Levofloxacin

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

Levofloxacin contains NLT 98.0% and NMT 102.0% of C₁₈H₂₀FN₃O₄, 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

Buffer: 8.5 g/L of ammonium acetate, 1.25 g/L of cupric sulfate, pentahydrate, and 1.3 g/L of l-isoleucine in water

Mobile phase: Methanol and Buffer (3:7)

Standard solution: 1 mg/mL of USP Levofloxacin RS in Mobile phase

Sample solution: 1 mg/mL of Levofloxacin in Mobile phase

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: UV 360 nm

Column: 4.6-mm × 25-cm; 5-μm packing L1

Column temperature: 45°

Flow rate: 0.8 mL/min

Injection size: 25 μL

System suitability

Sample: Standard solution

Suitability requirements

Tailing factor: 0.5–1.5

Relative standard deviation: NMT 1.0%

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of levofloxacin (C₁₈H₂₀FN₃O₄) in the portion of Levofloxacin taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak response of levofloxacin from the Sample solution

r_S = peak response of levofloxacin from the Standard solution

C_S = concentration of USP Levofloxacin RS in the Standard solution (mg/mL)

C_U = concentration of Levofloxacin in the Sample solution (mg/mL)

Acceptance criteria: 98.0%–102.0% on the anhydrous basis

4 IMPURITIES

Residue on Ignition 〈281〉: NMT 0.2%. Use a platinum crucible.

Change to read:

Organic Impurities, Procedure 1

[Note—Procedure 1 is recommended if levofloxacin N-oxide is a potential organic impurity. Procedure 2 and Procedure 3 are recommended if levofloxacin related compound B is a potential organic impurity.]

Buffer, (ERR 1-Feb-2022) Mobile phase, Sample solution, and Chromatographic system: Proceed as directed in the Assay.

System suitability solution: 1 mg/mL of USP Levofloxacin RS in Mobile phase

Sensitivity solution: 0.3 μg/mL of USP Levofloxacin RS in Mobile phase

System suitability

Samples: System suitability solution and Sensitivity solution

Suitability requirements

Relative standard deviation: NMT 1.0%, System suitability solution

Signal-to-noise ratio: NLT 10, Sensitivity solution

Analysis

Sample: Sample solution

Calculate the percentage of each individual impurity in the portion of Levofloxacin taken:

Result = (r_U/r_S) × (1/F) × 100

r_U = peak response of each impurity

r_S = peak response of levofloxacin

F = relative response factor (see Table 1)

Acceptance criteria: See Table 1.

Table 1

* Do not include the d-isomer in the calculation for total impurities.

^a (S)-9-Fluoro-2,3-dihydro-3-methyl-10-(piperazin-1-yl)-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic acid.

^b (S)-9-Fluoro-2,3-dihydro-3-methyl-10-[2-(methylamino)ethylamino]-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic acid.

^c (S)-4-(6-Carboxy-9-fluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido-[1,2,3-de][1,4]benzoxazine-10-yl)-1-methyl-piperazine-1-oxide.

d (S)-2,3-Dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic acid.

^e (R)-9-Fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic acid.

Organic Impurities, Procedure 2

[Note—Solutions of levofloxacin are not stable in light; use amber bottles.]

Buffer: Dissolve 3.08 g/L of ammonium acetate and 8.43 g/L of sodium perchlorate monohydrate in water. Adjust with phosphoric acid to a pH of 2.2.

Solution A: Acetonitrile and Buffer (16:84)

Solution B: Acetonitrile, methanol, and Buffer (30:20:50)

Solution C: 0.4 mg/mL of USP Levofloxacin RS by dissolving in acetonitrile at about 8% of volume and diluting with water to volume

Solution D: 0.05 mg/mL of USP Levofloxacin Related Compound A RS in 0.2% ammonium hydroxide in methanol

Mobile phase: See Table 2.

Table 2

System suitability solution: 0.1 mg/mL of USP Levofloxacin RS and 5 μg/mL of USP Levofloxacin Related Compound A RS in water from Solution C and Solution D

Levofloxacin stock solution: 0.4 mg/mL of USP Levofloxacin RS. Dissolve USP Levofloxacin RS in acetonitrile at about 8% of final volume, sonicate, and dilute with water to volume.

Levofloxacin standard solution: 0.02 mg/mL of USP Levofloxacin RS in acetonitrile and water (1:10) from Levofloxacin stock solution

Levofloxacin related compound B stock solution: 0.2 mg/mL USP Levofloxacin Related Compound B RS in methanol. [Note—Sonicate if necessary.]

