INTRODUCTION:

Levofloxacin (LEV), 7-fluoro-6-(4-methylpiperazin-1-yl) -10-oxo-4-thia-1-azatricyclo[7.3.1.0] trideca-5(13),6,8,11-tetraene-11-carboxylic acid, is a synthetic chemotherapeutic agent used to treat severe and life threatening bacterial infections and Ornidazole (ORZ), 1-chloro-3-(2-methyl-5-nitro-1H-imidazol-1-yl)propan-2-ol, is a drug that cures some protozoan infections (Figure 1a and 1b). The literature survey reports many analytical methods for quantitative determination of levofloxacin (LEV) and ornidazole (ORZ) alone^1-5 or in combination^6-7 with other drugs including spectrophotometry, high-performance liquid chromatography (HPLC) and reverse phase (RP) HPLC⁸ where the same formulation has been analyzed. The present investigation reports ratiospectra derivative spectrophotometry and high-performance liquid chromatography assays of levofloxacin and ornidazole in two component mixture without a previous separation step.

The utility of the developed methods to determine the contents of both drugs in pharmaceutical formulations is also demonstrated.

MATERIALS AND METHODS:

Apparatus:

Spectrophotometric analysis was carried out on a Shimadzu 1700 double beam spectrophotometer with a fixed slit width (2 nm). The system software of the instrument was used for obtaining the ratio spectra and tracing the 1^st derivative of the ratiospectra. The HPLC system consisted of Lichrom Hitachi Merck model with D-7000 Interface, L-7200 auto sampler, L-7400 UV detector, L-7100 pump and a detector set at 295 nm.

Chemicals and Pharmaceutical preparations:

Pure drug samples of LEV and ORZ was kindly gifted by M/s Zydus Cadila Ltd., Ahemdabad and M/s Alembic Ltd., Vadodara respectively. Chromatographic grade doubly distilled water was used for both the methods. HPLC grade acetonitrile (Rankem), analytical reagent grade triethylamine (Rankem) and orthophosphoric acid were used for HPLC. Pharmaceutical preparations Fynal-OZ^®tablet of Mankind Pharma and Ovel-O^® of Leolife Sciences Pvt. Ltd. were procured from commercial sources. The declared content was 250mg for LEV and 500 mg for ORZ.

Figure 1 (a) Levofloxacin

Figure 1 (b) Ornidazole

Procedure for Ratiospectra Derivative Spectrophotometry:

Preparation of standard stock solutions of LEV and ORZ

LEV and ORZ, 50 mg each, were accurately weighed and dissolved separately in 50 ml of distilled water. Five ml of the above solution was separately diluted to 50 ml with distilled water to produce 100 µg/ml each of LEV and ORZ in distilled water.

Preparation of standard stock solutions of LEV and ORZ for divisor spectra:

Suitable aliquots of the standard stock solutions were diluted with distilled water to produce standard solutions of 5µg/ml for LEV and ORZ to obtain the divisor spectra.

Preparation of binary mixture of LEV and ORZ:

Different binary mixture solutions containing LEV and ORZ in 1:2 ratio (same as commercial preparation) were prepared for calibration curve by diluting different aliquots of the standard stock solutions with doubly distilled water.

Preparation of calibration curve:

The absorption spectra of 5 µg/ml of LEV and 5 µg/ml of ORZ were recorded in the range of 200 nm to 400 nm and stored in the memory of the instrument as divisor spectra. The absorption spectra of the binary mixture solutions of LEV and ORZ were recorded in the range of 200 nm to 400 nm and were stored in the memory of the instrument. The stored spectra of the binary mixture solutions were divided by a previously stored standard divisor spectrum of 5µg/mL of ORZ to get the ratiospectra of LEV. The first derivative of the ratiospectra of LEV were traced with Δ l=3 interval multiplication factor = 3 and the amplitude at 292.8 nm were plotted against the respective concentrations of LEV (Fig. 2a and 2b). Similarly, the absorption spectra of binary mixture were divided by a previously stored standard divisor spectrum of 5 µg/ml of LEV to get the ratiospectra of ORZ. The first derivative of the ratiospectra were traced with ∆l=3 interval, multiplication factor = 4 and the amplitude at 302 nm were then plotted against the respective concentrations of ORZ (Fig. 3a and 3b). The method was validated for linearity range, slope, intercept, regression coefficient the results are summarized in Table 1.

