INTRODUCTION:

The Levofloxacin hemihydrate (LEVO) 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 ¹ the bacteriologically active L-isomer of the racemic fluoroquinolone ofloxacin. is a broad-spectrum antimicrobial agent currently under investigation by the R.W. Johnson Pharmaceutical Research Institute for the management of a variety of bacterial infections and is official in Indian Pharmacopoeia 2007.²

Ornidazole 1-(3-chloro-2- hydroxypropyl)-2-methyl-5nitroimidazole is an antiamoebic to Entamoeba histolytica.¹ Both the drugs are formulated in a binary solid dosage form for the treatment of a wide variety of infections caused by susceptible gram-positive and gram-negative organisms along with anaerobes and protozoa. LEVO was determined by UV spectrophotometry^3-4. flow injection analysis using UV detection. potentiometry and conductometry ^5.high-performance liquid chromatography.^6-10 ONZ was determined by UV spectroscopy¹¹ . polarography ¹² and high performance liquid chromatography^13-15. .

Literature survey revealed that single spectrophotometric ¹⁶.HPLC¹⁷ and HPTLC¹⁸ method has been reported for the simultaneous estimation of both these components in a combined dosage form. The aim of this paper was to develop the simultaneous equation (Vierodt’s) method and the absorbance ratio (Q-analysis) method for estimating LEVO and ONZ simultaneously in their mixture form. In the proposed methods no separation is required; the methods are fast and convenient.

MATERIALS AND METHODS:

Instrument .reagents and chemicals:

A dual-beam Shimadzu UV-visible spectrophotometer 1700 Pharmaspec was used. Freshly prepared 0.1M HCl (AR. E. Merck. Mumbai) in doubled distilled water was used as a solvent. Gift samples of LEVO and ONZ were procured from Cipla Pharmaceutical Pvt LTD. Mumbai. India and Suyash Laboratories LTD. Mumbai respectively.

Spectral and linearity characteristics of LEVO and ONZ:

Standard stock (100 µg/ ml) of LEVO and ONZ were prepared in 0.1 M HCl. The aliquot portions (0.2. 0.4. 0.6. 0.8. 1. 1.2. 1.4. 1.6.1.8. 2) from the 100 µg/ ml LEVO and ( 0.4.0.8.1.2.1.6.2.2.4.2.8.3.2.3.6.4) from the 100 µg/ ml ONZ working solutions were accurately transferred to 10-ml volumetric flasks. the volume was completed with 0.1 M HCl. The absorption spectra between 200- 400 nm of all solutions of LEVO and ONZ were measured at 293 nm

Table-I: Result of tablet assay for content of LEVO and ONZ by using the proposed methods.

	Simultaneous equation method	Graphical absorbance ratio method
	Recovery(%)+S.D.(n=6) LEVO ONZ	Recovery(%)+S.D.(n=6) LEVO ONZ
Tablet	100.02 + 0.01846 98.27 + 0.4248	99.75 + 1.43 98.37 +1.17
RSD (%)	0.0.01841 0.4241	1.41 1.15

S.D.: Standard deviation. RSD: Relative Standard Deviation

Table-II: Result of recovery study for the simultaneous estimation of LEVO and ONZ in tablet by using the proposed methods. (n=6)

Level of %recovery	Recovery of added standard (%) + S.D. LEVO ONZ
	SEM	GRM	SEM	GRM
80	99.96 +0.0076	98.19 +0.0124	98.04 +0.0051	99.85 +0.0089
100	100.23 +0.0031	98.71 +0.0146	100.05 +0.0035	99.49 +0.0030
120	99.26 +0.0043	99.25 +0.0228	99.18 +0.0761	99.66 +0.0069
% Mean recovery (Mean S.D)	99.82+ 0.0050	98.72+ 0.0166	99.09+ 0.0284	99.67 +0.0063

SEM- Simultaneous equation method, GRM-Graphical absorbance ratio method

Table-III: Data for calibration graph for LEVO and ONZ using Simultaneous equation method. Absorbance ratio method.

