A Stability Indicating UPLC Method for the Determination of Levofloxacin Hemihydrate in Pharmaceutical Dosage Form : Application to Pharmaceutical Analysis

A reliable and sensitive isocratic stability indicating RP-UPLC method has been developed and validated for quantitative analysis and content uniformity study of levofloxacin hemihydrate in tablets. An isocratic method for analysis of levofloxacin hemihydrate was archived on ACQUITY UPLC BEH C 18 (100∗2.1)mm particle size 1.7μ columns within shorter runtime of 4 min with a flow rate of 0.400mL/min and using a photodiode array detector to monitor the eluate at 294 nm. The mobile phase consisted of acetonitrile-buffer (23 : 77 v/v), (buffer: 20mM K 2 HPO 4 + 1mL triethylamine in 1 L water, pH = 2.50 by orthophosphoric acid). Response was a liner function of drug concentration in the range of 0.5–80 μg/mL (r = 0.999) with a limit of detection and quantification of 0.1 and 0.5 μg/mL, respectively. Accuracy (recovery) was between 99.77% and 101.55%.The drug was subjected to oxidation, hydrolysis, photolysis, and thermal degradation. Degradation products resulting from the stress studies did not interfere with the detection of levofloxacin hemihydrate, and the assay is stability indicating.


Introduction
Levofloxacin hemihydrate (Figure 1) is a synthetic chemotherapeutic antibiotic of the fluoroquinolone drug class and is used to treat severe life-threatening bacterial infection or bacterial infection that have failed to respond to other antibiotic classes.
Levofloxacin hemihydrate is highly water soluble in nature.It is also soluble in organic solvents, soluble in glacial acetic acid and chloroform, sparingly soluble in methanol, slightly soluble in ethanol, and practically insoluble in ether.Levofloxacin hemihydrate is odourless drug.Methods for quantitative analysis of Levofloxacin by HPLC [1][2][3][4][5][6][7], by UV [8][9][10] spectroscopy in single as well as in combination, are available in the literature.There are no methods available for quantitative analysis by UPLC.The method was developed and validated as per ICH [11][12][13] and USP [14] guideline.

Experimental
2.1.Chemicals and Reagents.Levofloxacin hemihydrates reference standard (claim 99.48%) was provided by Cipla pharmaceuticals.Tablets (500 mg) of Levofloxacin were produced from a pharmacy.HPLC grade methanol and ortho phosphoric acid were purchased from Merck India Limited, Mumbai, India.Analytical grade hydrochloric acid, sodium hydroxide pellets, and hydrogen peroxide solution 30% (v/v) were obtained from Ranbaxy Fine Chemicals, New Delhi, India.High purity water was obtained using Milli-Q (Millipore, Milford, MA, USA) water purification system.

Instruments and Condition.
The fast liquid chromatography was performed using waters UPLC system with photodiode array detector.Chromatogram and data were recorded by means of Empower software.The chromatographic system was performed using an Acquity BEH C 18 (100 × 2.1 mm) id., 1.7 m column.Separation was achieved using a mobile phase consisted of buffer : acetonitrile (77 : 23 v/v) (buffer: 20 mM KH 2 PO 4 + 1 mL triethylamine in 1 litre water, pH = 2.5 adjust with orthophosphoric acid) at a flow rate of 0.4 mL/min in only 4 minute runtime.The column temperature was maintained at 30 ∘ C, injection volume was 2 L, and detection wavelength was set at 294 nm for determination of levofloxacin hemihydrate (Figure 2).

Preparation of Standard Solution.
The stock solution of levofloxacin hemihydrate (500 g/mL) was prepared by accurately weighing 25 mg of levofloxacin hemihydrate reference standard and transferring to 50 mL standard volumetric flask containing approximately 25 mL of water.The flask was sonicated for 10 minutes to dissolve the solids.Volumes were made up to the mark with water (500 g/mL stock solution-A).Transferred 5 mL of above stock solution-A to a 50 mL volumetric flask and diluted up to the mark with mobile phase to obtained working standard solution (50 g/mL) of drug.