Figure 2 (a) Ratiospectra of LEV obtained when mixture spectra of LEV and ORZ in ratios 1:2, 2:4, 4:8, 6:12, 8:16, and 10:20 were divided by a standard spectrum of 5 µg/ml of ORZ

Figure 2 (b) First derivative of Ratiospectra of LEV obtained when mixture spectra of LEV and ORZ in ratios 1:2, 2:4, 4:8, 6:12, 8:16, and 10:20 were divided by a standard spectrum of 5 µg/ml of ORZ

Table 1 Analytical data from calibration curve for the determination of LEV and ORZ

Parameter	Method A		Method B
Parameter	LEV	ORZ	LEV	ORZ
Linearity range	1-10 µg/ml	2-20 µg/ml	50-5000 ng/ml	5-500 ng/ml
Slope	0.1246	0.0612	0.0007	0.0006
Intercept	0.0091	0.0074	-0.0289	-0.0108
Regression coefficient*	0.9994	0.9999	0.9994	0.9997
Parameter M	292.8	302.0	2.8 min	4.4 min

*n=6; Parameter M means analytical wavelength for Method A and retention time for Method B.

Method A - Ratio spectra derivative spectrophotometry and Method B – HPLC

Table 2 Analysis of tablet Formulation

Tablet	Label Claim (mg)		Amount Found (mg)			% Recovery
Tablet	LEV	ORZ	LEV	ORZ		LEV	ORZ
Method A
Fynal OZ ®	250	500	245.3	491.3	98.13		98.26
Ovel-O ®	250	500	241.3	489.3	96.53		97.86
Method B
Fynal OZ ®	250	500	246.9	495.8	98.76		99.15
Ovel-O®	250	500	245.5	489.8	98.19		97.96

Method A - Ratio spectra derivative spectrophotometry and Method B – HPLC

Figure 3 (a) Ratiospectra of ORZ obtained when mixture spectra of LEV and ORZ in ratios1:2,2:4, 4:8, 6:12, 8:16, and 10:20 were divided by a standard spectrum of 5 µg/ml of LEV

Figure 3 (b) First derivative of Ratiospectra of ORZ obtained when mixture spectra of LEV and ORZ in ratios1:2,2:4, 4:8, 6:12, 8:16, and 10:20 were divided by a standard spectrum of 5 µg/ml of LEV

Analysis of tablets:

A total of twenty tablets were accurately weighed and powdered in a mortar. A quantity equivalent to one tablet was accurately weighed and dissolved in 10 ml of doubly distilled water by magnetically stirring it for 5 minutes. About 10 ml of distilled water was then added and the solution was stirred again for 5 minutes. The mixture was then centrifuged for five minutes and the supernatant was transferred to a 100 ml volumetric flask through Whatman No. 42 filter paper. The residue was washed thrice with 10 ml of doubly distilled water and the combined filtrate was made up to the mark with doubly distilled water. The sample solution thus prepared was diluted with distilled water to get the solution containing about 3.75 µg/ml of LEV and 7.5 µg/ml of ORZ. The above solution was analyzed for the contents of LEV and ORZ using the method described in preparation of calibration curve. The results are summarized in Table 2.

Procedure for HPLC:

Chromatographic conditions:

Analysis was carried out isocratically on a Princeton RP-C₁₈ column (5µ, 150 x 4.6mm) using 0.1%v/v triethylamine-acetonitrile (80:20, v/v)/, (pH 6.0) as a mobile phase at a flow rate of 1.2 ml/min. All solvents were filtered through 0.45 µm Millipore filter paper and degassed in an ultrasonic bath.

Preparation of standard stock solutions of LEV and ORZ:

Standard solutions of 1 mg/ml each of LEV and ORZ were prepared in doubly distilled water. Working solutions have been prepared by further diluting these solutions with doubly distilled water.

Internal standard (ISTD) preparations:

The stock solution of internal standard (enrofloxacin) was prepared by dissolving enrofloxacin in doubly distilled water so as to achieve a concentration of 1 mg/ml (stock solution). A suitable aliquot from this stock solution was taken and diluted with doubly distilled water to achieve 100 µg/ml working solution.

Preparation of Calibration Curve:

Different binary mixture solutions containing LEV and ORZ in 1:2 ratio were prepared by mixing suitable aliquots of the standard stock solutions and diluting it with mobile phase. These binary mixture solutions were chromatographed and the peak areas of these solutions were measured at 2.8 and 4.4 min. corresponding to the retention time of LEV and ORZ respectively (Fig. 4). Calibration curves for both the drugs were plotted separately against the corresponding concentration to obtain the calibration graph. The method was validated for linearity range, slope, intercept, regression coefficient the results are summarized in Table - 2.

