hydroxide to a pH of 6.5 ± 0.1.

System suitability solution:

Dissolve about 10 mg of Rifabutin in 2 mL of methanol, add 1 mL of 2 N sodium hydroxide, stand 4 min.

Add 1 mL of 2 N hydrochloric acid and dilute with Mobile phase to 50 mL.

[Note—Portions may be stored frozen.]

Standard solution: 0.5 mg/mL USP Rifabutin RS.

Add acetonitrile to fill 10% of flask volume, dilute with Mobile phase.

Sample solution: 0.5 mg/mL Rifabutin prepared similarly.

Chromatographic system

Mode: LC

Detector: UV 254 nm

Column: 4.6 mm × 12.5 cm, 5-µm L7

Flow rate: 1 mL/min

Injection volume: 10 µL

Run time: NLT 2× rifabutin peak retention time

System suitability

System suitability solution and Standard solution.

Chromatogram of suitability solution exhibits:

major degradant peak

two minor degradant peaks

rifabutin peak

with RRT ≈ 0.5, 0.6, 0.8, 1.0.

Requirements:

Resolution ≥ 1.3 (degradant at RRT 0.8 vs rifabutin)

RSD ≤ 2.0%, Standard solution

Analysis

Calculate rifabutin (µg/mg):

Result = (rᵤ / rₛ) × (Cₛ / Cᵤ) × P

rᵤ = peak response of Sample solution

rₛ = peak response of Standard solution

Cₛ = concentration of USP Rifabutin RS (mg/mL)

Cᵤ = concentration of Rifabutin (mg/mL)

P = potency of USP Rifabutin RS (µg/mg)

Acceptance criteria: 950–1020 µg/mg (anhydrous)

4 IMPURITIES

Organic Impurities – Procedure 1 (Limit of N-Isobutylpiperidone)

Diluent: Chloroform:methanol = 1:1

Standard solution 1: 0.1 mg/mL

Standard solution 2: 0.05 mg/mL

Standard solution 3: 0.02 mg/mL

Standard solution 4: 0.01 mg/mL

Standard solution 5: 0.005 mg/mL

Sample solution: 10 mg/mL Rifabutin in Diluent

TLC system:

Adsorbent: silica gel 0.25 mm

Application: 10 µL

Solvent: hexanes:acetone (100:30)

Visualization: iodine vapor → starch TS spray.

Acceptance criteria:

NMT 0.5%.

No Sample spot at matching Rf > Standard 2.

Organic Impurities – Procedure 2

Solution A: 0.1% triethylamine in water

Solution B: ACN + A (20:80), pH 2.5 (TFA adjusted)

Solution C: ACN + A (90:10), pH 2.5

Mobile phase: Table 1.

Diluent: ACN:A = 20:80, pH 6.0

System suitability solution: prepared as in Assay

Standard stock solution: 1 mg/mL USP Rifabutin RS

Standard solution: 0.01 mg/mL

Sensitivity solution: 0.5 µg/mL

Sample solution: 1 mg/mL Rifabutin (prepare fresh)

Chromatographic system (Procedure 2)

Mode: LC

Detector: UV 254 nm

Column: 4.6 mm × 25 cm, 5-µm L1

Column temp: 50 °C

Flow: 1 mL/min

Injection: 20 µL

System suitability requirements:

Resolution ≥ 1.5 (rifabutin 14R vs rifabutin)

RSD ≤ 5.0%

S/N ≥ 10 (Sensitivity solution)

Analysis

% impurity = (rᵤ / rₛ) × (Cₛ / Cᵤ) × P × (F₁ / F₂) × 100

rᵤ = peak response of impurity

rₛ = peak response of Standard solution

Cₛ = concentration of USP Rifabutin RS

Cᵤ = concentration of Sample

P = potency of USP Rifabutin RS

F₁ = conversion factor (0.001 mg/µg)

F₂ = relative response factor

Reporting threshold: 0.05%

a (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]naphtho[1,2-d]imidazole-2,4′-piperidine]-5,10,26(3H,9H)-trione.
b (9S,12E,14R,15R,16S,17R,18R,19R,20S,21S,22E,24Z)-6,18,20-Trihydroxy-1′-isobutyl-14-methoxy-7,9,15,17,19,21,25-heptamethyl-5,10,26-trioxo-3,5,9,10-tetrahydrospiro[9,4-(epoxypentadeca[1,11,13]trienimino)-2H-furo[2′,3′:7,8]naphtho[1,2-d]imidazole-2,4′-piperidine]-16-yl acetate.
c (9S,12E,14S,15R,16S,17R,18R,19R,20S,21S,22E,24Z)-16-Acetyloxy-6,18,20-trihydroxy-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]naphtho[1,2-d]imidazole-2,4′-piperidine]-5,10,26(3H,9H)-trione 1′-oxide.
d (9S,12E,14S,15R,16S,17R,18R,19R,20S,21R,22E,24Z)-6,18,20-Trihydroxy-1′-isobutyl-14-methoxy-7,9,15,17,19,21,25-heptamethyl-5,10,26-trioxo-3,5,9,10-tetrahydrospiro[9,4-(epoxypentadeca[1,11,13]trienimino)-2H-furo[2′,3′:7,8]naphtho[1,2-d]imidazole-2,4′-piperidine]-16-yl acetate.
e (9S,12E,14S,15R,16S,17R,18R,19R,20S,21S,22E,24Z)-6,18,20-Trihydroxy-1′-isobutyl-14-methoxy-7,9,15,17,19,21,25-heptamethyl-21-methylene-5,10,26-trioxo-3,5,9,10-tetrahydrospiro[9,4-(epoxypentadeca[1,11,13]trienimino)-2H-furo[2′,3′:7,8]naphtho[1,2-d]imidazole-2,4′-piperidine]-16-yl acetate.
f (11R,13E,16S,17R,18S,19R,20R,21S,22S,23E,25Z)-17-Acetyloxy-6,8,11,19,21-pentahydroxy-15-methoxy-7,11,16,18,20,22,26-heptamethyl-1′-(2-methylpropyl)-5,10,27-trioxo-3,5-dihydrospiro[4,9-(epiminopentadeca[2,4,14]trienoxyethano)naphtho[1,2-d]imidazole-2,4′-piperidine] N¹′-oxide.

5 SPECIFIC TESTS

Water Determination 〈921〉, Method I: NMT 2.5%

6 ADDITIONAL REQUIREMENTS

Packaging and Storage:

Preserve in well-closed containers, protected from light and excessive heat.

USP Reference Standards 〈11〉

USP Rifabutin RS