Preparation of Sample Solution.
For analysis of the tablet dosage form, ten tablets were weighed individually, and their average weight was determined.The tablets were crushed to fine homogenous powder, and a quantity equivalent to 25 mg levofloxacin was transferred in a 50 mL volumetric flask.Added about 25 mL of water to the flask, shaken for 10 minutes, and then sonicated for 15 minutes.The solution was allowed to stand at room temperature and filtered through Whatman no.41 filter paper.The residue was washed with water, and the combined filtrate was made up to the mark with water (stock solution-B 500 g/mL), transferred quantitatively 5 mL of above stock solution-B in 50 mL volumetric flask and diluted up to the mark with mobile phase (50 g/mL).
2.5.Specificity/Force Degradation Study.Force degradation studies were performed to evaluate the stability indicating properties and specificity of the method.All solutions used in stress studies were prepared at an initial concentration of 500 g/mL of levofloxacin hemihydrate and heat for 1 hr at 80 ∘ C. All samples were then diluted in mobile phase to give a final concentration of 50 g/mL and filtered before injection.

Acid Degradation Study.
Acid decomposition was carried out in 0.1 M HCl at a concentration of 500 g/mL of levofloxacin hemihydrate and after heat for 1 hr at 80 ∘ C. The stressed sample was cooled, neutralized, and diluted with mobile phase.

Base Degradation Study.
Base decomposition was carried out using 0.1 M NaOH at a concentration of 500 g/mL and heat for 1 hr at 80 ∘ C.After cooling, the solution was neutralized and diluted with mobile phase.

Oxidative Degradation.
Oxidation solutions for oxidative stress studies were prepared using 3% H 2 O 2 at a concentration of 500 g/mL of levofloxacin and after heat for 1 hr at 80 ∘ C. The sample solution was cooled and diluted accordingly with the mobile phase.

Thermal Degradation.
For thermal stress testing, the drug solution 500 g/mL was heated for 1 hr at 80 ∘ C, cooled, and used for the study.

Photo Degradation.
For Photolytic degradation, the powder drug has been exposed to sunlight for 48 hrs and used for the study.

Method Validation
The method was validated as per ICH guideline.The method was validated by performing system suitability, linearity, limit of Detection (LOD), limit of quantification (LOQ), precision, accuracy, selectivity, and robustness.

Precision.
Method precision of the analytical method was determined by analyzing six sets of sample solution preparation.Assay of all six replicate sample preparations was determined, and mean percentage of assay value, standard deviation and percentage of relative standard deviation for the same were calculated.Intermediate precision of the analytical method was determined by performing method precision on another day by another analyst using different make of raw materials under same experimental condition.Overall assay value of method precision and intermediate precision was compared and percentage of difference and overall percentage of relative standard deviation were calculated.

LOD and LOQ.
Sensitivity of the method was determined by establishing the LOD and LOQ.The LOD and LOQ were established at signal-to-noise ratio of 3 : 1 and 10 : 1, respectively.
3.3.Linearity.Linearity test solutions for the assay method were prepared from a stock solution at 7 concentration levels of the assay analyte concentration (20, 30, 40, 50, 60, 70, and 80 g/mL).The peak area versus concentration data was analyzed with least squares linear regression.The slope and -intercept of the calibration curve were reported.
3.4.Accuracy.Accuracy of the developed method was confirmed by doing recovery study as per ICH guidelines at three different concentration levels 50%, 100%, and 150% by replicate analysis ( = 3).The results indicated that the method is highly accurate for the determination of levofloxacin hemihydrate.

3.5.
Robustness.The robustness of the assay method was established by introducing small changes in the chromatographic condition which included percentage of methanol in mobile phase (58% and 62%), flow rate (0.38 and 0.42 mL/min), and column oven temperature (25 ∘ C and 35 ∘ C).Robustness of the method was studied using six replicates at a concentration level of 50 g/mL of levofloxacin hemihydrate.