Figure 4 HPLC spectra for simultaneous estimation of LEV and ORZ

Analysis of tablets:

A total of 20 tablets were accurately weighed and powdered in a mortar. A quantity equivalent to one tablet was accurately weighed and dissolved in 10 ml of doubly distilled water by magnetically stirring it for five minutes. About 10 ml of distilled water was then added and stirred for further five minutes. The solution was then centrifuged for 5 min at 1000 rpm and the supernatant was filtered into a 100 ml flask through Whatman No. 42 filter paper. The residue was washed with 10 ml of solvent and the volume was made up to 100 ml with doubly distilled water. The sample solution thus prepared was diluted with distilled water to obtain a solution containing 10 µg/ml of LEV and 20 µg/ml of ORZ. The above solution was analyzed for the content of LEV and ORZ using the method described in preparation of calibration curve. The results are summarized in Table-2.

RESULTS AND DISCUSSION:

Ratiospectra derivative spectrophotometry and HPLC methods for the simultaneous determination of LEV and ORZ from their binary mixture were successfully developed.

The ratiospectra derivative spectrophotometry showed good linearity for LEV in the range of 1 to 10 µg/ml with correlation coefficient of 0.9994 and 2 to 20 µg/ml and 0.9999 for ORZ. The results are summarized in Table – 1.

The instrumental parameters like divisor spectra and smoothing factor (∆l) of tracing the first derivative of ratiospectra were optimized for the reliable determination of subject components. Some divisor concentrations were tested selecting the standard solution as divisor at an appropriate concentration, and it was observed that the standard solution of 5µg/mL of LEV and ORZ was suitable for determination of ORZ and LEV. The smoothing factors ∆l=3 nm for LEV and ∆l=4 nm for ORZ were found to be optimum for tracing the first derivatives of ratiospectra as far as linearity and sensitivity is concerned. The results are summarized in Table -3. The experiment was repeated six times in a day for intra-day and on six different days for inter-day precision. The method was found to be precise as % RSD for intra-day and inter-day precision were 0.864 and 0.661 for LEV and 0.938 and 0.834 for ORZ respectively as shown in Table - 4 The accuracy of the method was determined by performing recovery studies by standard addition method in which pre-analyzed samples were taken and standard drug was added at three different levels. As evident from the results shown in Table – 5 the % recovery±SD lies in the range of 96.09±0.612 to 102.48±1.149 for LEV and 94.67±0.756 to 97.47±0.543 for ORZ. The analysis of commercially available tablet formulations reveals satisfactory results of % recovery i.e., 98.13, 96.53 for LEV and 98.26, 97.86 for ORZ as evident from the results shown in Table - 2.

A simple and rapid HPLC method was also developed for the simultaneous estimation of LEV and ORZ using Princeton RP-C₁₈(5µ, 150 x 4.6 mm) column. The linearity was observed in the range of 50-5000 ng/ml with correlation coefficient of 0.9994 for LEV and 5-500 ng/ml with correlation coefficient of 0.9997 for ORZ. Accuracy of the method was determined by recovery studies (n = 5). Recovery data from the study are reported in Table - V and the % recovery was found to be satisfactory in the range of 98.10±0.438 to 100.18±0.546 for LEV and 96.73±0.568 to 100.19±0.543 for ORZ. The method was found to be precise as % RSD for intra-day and inter-day precision were 2.466 and 0.8856 for LEV and 1.9585 and 1.647 for ORZ respectively as shown in Table – 6. The content of drugs in the commercial formulations was found to be 98.76% and 98.19% for LEV and 99.15% and 97.96% for ORZ (Table - 2). The estimated amount was within the acceptable limits of the labeled claim of the formulation.

Table 3 Optimization of Method Parameters

Method A		Method B
Method Parameter	Optimized Values	Method Parameter	Optimized Values
Scanning Parameter	200-400 nm	Column	Princeton RP-C₁₈column (5µ, 150 x 4.6 mm)
Slit Width	2 nm	Mobile Phase	0.1%v/v triethylamine:acetonitrile (80:20) (pH 6)
Scan Speed	Fast	Flow rate	1.2 ml / min
∆ λ for tracing 1^st derivative	3 nm	Detection	U.V. 295 nm
Multiplication factor	3 for LEV	Injection volume	50 µl
Multiplication factor	4 for ORZ	Injection volume	50 µl
Divisor spectra for determination of LEV and ORZ	5µg/ml	Retention time	2.8 min for LEV, 4.4. min for ORZ (ISTD 5.5. min)
Analytical Wavelength for determination of LEV	292.8 nm for LEV	Run time	9 min
Analytical Wavelength for determination of LEV	302 nm for ORZ	Run time	9 min

