4Department of Pharmacy, B. S. Anangpuria Institute of Pharmacy, Faridabad, Haryana.
*Corresponding Author E-mail: manishdevgun@gmail.com; manishdevgun@yahoo.co.in
ABSTRACT:
INTRODUCTION:
Levofloxacin hemihydrate is chemically (–)–(S)–9–fluro–2,3–dihydro–3–methyl–10–(4–methyl–1–piperazinyl)–7–oxo–7H–pyrido [1,2,3-de]–1,4–benzoxazine–6–carboxylic acid. It is the optically active S–(–) isomer of the fluoroquinolone antibacterial agent Ofloxacin. It inhibits bacterial topoisomerase IV and DNA gyrase enzyme required for DNA replication, transcription, repair and recombination. It is used in variety of indications like acute maxillary sinusitis, acute exacerbation of chronic bronchitis, community acquired pneumonia, uncomplicated skin and skin structure infection, acute pyelonephritis, complicated urinary tract infections1-2.
Ornidazole is chemically 1–chloro–3-(2 methyl–5–nitroimidazol–1–yl) propan–2–ol. Ornidazole is active against protozoa and anaerobic bacteria. It is used in variety of diseases like amebiasis, giardiasis, trichomoniasis and bacterial vaginosis. Ornidazole is readily absorbed from the gastrointestinal tract. It is widely distributed in body tissues and fluids. It is metabolized in liver and is excreted in the urine mainly as conjugate and metabolites3.
There are a few combination dosages of Levofloxacin hemihydrate and Ornidazole in the market these days. There are various reports of the determination of the Levofloxacin hemihydrate by High Performance Liquid Chromatography (HPLC)4-9. Ornidazole has also been estimated by HPLC10-13. The simultaneous estimation of levofloxacin hemihydrate and ornidazole was reported by using HPTLC method14. An attempt was, therefore, made to develop new, rapid, and sensitive method for the simultaneous estimation of Levofloxacin hemihydrate and Ornidazole in pharmaceutical preparations.
MATERIALS AND METHODS:
Equipment:
The separation was performed using HPLC system (Waters) HPLC Waters 515 pump, 2487 Water detector enabled with Empower software. The Kromacil 5 µ, 100 A, C18, 150 x 4.6 mm column was used. The Dhona-200 D balance and Eutech pH 510 pH meter were used. The UV 1700-Shimadzo UV spectrophotometer was employed.
Chemicals:
All the reagents and solvents used were of analytical and HPLC grade. The acetonitrile was procured from Qualigens, triethylamine from S. D. Fine Chem. Ltd. and dil. Orthophasphoric acid from Merck. The commercial tablets, Fynal-Oz (Mankind), containing Levofloxacin (250 mg) and Ornidazole (500 mg) were purchased from the local market. The reference standards of Levofloxacin hemihydrate and Ornidazole were obtained from Vintech Formulation, Karnal, Haryana. Their authenticity and purity (Levofloxacin hemihydrate 98 % pure and Ornidazole 99.56 % pure) were certified.
Identification of drug samples:
(a) Melting Point:
The melting points of both Levofloxacin hemihydrate and Ornidazole were determined and compared to that reported in literature.
(b) UV Scan:
Mobile phase and Stationary phase:
Mobile phase consisted of acetonitrile:water in 40:60, to this 1ml triethylamine was added and the pH was adjusted to 3.0 ± 0.05 with dil. Orthophosphoric acid. The column used was Kromacil 5 µ 100 A C18, 150 x 4.6 mm.
At first the mobile phase acetonitrile:water (60:40) was tried and the flow rate was 1.2 ml/min at 4.0 pH. Peak splitting was seen. In order to improve the peak shape, the mobile phase composition was changed to acetonitrile:water (40:60), the flow rate was changed to 1.3 ml/min and the pH was changed to 3.0. The two components were separated at different time intervals with good resolution and good peak shapes.
HPLC method validation:
Specificity: The specificity was assessed by comparing results obtained from reference containing the analyte only with results obtained from samples containing excipients, related substances or degradation products, and including or excluding the analyte.
Linearity: For the establishment of linearity, five solutions of different concentrations (µg/ml) of Levofloxacin hemihydrate and Ornidazole (3.125, 6.25; 6.25, 12.5; 12.5, 25; 25, 50; 50, 100) respectively were injected in triplicate. Chromatograms obtained and the calibration plot was drawn between concentration and peak height. The linearity was evaluated by the visual inspection of the plot and also by calculating correlation coefficient.
Calibration Range: The range of an analytical procedure is the interval between the upper and lower concentration (amounts) of analytes in the sample (including these concentrations) for which it has been demonstrated that the analytical procedure has a suitable level of precision, accuracy and linearity. The specified range was derived from linearity studies and depends on the intended application of the procedure.
Number of Theoretical Plates: The number of theoretical plates is the measure of the sharpness of the peaks and therefore the efficiency of the column.