Solution Stability.
The solution stability of levofloxacin hemihydrate in the assay method was carried out by leaving both the sample and reference standard solutions in tightly capped volumetric flasks at room temperature for 48 hr.The same sample solution was assayed at 6 hr intervals over the study period.The percentage of RSD of the levofloxacin hemihydrate assay was calculated for solution stability experiments.An additional study was carried out using the stock solution by storing it in a tightly capped volumetric flask at 4 ∘ C.

Method Validation
4.1.1.Precision.The precision of the method was determined by repeatability (intraday precision) and intermediate precision (interday precision) of the levofloxacin hemihydrate standard solution.The precision of the assay method was evaluated by carrying out six independent assays.For assay method ( = 6), percentage of RSD for system precision was 0.45% on the same day (intra-day) and 0.41% on different days (inter-day).The mean values of method precision (repeatability) were 101.2% (RSD = 0.26%) for assay on the same day (intra-day) and 101.58% (RSD 0.57%) for assay on different days (inter day).Intermediate precision was

LOD and LOQ.
All the results of LOD and LOQ data were within the acceptance criteria; hence, it can be concluded that the LOD and LOQ of the method were 0.1 g/mL and 0.5 g/mL, respectively.The signal-to-noise ratio for the LOD was well within the acceptance criteria which means more than 3.3, and for the LOQ it was more than 10.0.Furthermore, the data of linearity extension chart up to LOQ level also suggest that the levofloxacin can be quantified up to 0.5 g/mL with well precisely and accurately.

Linearity.
The calibration curve for the levofloxacin hemihydrate was linear over the concentration range of 0.5-80 g/mL.The data for the peak area versus concentration were treated by linear regression analysis, and the correlation coefficient ( 2 ) was obtained (0.9994).The regression equation for the calibration curve was found to be  = 46, 965 + 85, 486 (Table 1).

Accuracy.
The percentage recovery of levofloxacin hemihydrate in the drug tablets samples was obtained in a range from 99.97% to 101.55%, respectively (Table 2).Percentage of RSD value of replicated sets was less than 2% which indicates that this method is highly accurate.
4.1.5.Robustness.The robustness of an analytical procedure refers to its ability to remain unaffected by small and deliberate variations in method parameters and provides an indication of its reliability for routine analysis.The robustness of the method was evaluated by assaying the same sample under different analytical conditions deliberately changing from the original condition.The results obtained from assay of the test solutions were not affected by varying the conditions and were in accordance with the results for original conditions (Table 3).The percentage of RSD value of assay determined for the same sample under original conditions and robustness conditions was less than 2.0%, indicating that the developed method was robust.separation of levofloxacin peak from the degraded samples derived from the empower software.The degradation of levofloxacin hemihydrate was found to be very similar for both the tablets and standard.Typical chromatograms obtained following the assay of stressed samples are shown in Figures 3, 4, and 5. Levofloxacin hemihydrate standard and tablet powder were found to be quite stable under light and heat conditions.A slight decomposition was seen on exposure of levofloxacin drug solution to acid and heat.On the other hand, drug   decomposition under oxidative and alkaline degradation was found to be 66.44% and 12.38%, respectively.The results of force degradation study are explained in Table 4.

Conclusion
A stability indicating UPLC method was developed, validated, and applied for the determination of levofloxacin hemihydrate in pharmaceutical dosage forms.The developed method was validated as per ICH guidelines and was found to be accurate, precise, robust, and specific.The chromatographic elution step was under taken in a short time (4 min).
No interference from any components of pharmaceutical dosage form or degradation products was observed, and the method has been successfully used to perform long term and accelerate stability studies of levofloxacin hemihydrate.

Figure 5 :
Figure 5: Peak purity result of base degradation.
4.1.7.Specificity.The specificity of the developed method was determined by injecting sample solutions (50 g/mL) which were prepared by forcibly degrading under such stress conditions as heat, light, oxidative agent, acid, and base under proposed chromatographic condition.The stability indicating capability of the method was established from the