(lee'' voe flox' a sin).
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C18H20FN3O4·½H2O 370.38
7H-Pyrido[1,2,3-de]-1,4-benzoxazine-6-carboxylic acid, 9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-hydrate (2:1), (S)-;    
()-(S)-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, hemihydrate     [138199-71-0].
Anhydrous     [100986-85-41].
Levofloxacin contains NLT 98.5% and NMT 102.0% of C18H20FN3O4, calculated on the anhydrous basis.
•  B. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay.
•  Procedure
Solution A:  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 Solution A (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  
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%
Samples:  Standard solution and Sample solution
Calculate the percentage of C18H20FN3O4 in the portion of Levofloxacin taken:
Result = (rU/rS) × (CS/CU) × 100
rU== peak response of Levofloxacin from the Sample solution
rS== peak response of levofloxacin from the Standard solution
CS== concentration of USP Levofloxacin RS in the Standard solution (mg/mL)
CU== concentration of Levofloxacin in the Sample solution (mg/mL)
Acceptance criteria:  98.5%–102.0% on the anhydrous basis
Inorganic Impurities 
•  Residue on Ignition 281: NMT 0.2%. Use a platinum crucible.
•  Heavy Metals, Method II 231: NMT 10 ppm
Organic Impurities 
•  Procedure
Solution A, 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
Sample:  Sample solution
Calculate the percentage of each individual impurity in the portion of Levofloxacin taken:
Result = (rU/rS) × (1/F) × 100
rU== peak area response of each impurity
rS== peak area response of levofloxacin
F== relative response factor (see Impurity Table 1)
Acceptance criteria 
Individual impurities:  See Impurity Table 1.
Total impurities:  NMT 0.5%. [Note—Do not include the d-isomer in the calculation for Total impurities. ]
Impurity Table 1
Name Relative
NMT (%)
N-Desmethyl levofloxacina 0.47 1.0 0.3
Diamine derivativeb 0.52 0.9 0.3
Levofloxacin N-oxidec 0.63 1.1 0.3
9-Desfluoro levofloxacind 0.73 1.0 0.3
Levofloxacin 1.0
d-Isomere 1.23 1.0 0.8
Any unknown
1.0 0.1
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.
•  Optical Rotation, Specific Rotation 781S
Solvent:  Methanol
Sample solution:  5 mg/mL in Solvent
Acceptance criteria:  92 to 106, at 20
•  Packaging and Storage: Preserve in tight and light-resistant containers. Store at room temperature.
•  USP Reference Standards 11
USP Levofloxacin RS Click to View Structure
Auxiliary Information— Please check for your question in the FAQs before contacting USP.
Topic/Question Contact Expert Committee
Monograph Behnam Davani, Ph.D., M.B.A.
Senior Scientific Liaison
(SM12010) Monographs - Small Molecules 1
Reference Standards RS Technical Services
USP35–NF30 Page 3671
Pharmacopeial Forum: Volume No. 35(6) Page 1459