Tailing Factor: Tailing factor is the distance from the front slope of the peak to the back slope divided by twice the distance from the centre line of the peak to the front slope, with all measurements made at 5 % of the maximum peak height.
Robustness:The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small, but deliberate variations in method parameters and provides an indication of its reliability during normal usage.
Relative Standard Deviation or Precision: The Relative Standard Deviation was calculated to determine the repeatability of the system.
Sample preparation and evaluation:
Twenty tablets were weighed accurately and finely powdered. A portion of it which was equivalent of 125 mg of Levofloxacin and 250 mg of Ornidazole (half tablet) was dissolved in 200 ml of diluent (acetonitrile:water, 40:60). The solution was filtered through 0.45 µ-membrane filter paper. One ml of this solution was taken and dissolved in 50 ml of the above-mentioned diluent to get a solution having a concentration of 12.5 µg of Levofloxacin and 25 µg of Ornidazole. Twenty microlitres of this solution was injected in triplicate in the specified column of the HPLC.
Peak identification:
The peaks obtained by applying this method on the sample were identified by applying the above optimized method on the individual standards and the retention times were compared.
RESULTS AND DISCUSSION:
Identification of drug samples:
a) Levofloxacin hemihydrate: The observed melting point came out to be 216-217 °C which is close to the reported 218 °C. The peak of 0.7164 was observed in the UV spectrum at absorption maximum, λmax 288 nm, which confirms the drug, Fig. 1.
b) Ornidazole: The observed melting point came out to be 84-88 °C which is close to the reported 85-90 °C. The peak of 0.3 was observed in the UV spectrum at absorption maximum, λmax 276.8 nm, which confirms the drug, Fig. 2.
Fig. 1: UV spectrum of Levofloxacin hemihydrate (λmax 288 nm)
Fig. 2: UV spectrum of Ornidazole (λmax 276.8 nm)
Optimized chromatographic parameters:
The present method was aimed at developing a simple HPLC method for simultaneous estimation of Levofloxacin hemihydrate and Ornidazole. The optimized conditions were obtained using Waters HPLC with Waters 515 pump, 2487 Water detector enabled with Empower software. The Kromacil 5 µ, 100 A, C18, 150 x 4.6 mm column was used. Mobile phase used was a mixture of acetonitrile and water (40:60), triethylamine (1 ml) and pH adjusted to 3.0 ± 0.05 with dil. Orthophosphoric acid and run time was just 3 min with the flow rate of 1.3 ml/min, the injection volume was 20 µl. The retention time observed in the case of Levofloxacin hemihydrate and Ornidazole comes out to be 1.051 min and 1.777 min respectively, which made the method rapid and economical and both of the drugs were clearly separated.
HPLC Method Validation:
Specificity: To test the specificity, a blank run was conducted which showed no peak at 1.051 and 1.777 retention time. In other words, there was no other interference at the retention time of, in the chromatogram of placebo solution.
Linearity: The Linearity of the method was determined. The chromatograms of the five solutions with different concentrations of pterostilbene were plotted. The peak height was calculated (Table 1) and was plotted against concentration. A linear relationship was observed, and correlation coefficient was calculated to be 0.999, Fig. 3 and Fig. 4.
Table 1: Mean of Peak Height of Drugs.
|
Levofloxacin hemihydrate Eq. To Levofloxacin |
Ornidazole |
||
|
Concentration (µg/ml) |
Mean of Peak Height |
Concentration (µg/ml) |
Mean of Peak Height |
|
3.125 |
47428.66 |
6.25 |
36717 |
|
6.25 |
91070.33 |
12.5 |
70967 |
|
12.5 |
187478.66 |
25 |
146348.66 |
|
25 |
383848.33 |
50 |
291204.66 |
|
50 |
790578 |
100 |
575751.66 |
Fig. 3: Linear relationship of Levofloxacin hemihydrate.
Fig. 4: Linear relationship of Ornidazole.
Calibration Range: The calibration range of Levofloxacin hemihydrate lied between 3.125 mcg-50 mcg and that of Ornidazole lied between 6.25 mcg- 100 mcg. In between these ranges, the system had a suitable level of precision, accuracy and linearity.
Number of Theoretical Plates: The number of theoretical plates came out to be fairly high, i.e., 1082 and 2051 for the Levofloxacin hemihydrate and Ornidazole respectively, ensuring that the column was reasonably efficient.
Tailing Factor: The tailing factor came out to be 1.26 and 1.11 for Levofloxacin hemihydrate and Ornidazole respectively, indicating that the peaks were sufficiently asymmetric.
Robustness: One consequence of the evaluation of robustness is that a resolution test is established, a resolution of 5.09 ensures that the validity of the analytical procedure is maintained whenever used, Fig. 5.
Fig. 5: Theoretical plate count, Tailing factor and Resolution.
Relative Standard Deviation or Precision: The relative standard deviation or precision came out to be 0.5 and 0.3 for Levofloxacin hemihydrate and Ornidazole respectively. This ensured the repeatability of the method, Fig. 6.