Method A - Ratio spectra derivative spectrophotometry and Method B – HPLC

Table 4 Intraday and Inter day precision for determination of LEV and ORZ for Ratio Spectra Derivative Spectrophotometry

Sr. No.	Conc. of LEV µg/ml	*Amplitude at 292.8 nm	% RSD	Conc. of ORZ µg/ml	*Amplitude at 292.8 nm	% RSD
Intraday Precision
1.	1	0.134	1.542	2	0.135	1.118
2.	2	0.266	1.527	4	0.257	0.928
3.	4	0.506	0.799	8	0.495	0.839
4.	6	0.745	0.511	12	0.741	0.607
5.	8	1.029	0.483	16	0.992	1.195
6.	10	1.250	0.322	20	1.234	0.839
		Average of % RSD	0.864			0.938
Inter day Precision
1.	1	0.134	0.847	2	0.133	2.083
2	2	0.264	0.494	4	0.253	0.761
3	4	0.504	0.556	8	0.495	0.438
4	6	0.747	0.658	12	0.741	0.528
5	8	1.025	1.175	16	0.990	0.429
6	10	1.257	0.336	20	1.246	0.765
		Average of % RSD	0.661			0.834

*Average of five readings

Table 5 Recovery Study from Fynal-OZ® Tablet

S. No.	% Standard Addition	Sample Concentration LEV : ORZ µg/ml	%Recovery±SD LEV	%Recovery±SD ORZ
Method A
1	80	0.00375:0.0075	96.09±0.612	96.67±0.958
2	90	0.00375:0.0075	101.17±0.694	97.47±0.543
3	100	0.00375:0.0075	102.48±1.149	95.79±0.943
4	110	0.00375:0.0075	98.80±0.978	95.75±0.513
5	120	0.00375:0.0075	98.66±0.845	94.67±0.756
Method B
1	80	1000:2000	98.10±0.438	100.19±0.543
2	90	1000:2000	98.62±0.677	99.61±0.629
3	100	1000:2000	100.18±0.546	96.73±0.568
4	110	1000:2000	98.63±0.753	97.33±0.573
5	120	1000:2000	98.69±0.642	97.20±0.342

Method A - Ratio spectra derivative spectrophotometry and Method B – HPLC

Table 6 Intraday and Inter day precision for determination of LEV and ORZ for High Performance Liquid Chromatography

Sr. No.	Conc. of LEV ng/ml	Height/Height (ISTD)*	% RSD	Conc. of ORZ µg/ml	*Height/Height (ISTD)	% RSD
Intraday Precision
1.	50	0.027	3.075	50	0.028	2.525
2.	100	0.046	2.822	100	0.042	2.689
3.	150	0.097	3.813	150	0.090	2.760
4.	300	0.200	2.803	300	0.158	2.550
5.	500	0.323	2.267	500	0.264	1.276
6.	1000	0.681	1.221	1000	0.553	1.373
7.	3000	2.164	1.115	3000	1.678	1.218
8.	5000	3.666	2.614	5000	2.829	1.277
		Average % RSD	2.466			1.9585
Inter day Precision
1.	50	0.026	2.0747	50	0.026	2.719
2.	100	0.042	2.381	100	0.043	1.936
3.	150	0.098	2.837	150	0.084	2.284
4.	300	0.190	2.317	300	0.158	1.973
5.	500	0.328	2.537	500	0.260	0.884
6.	1000	0.654	0.983	1000	0.551	1.407
7.	3000	2.166	0.558	3000	1.678	0.692
8.	5000	3.581	3.610	5000	2.637	1.283
		Average % RSD	0.8856			1.647

*Average of five readings

CONCLUSION:

Ratiospectra derivative spectrophotometry and high-performance liquid chromatography are suitable techniques for the reliable analysis of commercial formulation containing combinations of levofloxacin and ornidazole

The most striking features of the ratiospectra derivative spectrophotometry are its simplicity and rapidity which renders it suitable for routine analysis in control laboratories. The HPLC method gives a good resolution between levofloxacin and ornidazole within a short analysis time of 9 min and with sensitivity of 50 ng/ml. The HPLC method was shown to be a versatile method and may offer advantages over the derivative method for selective determination of the drug in their combinations.

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Received on 30.04.2010 Modified on 16.05.2010

Asian J. Research Chem. 3(4): Oct. - Dec. 2010; Page 922-927

Parag R. Patel1, Vandana B. Patel2* and Mayank Bapna2

INTRODUCTION:

REFERENCES:

Parag R. Patel¹, Vandana B. Patel²*and Mayank Bapna²