Application Note: Levofloxacin Assay

Peter M. Lambert1

Impact Analytical, Inc., Midland, Michigan

Levofloxacin is an antibiotic2, the structure of which is shown in Structure 1.

<B>Structure 1</b> Levofloxacin Hemihydrate

Structure 1 Levofloxacin Hemihydrate

Levofloxacin was analyzed by high performance liquid chromatography (HPLC) following the USP Levofloxacin monograph3.

Experimental A buffer was prepared by weighing 8.4561 g ammonium acetate, 1.2481 g copper(II) sulfate, pentahydrate, and 1.3062 g L-isoleucine into a 1000 mL volumetric flask and filling to volume with water.

The mobile phase solution was prepared by mixing 700 mL buffer with 300 mL methanol.

A reference standard solution was prepared by weighing 51.86 mg levofloxacin USP standard into a 50 mL volumetric flask and filling to volume with mobile phase.

System suitability was established by five replicate injections of the standard solution. The following conditions were used.

System: Waters Alliance HPLC
Column: Phenomenex Luna C18(2), 5 μm particle size, 250 x 4.6 mm
Column Temp.: 45 °C
Injection volume: 25 μL
Run time: 25 minutes
Flow rate: 0.8 mL/minute
UV Wavelength: 360 nm

Results The system suitability passed, with an average tailing factor of 0.7 (criteria: 0.5 – 1.5) and a relative standard deviation of 0.1% (criteria: NMT 1.0%). A representative HPLC chromatogram of the levofloxacin standard is shown in Figure 1. The retention time of levofloxacin was about 16.8 minutes.

<b>Figure 1.</b> HPLC chromatogram of Levofloxacin USP standard.

Figure 1. HPLC chromatogram of Levofloxacin USP standard.

  1. Correspondence can be sent to: []
  2. Anderson VR, Perry CM, Levofloxacin : a review of its use as a high-dose, short-course treatment for bacterial infection, Drugs. 2008;68(4):535-65. []
  3. United States Pharmacopeia, USP Monograph: Levofloxacin, USP41-NF36. []

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