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 C18 (100*2.1) mm particle size 1.7
Levofloxacin hemihydrate (Figure
Levofloxacin hemihydrate.
IUPAC name is (
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 [
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.
The fast liquid chromatography was performed using waters UPLC system with photo-diode array detector. Chromatogram and data were recorded by means of Empower software. The chromatographic system was performed using an Acquity BEH C18 (100 × 2.1 mm) id., 1.7
Chromatogram of levofloxacin hemihydrates reference standard.
The stock solution of levofloxacin hemihydrate (500
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
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
Acid decomposition was carried out in 0.1 M HCl at a concentration of 500
Base decomposition was carried out using 0.1 M NaOH at a concentration of 500
Oxidation solutions for oxidative stress studies were prepared using 3% H2O2 at a concentration of 500
For thermal stress testing, the drug solution 500
For Photolytic degradation, the powder drug has been exposed to sunlight for 48 hrs and used for the study.
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.
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.
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.
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
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 (
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
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.
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 (
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
The calibration curve for the levofloxacin hemihydrate was linear over the concentration range of 0.5–80
Result of precision, linearity, LOD, and LOQ study.
Parameter | UPLC |
---|---|
Linearity (µg/mL) | 0.5–80 |
Correlation coefficient ( |
0.9994 |
Slope (m) | 46965 |
Intercept ( |
85486 |
LOD (µg/mL) | 0.1 |
LOQ (µg/mL) | 0.5 |
Accuracy (% RSD) | 0.21–0.76 |
Interday precision (RSD, |
0.57 |
Intraday precision (RSD, |
0.26 |
The percentage recovery of levofloxacin hemihydrate in the drug tablets samples was obtained in a range from 99.97% to 101.55%, respectively (Table
Accuracy study.
Level % | No. | Amount of drug added ( |
Amount of drug found ( |
Recovery (%) | Mean recovery (%) | RSD (%) |
---|---|---|---|---|---|---|
50 | 1 | 25.23 | 25.15 | 99.87 | 100.63 | 0.76 |
2 | 25.29 | 25.64 | 101.40 | |||
3 | 25.34 | 25.63 | 100.62 | |||
100 | 1 | 50.82 | 50.98 | 100.32 | 99.97 | 0.35 |
2 | 50.60 | 50.40 | 99.62 | |||
3 | 50.27 | 50.52 | 99.97 | |||
150 | 1 | 75.21 | 76.21 | 101.34 | 101.55 | 0.21 |
2 | 75.39 | 76.72 | 101.77 | |||
3 | 75.06 | 76.61 | 101.54 |
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
Robustness study.
Conditions | Assay (%) | RT (min) | System suitability data | |
---|---|---|---|---|
Theoretical plates | Asymmetry | |||
Flow mL/min | ||||
0.38 | 98.89 | 1.27 | 5244 | 1.14 |
0.40 | 99.08 | 1.24 | 5253 | 1.11 |
0.42 | 99.21 | 1.25 | 5276 | 1.10 |
Column temperature | ||||
25°C |
98.93 |
1.46 |
5378 |
1.12 |
ACN: buffer ratio | ||||
21 : 79 V/V | 99.87 | 1.40 | 5402 | 1.10 |
23 : 77 V/V | 99.08 | 1.24 | 5253 | 1.11 |
25 : 75 V/V | 100.27 | 1.16 | 5185 | 1.13 |
The specificity of the developed method was determined by injecting sample solutions (50
H2O2 degradation study of levofloxacin.
Base Degradation Study of Levofloxacin.
Peak purity result of base degradation.
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, the 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
Specificity Study.
Stress condition | Time | Purity angle | Purity threshold | % Degradation |
---|---|---|---|---|
Acid Hydrolysis | 1 hr | 0.409 | 0.874 | 8.12 |
Base Hydrolysis | 1 hr | 1.107 | 13.28 | 12.38 |
Oxidation | 1 hr | 4.578 | 12.82 | 66.44 |
Thermal | 1 hr | 0.854 | 1.057 | 24.37 |
Photo | 5 days | 0.748 | 1.682 | 27.82 |
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.
The authors are grateful to the Department of Chemistry, Saurashtra University (UGC-SAP Sponsored and DST-FIST Funded) Rajkot, Gujarat, India, for providing the instrumental facilities. Special thanks to the “National Facility for Drug Discovery through New Chemicals Entities (NCE’s) Development and Instrumentation Support to Small Manufacturing Pharma Entities” Program under the Drug and Pharma Research Support (DPRS) jointly funded by Department of Science & Technology, New Delhi, Government of Gujarat Industries Commissionerate and Saurashtra University, Rajkot, India.