Parameters

Simultaneous equation method

Absorbance ratio method

Levofloxacin

Slope(m)*

Intercept(c)*

Correlation coefficient

Beer’s law limit(μg/ml)

LOD µg/ ml)

LOQ µg/ ml

0.800 +0.00004

0.02781 +0.00012

0.9994

2-14

1.41

4.29

0.2828 +0.00005

0.00726 + 0.00015

0.9992

2-20

6.3

19.14

Ornidazole

Slope

Intercept

Correlation coefficient

Beer’s law limit(μg/ml)

LOD(µg/ ml)

LOQ(µg/ ml)

0.268 + 0.00008

0.05785 +0.0035

0.9993

4-36

3.59

10.87

0.265 + 0.0006

0.01798 + 0.00031

0.9992

4-40

2.24

6.80

*Mean + standard deviation

Table-IV: Precision of spectrophotometric methods developed for analysis of tablet (n=6)

Simultaneous equation method

Absorbance ratio method

Levofloxacin

Intra day

Amount found(mean %+S.D)

Precision. RSD (%)

Inter day

Amount found(mean %+S.D)

Precision. RSD (%)

99.8+ 0.0047

0.6289

99.35 +0.0088

1.1746

99.69 + 0.0092

0.7778

98.64 + 0.0064

0.5371

Ornidazole

Intra day

Amount found(mean %+S.D)

Precision. RSD (%0

Inter day

Amount found(mean %+S.D)

Precision. RSD (%0

99.9 + 0.0039

0.6372

99.8 + 0.0026

0.4216

99.36 + 0.0028

0.3553

99.45 +0.0070

0.9047

(λ max for LEVO). 277 nm (λmax for ONZ). 270nm (isobestic wavelength).

Application of the proposed procedures for the determination of LEVO and ONZ in tablets:

The average weight quantity (0.123 gm) of twenty tablets (FINAL-OZ. Mankind Pharma. Ltd; content- Levofloxacin 250 mg and Ornidazole 500 mg) was transferred to 100 ml volumetric flask and dissolved in 0.1M HCl by intermittent shaking and volume was made up to 100 ml with the same solvent. The solution was then filtered through a Whatmann filter paper (No. 41). The solution was diluted with 0.1M HCl to obtain 2 μg/ml of LEVO and 4 μg/ml of ONZ .This sample was scanned over the range of 200nm to 400nm in spectrum mode for the estimation of both these drugs by using simultaneous equation. absorbance ratio method (table-I). The analysis procedure was repeated six times (n=6).The selectivity of the proposed procedures was examined by determining the recovery of the two drugs by standard addition method (table-II).

Fig.1: Overlain spectra of Levofloxacin (LEVO) and Ornidazole (ONZ)

RESULTS AND DISCUSSION:

Simultaneous equations method:¹⁹

The absorptivity values of the drugs were determined at the λ max of LEVO and ONZ respectively. The absorptivity value of the drugs is the ratio of absorbance at selected wavelengths with concentration of drugs in µg/ml. A set of two simultaneous equations were framed using these absorptivity values.

A1 = 8502 C1 + 2019C2 ---------- (at -293)

A2 = 4271C1 + 3049 C2---------- (at -277)

Where, A1 and A2 are absorbance of the sample solution at 293nm and 277 nm respectively. 8502 and 4271 are absorptivities of LEVO at 293 nm and 277 nm. respectively. 2019 and 3049 are the absorptivities of ONZ at 293 nm and 277 nm. respectively. C1 is the concentration of the LEVO and C2 is the concentration of the ONZ in µg/ml.

The graphical absorption ratio method (Q- analysis): ¹⁹

In quantitative assay of two components by Q-analysis method. absorbances were measured at the isobestic wavelength and maximum absorption of one of the two components. From overlain spectra of LEVO and ONZ shown in figure (Fig.1). absorbances were measured at the selected wavelengths i.e. 270 nm (isobestic wavelength) and 293nm (wavelength of maximum absorption of LEVO). From the following sets of equations. the concentration of each component in sample can be calculated.

Concentration of LEVO = (Q_M– 0.7272) A / 2.2145*ax₁

Concentration of ONZ = (2.9418- Q_M) A / 2.2145*ay₁

Where, A = Absorbance of sample solution at isobestic wavelength 270 nm. Q_M = Ratio of absorbance of sample solution at 293 nm to absorbance of sample solution at isobestic wavelength 270 nm. ax₁= absorptivity of LEVO at 270nm. (Isobestic wavelength); ay₁= absorptivity of ONZ at 270nm (isobestic wavelength)

Method Validation 20

The methods were validated with respects to linearity. limit of detection (LOD). limit of quantification (LOQ). precision. accuracy (recovery) and sensitivity. The results obtained are summarized in table-II. table-III and IV. respectively.

CONCLUSION:

The proposed methods were found to be simple. accurate. economical and rapid for routine simultaneous estimation of two drugs. All the methods were found to be economical. as they require only 0.1 M HCl as a solvent. The results obtained for tablets and recovery study is summarized in table I. and II respectively. The results of validation parameters shown in table III and IV are satisfactory. indicates the accuracy of proposed methods for estimation of LEVO and ONZ. These methods also gave excellent result and can be employed for routine analysis of these two drugs in combined dosage form.

