The technique of ultraviolet-visible spectroscopy is one of the most frequently employed techniques in pharmaceutical analysis. Molecular absorption in ultraviolet & visible region of spectrum is depending on the electronic structure of molecules. Absorption of energy as quantized, resulting in the elevation of electrons from orbital in the ground state to higher orbital in the exited state. The wavelength range of UV radiation starts at the blue end of the visible light and ends at 2000Å. The ultraviolet region is subdivided in to two spectral regions:

1. The region between 2000 Å -4000 Å is known as near as UV region
2. The region below 2000 Å is called far or vaccum UV region.

Wavelengths in the ultraviolet region are usually expressed in nanometers (1nm =10-7 cm) or angstroms (Å) (1 Å=10-8cm). Occasionally, absorption is reported in wave numbers (?). 

Three distinct types of electrons are involved in organic molecules. These are as follows:

? electrons:

 These electrons are involved in saturated bonds, such as those between carbons and hydrogens in paraffins.

? Electrons:

These electrons are involved in unsaturated hydrocarbons. Typical compounds with ? bonds are trienes and aromatic compounds. 

n-electrons:

These are the electrons which are not involved in the binding between atoms in molecules. Examples are organic compounds containing, nitrogen, oxygen or halogens. Levofloxacin or L-ofloxacin, the bacteriologically active L-isomer of the racemic fluoroquinolone ofloxacin, is a broad-spectrum antimicrobial agent. Levofloxacin acts by inhibiting bacterial DNA gyrase which is required for DNA replication and thus causes bacterial lysis. The addition of 6-fluoro and 7-piper- azinyl groups to the molecule greatly increases their antibacterial activity. They are commonly referred to as the second-generation fluoroquinolone antibacterial agents and are greatly effective against both gram-negative and gram-positive bacteria that are resistant to other antibacterials. Several HPLC assay methods have been reported for the determination of ofloxacin or its stereoisomers. Litera- ture survey revealed that various analytical methods such as high performance thin layer chromatography (HPTLC) and conductometry have been reported for the estimation of levofloxacin. Recently some UV spectrophotometric meth- ods were also reported for estimating levofloxacin using various solvents like 0.1 M hydrochloric acid, 100% methanol  or acetonitrile.

In this study, efforts were made to develop a simple, easy and economic UV spectrophotometric method using a diluent composed of water:methanol:acetonitrile (9:0.5:0.5) for the determination of levofloxacin in the raw materials as well as in the marketed dosage formulations. The developed method was optimized and validated as per the guidelines of International Conference on Harmonization (ICH) and demonstrated excellent specificity, linearity, precision and accuracy for levofloxacin.

MATERIALS AND METHODS

Apparatus

A Shimadzu UV–visible spectrophotometer (UV mini-1700, Shimadzu Corporation, Kyoto, Japan) was used for all absorbance measurements with matched quartz cells.

Materials

All chemicals and reagents were of analytical or HPLC grade. Levofloxacin in the form of levofloxacin hemihydrate powder was provided by Incepta Pharmaceuticals Ltd., Bangladesh, which was used as the reference standard. Pharmaceutical grade excipients were obtained from Pharmaceutical Technology Lab. of State University of Bangladesh.

Determination of wavelength of maximum absorption

A standard stock solution (LS) of levofloxacin (20 mg/mL) was prepared using diluents and 3 mL of LS was then diluted to 10 mL with the same diluent to obtain 6 mg/mL levofloxacin reference solution (LR). An UV spectroscopic scanning (190– 400 nm) was carried out with the LR to determine the lmax for the detection of levofloxacin using diluent as blank.

Linearity and range

For linearity study, seven solutions at different concentrations (1, 2, 4, 6, 8, 10 and 12 mg/mL) were prepared using seven different aliquots of LS, and the obtained data were used for

Intra-day precision (repeatability) and inter-day precision study (intermediate precision)

Levofloxacin tablets were finely powdered and the sample stock solution (LP) of 20 mg/mL was prepared following the same dilution pattern of LS. Three different aliquots of LP were then diluted to 10 mL to obtain the concentrations of 4, 6 and 8 mg/mL. This procedure was repeated in the following days.

Stability study

Samples prepared for repeatability study were preserved for 24 h at room temperature and analyzed on the following day to test for short-term stability.

Accuracy/recovery study

This study was carried out using pre-formulated granules containing pure levofloxacin hemihydrate and common excipients. Calculation was done from the label claim and the average weight of the final product. Previously used dilution pattern was followed for the granules to obtain five concentrations—80%, 90%, 100%, 110% and 120% of reference solution.

Specificity in the presence of excipients

The test for the specificity was carried out using only excipients. Spectra for placebo granules, blank, and sample were com- pared. Secondly the specificity was determined by subjecting the sample solution to accelerated degradation by heat (60 1C) for 72 h in order to verify that none of the degradation products interfered with the quantification of the drug.

Assay of content of levofloxacin in selected marketed brands

Five market brands of levofloxacin tablet from Bangladesh were randomly selected and analyzed using the newly developed and validated method. 3 mL of LS was diluted to 10 mL to obtain 6 mg/mL levofloxacin reference standard solution. Sample solutions of each brand (6 mg/mL) were also prepared and assayed for content of levofloxacin against the reference standard. The content of levofloxacin in the marketed brands was deter- mined using

As                   

Wst × 5 × 3 Content of levofloxacinð%) per tablet =        ×

 the linearity calibration plot. Limit of detection (LOD) and limit of quantification (LOQ) for the assay were also calculated.

where As is the absorbance of generic sample solution, Ast is the absorbance of levofloxacin reference standard solution, Ws is the weight of generic sample powder (mg), Wst is the weight of levofloxacin reference standard powder (mg), W is the average weight of tablet (mg), P is the potency of standard levofloxacin hemihydrates and CF is the conversion factor of levofloxacin hemihydrate to levofloxacin (0.976).

Table 3 Recovery/accuracy for five different concentrations of levofloxacin by the proposed method.

Figure 1 Specificity of the method determined by comparing the spectra of accuracy sample, placebo and degradation products.

Method development and optimization

Levofloxacin is almost insoluble in aqueous medium and freely soluble in organic solvents like methanol and acetonitrile. During the development phase, the use of a few milliliters of acetonitrile and methanol with water as the diluent resulted in preferable outcome in UV analysis. The solvent composition was optimized to water (9):methanol (0.5):acetonitrile (0.5). The pre-determined wavelength of maximum absorption (lmax) was 292 nm.

Method validation

Linearity and range

The calibration curve obtained was evaluated by its correla- tion coefficient. The absorbance of the samples in the range of 1.0–12.0 mg/mL was linear with a correlation coefficient (R2) greater than 0.999. The LOD and LOQ were calculated as

0.021 mg/mL and 0.064 mg/mL respectively.

Intra-day and inter-day precision

The intra-day and inter-day precision study (Table 1) of the developed method confirmed adequate sample stability and method reliability where all the RSDs were o2%.

Stability

Stability study’s results were within the acceptance range (Table 2) and indicated the samples stability over 24 h (short-term).

Accuracy/recovery

Results within the range of 99.00–100.07% ensure an accurate method (Table 3) as well as indicate non-interference with the excipients of formulation.

Specificity in the presence of excipients

The specificity of the analytical method was proved by comparing the spectra of placebo and degradation product of sample solution with that of accuracy sample (Fig. 1).

Content of levofloxacin in marketed brands

Levofloxacin content of five marketed products determined by the proposed method (Table 4) was in good agreement with the label claims and was in the range of 98.05–99.47% with the RSD values of 0.067–0.140% respectively.