Sample evaluation and peak identification:
The chromatogram of the sample tablets were obtained by the optimized method, Fig. 8. The average retention time of Levofloxacin hemihydrate and Ornidazole came out to be 1.051 and 1.777 respectively. This nearly equals the retention time (Levofloxacin hemihydrate: 1.058 and Ornidazole: 1.775) obtained when the standard compounds were treated with the same optimized HPLC technique, Fig. 9 and Fig. 10.
Fig. 6: Relative Standard Deviation or Precision of the System.
Fig. 8: HPLC chromatogram of Fynal-Oz tab.
Fig. 9: HPLC chromatogram of standard Levofloxacin hemihydrate.
Fig. 10: HPLC chromatogram of standard Ornidazole.
CONCLUSION:
The present method was aimed at developing a simple HPLC method for simultaneous estimation of Levofloxacin hemihydrate and Ornidazole in pharmaceutical dosage form. Both drugs were clearly separated. The retention times for these drugs were 1.051 min and 1.777 min, respectively. The method was found to be linear with a correlation coefficient of 0.999. The number of theoretical plates came out to be fairly high, i.e., 1082 and 2051 for the Levofloxacin hemidrate and Ornidazole respectively. The tailing factor was 1.26 and 1.11 for Levofloxacin hemihydrate and Ornidazole respectively. The relative standard deviation or precision came out to be 0.5 and 0.3 for Levofloxacin hemihydrate and Ornidazole respectively. The present method offers a simple, robust, rapid and sensitive HPLC method for simultaneous estimation of Levofloxacin hemihydrate and Ornidazole in a combined pharmaceutical dosage form. The above method gave good and reproducible results which can be used for the routine analysis of Levofloxacin hemihydrate and Ornidazole in pharmaceutical dosage form.
ACKNOWLEDGEMENT:
The authors are thankful to Vintech Formulation, Karnal, Haryana for providing the gift samples of drugs for this work.
1. Sweetman SC. Martindale- he complete drug reference. Pharmaceutical Press, London. 2007; 35th ed: pp 261.
2. Petri WA Jr. Sulphonamides, trimethoprim-sulfamethoxazole, quinolones, and agents for urinary tract infections. In Goodman and Gillman’s. The pharmacological basis of therapeutics, edited by Brunton LL, Lazo JS and Parker KL. McGraw-Hill, New York. 2006; 11th ed: pp. 1111-1126.
3. Sweetman SC. Martindale- he complete drug reference. Pharmaceutical Press, London. 2007; 35th ed: pp 759.
4. Lyon DJ et al. Rapid HPLC assay of clinafloxacin, fleroxacin, levofloxacin, sparfloxacin and tosufloxacin. Journal of Antimicrobial Chemotherapy. 34(3); 1994: 446-448.
5. Wong FA, Juzwin SJ and Flor SC. Rapid stereospecific high-performance liquid chromatographic determination of levofloxacin in human plasma and urine. Journal of Pharmaceutical and Biomedical Analysis. 15(6); 1997: 765-771.
6. Tobin CM et al. A reverse-phase, isocratic high performance liquid chromatography assay for levofloxacin. Journal of Antimicrobial Chemotherapy. 43(3); 1999: 434-435.
7. Böttcher S et al. An HPLC assay and a microbial assay to determine levofloxacin in soft tissue, bone, bile and serum. Journal of Pharmaceutical and Biomedical Analysis. 25(2); 2001: 197- 203.
8. Cheng FC et al. Pharmacokinetic study of levofloxacin in rat blood and bile by microdialysis and high-performance liquid chromatography. Journal of Chromatography A. 961(1); 2002: 131-136.
9. Djabarouti S et al. Determination of levofloxacin in plasma, bronchoalveolar lavage and bone tissues by high-performance liquid chromatography with ultraviolet detection using a fully automated extraction method. Journal of Chromatography B. 799(1); 2004: 165-172.
10. Merdjan H et al. Measurement of ornidazole by high-performance liquid chromatography. Journal of Chromatography. 273(2); 1983: 475-480.
11. Groppi A, Papa P and Montagna M. Deterrmination of ornidazole in human plasma and red blood cells using high-performance liquid chromatography. Journal of Chromatography. 380(2); 1986: 437-442.
12. Róna K and Gachályi B. Simple liquid chromatographic method for the determination of ornidazole and meteronidazole in human serum. Journal of Chromatography. 420(1); 1987: 228-230.
13. Bakshi M et al. The ICH guidance in practice: stress degradation studies on ornidazole and development of a validated stability-indicating assay. Journal of Pharmaceutical and Biomedical Analysis. 26(5-6); 2001: 891-897.
14. Chepurwar SB et al. Validated HPTLC method for simultaneous estimation of levofloxacin hemihydrate and ornidazole in pharmaceutical dosage form. Journal of Chromatographic Science. 45(8); 2007: 531-536.
Received on 29.08.2011 Modified on 16.09.2011
Accepted on 25.09.2011 © AJRC All right reserved
Asian J. Research Chem. 4(11): Nov., 2011; Page 1784-1788