ACKNOWLEDGEMENT:

The authors wish to extend thanks to the Principal. Government college of Pharmacy. Karad for providing necessary facilities. The authors also thankful to Cipla Pharmaceutical Pvt LTD. Mumbai. India and Suyash Laboratories LTD. Mumbai for providing gift samples of drugs LEVO and ONZ.

REFERENCES

1. Sweetman S.C. Martindale: The complete drug reference; 34^thed. pharmaceutical press London. 2004: 225.612.

2. Indian Pharmacopoeia. Vol. II. Government of India. The controller of Publication. New Delhi. 2007: 1296.

3. Siva Subramanian L. Kasi S V. Raja T.K. Visible spectrophotometric determination of levofloxacin in tablet dosage forms. Pak J. Pharm. Sci. 2007:20(2): 100-106.

4. Amina Mohamed El-Brashy. Mohamed El-Sayed Metwally. Spectrophotometric Determination of Some Fluoroquinolone Antibacterial by Ion-pair Complex Formation with Cobalt (II) Tetrathiocyanate. Journal of the Chinese Chemical Society.2005:52:77-84

5. Altookka G. Atkosar Z. Can N.O. The determination of Levofloxacin by flow injection analysis using UV detection. potentiometry and conductometry in pharmaceutical preparations. Journal of Pharmaceutical and Biomedical Analysis. 2002:30: 881-885

6. Santoro R.M. Kassab N. M. Anil K. .Quantitative determination of gatifloxacin. Levofloxacin. lomefloxacin and pefloxacin fluoroqunolonic antibiotics in pharmaceutical preparations by high-performance liquid chromatography. Journal of Pharmaceutical and Biomedical Analysis. 2006:40:179-184

7. Siewert S. Validation of levofloxacin HPLC assay in plasma and dialysate for pharmacokinetic studies. Journal of Pharmaceutical and Biomedical Analysis. 2006:41:1360-1362

8. Wong F.A. Juzwin S.J. Flor S.C. Rapid Stereospecific high-performance liquid chromatographic determination of levofloxacin in human plasma and urine. Journal of Pharmaceutical and Biomedical Analysis. 1997:15:765-771

9. Giuseppe Carlucci. Analysis of fiuoroquinolones in biological fluids by high-performance liquid chromatography. Journal of Chromatography A. 1998: 812:343-367.

10. Bottche S. R. Baum H.V. C.Benz. Sonntag H.G. An HPLC assay and a microbiological assay to determine levofloxacin in soft tissue, bone, bile and serum. Journal of Pharmaceutical and Biomedical Analysis. 2001: 25: 197-203

11. Shingare M.S. Kale U.N. Spectrophotometric determination of ornidazole and norfolxacin on tablet. Indian journal of pharmaceutical sciences.2003:65:523-526.

12. Brooks M.A. D’arconte et al.Journal of pharmaceutical science.vol 1976:65:112

13. Han-Wen Sun. Feng-chi Wang. Lian-Feng Ai. Simultaneous Determination of seven nitriimidazole residues in meat by using HPLC-UV detection with solid-phase extraction Journal of Chromatography B. 2007:857:.296-300.

14. K.Rong B. Galchalyi. Simple liquid chromatographic method for determination of Ornidazole and Metrinidazole in human serum. Journal of Chromatography. 2005:420:228-230

15. A Groppi. P Papa. G. Carosi. Detrmination of Ornidazole in human plasma and red blood cells using HPLC. Journal of Chromatography.1986:380:437-442

16. Chepurwar S.B. Shirkhedkar A.A. Bari S.B. and Surana S.J. Simultaneous Spectrophotometric Determination of Levofloxacin and Ornidazole from Combined Dosage Form. Asian Journal of Chemistry.2007.19.1628-1630

17. Patel S.A. Prajapati. A M. Paresh U. P. Development and validation of column high-performance liquid chromatographic and derivative spectrophotometric methods for determination of levofloxacin and Ornidazole in combined dosage forms. AOAC Int 91:756-61

18. Chepurwar S.B.. Shirkhedkar A.A. Bari S.B. and Surana S.J. Validated HPTLC method for simultaneous estimation of Levofloxacin and Ornidazole in pharmaceutical dosage Form. Journal of chromatographic science. 2007:.45:. 531-536.

19. Beckett A.H. Stenlake J.B. Practical pharmaceutical chemistry part II. 4 ^thed New Delhi: CBS publishers and distributers.1997:281-306

20. ICH Topic Q2A. Validation of analytical procedures methodology. CPMP/ ICH/ 281/ 1995.