Levofloxacin related compound B standard solution: 0.04 mg/mL USP Levofloxacin Related Compound B RS in methanol from Levofloxacin related compound B stock solution

Standard solution: 0.4 μg/mL of levofloxacin and 0.8 μg/mL of levofloxacin related compound B in acetonitrile and water (1:10) from Levofloxacin standard solution and Levofloxacin related compound B standard solution

Sample solution: 0.4 mg/mL by dissolving the sample in acetonitrile at about 8% of final volume and diluting with water to volume. [Note—Sonicate if necessary.]

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: 280 nm

Column: 4.0-mm × 15-cm; 3.0-μm packing L1

Column temperature: 38°

Flow rate: 1.0 mL/min

Injection size: 10 μL

System suitability

Sample: System suitability solution

Suitability requirements

Relative standard deviation: NMT 2.0% for levofloxacin

Analysis

Samples: Standard solution and Sample solution

Calculate the percentage of levofloxacin related compound B in the portion of Levofloxacin taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

r_U = peak response for levofloxacin related compound B from the Sample solution

r_S = peak response for levofloxacin related compound B from the Standard solution

C_S = concentration of USP Levofloxacin Related Compound B RS in the Standard solution (mg/mL)

C_U = concentration of Levofloxacin in the Sample solution (mg/mL)

Calculate the percentage of other impurities in the portion of Levofloxacin taken:

Result = (r_U/r_S) × (C_S/C_U) × 100

_U = peak response of any other impurity from the Sample solution

r_S = peak response of levofloxacin from the Standard solution

C_S = concentration of USP Levofloxacin RS in the standard solution (mg/mL)

C_U = concentration of Levofloxacin in the Sample solution (mg/mL)

Acceptance criteria: See Table 3.

Table 3

^a (S)-9-Fluoro-3-methyl-10-(piperazin-1-yl)-7-oxo-2,3-dihydro-7H-pyrido[1,2,3-de][1,4-benzoxazine-6-carbocylic acid.

^b (S)-9,10-Difluoro-3-methyl-7-oxo-2,3-dihydro-7H-pyrido[1,2,3-de][1,4-benzoxazine-6-carbocylic acid.

Organic Impurities (Enantiomeric Purity), Procedure 3

Buffer: 1.32 g/L of d-phenylalanine and 0.75 g/L of copper(II)sulfate pentahydrate in water

Mobile phase: Methanol and Buffer (15:85)

System suitability solution: 0.01 mg/mL of USP Ofloxacin RS and 0.01 mg/mL of USP Levofloxacin RS in water

Sample solution: 0.08 mg/mL in water

Chromatographic system

(See Chromatography 〈621〉, System Suitability.)

Mode: LC

Detector: 294 nm

Column: 4.6-mm × 15-cm; 3.5-μm packing L1

Column temperature: 40°

Flow rate: 0.7 mL/min

Injection size: 10 μL

System suitability

Sample: System suitability solution

[Note—The relative retention times for d-ofloxacin and levofloxacin are 0.91 and 1.0, respectively.]

Suitability requirements

Resolution: NLT 2.0 between d-ofloxacin (d-isomer) and levofloxacin

Analysis

Sample: Sample solution

Calculate the percentage of d-ofloxacin in the portion of Levofloxacin taken:

Result = (r_U/r_T) × 100

r_U = peak response for d-ofloxacin

r_T = sum of responses of all peaks

Acceptance criteria: NMT 1.0%

5 SPECIFIC TESTS

Optical Rotation, Specific Rotation 〈781S〉

Solvent: Methanol

Sample solution: 5 mg/mL in Solvent

Acceptance criteria: −92° to −106°, at 20°

Water Determination, Method Ia〈921〉: 2.0%–3.0%

6 ADDITIONAL REQUIREMENTS

Packaging and Storage: Preserve in tight, light-resistant containers. Store at room temperature.

Labeling: If a procedure for Organic Impurities other than Procedure 1 is used, then the labeling states with which Organic Impurities procedure the article complies.

USP Reference Standards 〈11〉

USP Levofloxacin RS

USP Levofloxacin Related Compound A RS (S)-9-Fluoro-3-methyl-10-(piperazin-1-yl)-7-oxo-2,3-dihydro-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic acid.

C₁₇H₁₈FN₃O₄ 347.34

USP Levofloxacin Related Compound B RS(S)-9,10-Difluoro-3-methyl-7-oxo-2,3-dihydro-7H-pyrido[1,2,3-de][1,4]benzoxazine-6-carboxylic acid.

C₁₃H₉F₂NO₄ 281.21

USP Ofloxacin RS