SPECTROPHOTOMETRIC METHOD FOR SIMULTANEOUS ESTIMATION OF LEVOFLOXACIN AND ORNIDAZOLE IN TABLET DOSAGE FORM

Chepurwar S. B., Shirkhedkar A. A.*, Bari S. B., Surana S. J.

*Address for Correspondence R.C. Patel College of Pharmacy, Karvand Naka, Shirpur 425405 Dist: Dhule (M.S.) Ph.no. 09822357630 Email: atul_shir@indiatimes.com

ABSTRACT
Two simple, rapid, accurate and precise spectrophotometric methods have been developed for simultaneous estimation of levofloxacin and ornidazole from tablet dosage form. Method 1 involves, formation of Q-absorbance equation at 298(isoabsorptive point) and 320 nm ( max of Ornidazole); while Method 2 Multicomponent mode of analysis involves, the measurement of absorbances at two wavelengths 289 nm ( max of LEV) and 320 nm ( max of ORN) in distilled water. The linearity lies between 4 - 20 mcg/mL for levofloxacin and 4 - 40 mcg/mL for Ornidazole. The results of recovery studies confirm the accuracy of the proposed methods.

Keywords
Levofloxacin, Ornidazole, Spectrophotometry, Q- absorbance ratio method, Multicomponent mode of analysis

INTRODUCTION
Levofloxacin (LEV), (-)-(S)-9-fluro-2, 3-dihydro-3methyl-10-(4-methyl-1piperzinyl)-7-oxo-7H pyrido [1, 2, 3-de]-1, 4-benzoxazine-6 carboxylic acid hemihydrate is synthetic fluorinated quinoline derivative.1 It is used mainly as an antibacterial.2 Ornidazole (ORN), [1-chloro-3-(2-methyl-5-nitroimidazole -1-yl) 2-propanol] is 5- nitroimidazole derivative with antiprotozoal properties against anaerobic bacteria.3 Literature survey revealed that visible spectrophotometric4; HPLC5 methods are available for estimation of LEV from pharmaceutical formulations and spectrophotometric formulations. These two drugs are not official in any Pharmacopoeia; hence, no official method is available for the estimation of LEV and ORN in formulations. In present communication, we propose fast, simple and accurate
6, 7

methods for estimation of ORN from pharmaceutical

spectrophotometric methods for simultaneous estimation of both the drugs in tablet dosage form.

EXPERIMENTAL
Instrument 1. UV-Visible spectrophotometer (1601 Shimadzu). a. Spectral bandwidth 2nm b. Wavelength accuracy 0.5nm 2. 10mm matched quartz cells. Solvent Distilled water

Procedure Method 1: Q-absorbance method 8 Q-absorbance method uses the ratio of absorbances at two selected wavelengths; one at isoabsorptive point and other being the
max

of one of the two

components. The standard stock solution of strength 100 mcg/mL of LEV and ORN were prepared in distilled water separately. From the overlain spectra of LEV (10 mcg/mL) and ORN (20 mcg/mL), two wavelengths 298 nm (isoabsorptive point) and 320 nm (
max

of ORN) were selected for the formation of Q-absorbance equation

(fig.1). Different aliquots were taken from the stock solutions and diluted with the same solvent to prepare a series of concentrations. The calibration curves were found to be linear in the concentration range of 4 - 20 mcg/mL for LEV and 4 - 40 mcg/mL for ORN. The absorbances of LEV and ORN were measured at 298 nm (isoabsorptive point) and
max

of ORN is at 320 nm respectively and the absorptivity coefficients of

each drug at both wavelengths were determined and presented in Table I. The concentration of two drugs in the mixture can be calculated by, using equations CLEV = QM Qy/QX QY A1/ax1 --------------------------------------- ------ (1) CORN = QM X/QY Qx A 1 /ay1 ----------------------------------------------(2) Q Where, A1 and A2 are absorbances of mixture at 298 nm and 320 nm, and ax1 (606.17), ax2 (212.17) and ay1 (303.08), ay2 (361.5) are absorptivities E (1%, 1cm) of LEV and ORN at 298 nm and 320 nm respectively and QM = A2/A1, QY = ay2/ay1 and QX = ax2/ax1. Method 2: Employing Multicomponent Mode of Analysis of the Recording Spectrophotometer. (Shimadzu 1601)

The Seven mixed standard solutions with concentration of LEV and ORN in the ratio of 20:0, 0:40, 4:8, 8:16, 12:24, 16:32, 20:40 (mcg/mL) were prepared in distilled water. All the mixed standard solutions were scanned over the range of 400 200 nm, in the multicomponent mode; using two sampling wavelength 289 nm (( of LEV) and 320 nm ((
max max

of ORN). Overlain spectra of the mixed standard

solutions are given in fig.2. The spectral data from these scans was used to determine the concentration of two drugs in tablet sample solutions. Preparation and analysis of tablet formulations Twenty tablets were weighed and ground to a fine powder. A quantity equivalent to 10 mg of LEV and 20 mg of ORN was accurately weighed and transferred to a 100 mL volumetric flask; 20mL of distilled water was added and sonicated for 10 min.. The solution was filtered through whatman filter paper (no.41) and the volume was made up to 100 mL, using same solvent. The resulting solution was further diluted to get concentration of 8 mcg/mL of LEV and 16 mcg/mL of ORN. Absorbances of the sample solutions were recorded, at 298nm and 320nm i.e.A1 and A2 respectively. And, the concentrations of two drugs in sample were determined, by using equation 1 and 2. Also, these solutions obtained were subjected to analysis in the multicomponent mode of instrument. These solutions were scanned over the range of 400 - 200 nm wavelength and the concentration of each drug was determined by analysis of spectral data of the sample solutions with reference to the mixed standards. The analysis procedure was repeated five times with tablet formulations. The results of analysis of tablet formulations are presented in the Table II

Recovery studies The recovery studies were carried out by adding known amount of standard solution of LEV and ORN to preanalysed tablet solutions. The resulting solutions were then analysed by proposed methods. The results of recovery studies were found to be satisfactory and the results are presented in Table III.

RESULTS AND DISCUSSION

The proposed methods were found to be simple, accurate, economical and rapid for routine analysis of levofloxacin and ornidazole in combined dosage forms. The accuracy of the method was determined by calculating mean percentage recovery. Precision was calculated as repeatability (% R.S.D.) and inter and intra day variation (% R.S.D.) for both the drugs. The repeatability data, ruggedness data are presented in Table IV. Both the methods, were successfully used to estimate the amount of LEV and ORN present in the marketed formulation.

ACKNOWLEDGEMENTS
The authors are thankful to M/S. Glenmark Pharmaceuticals, M.I.D.C., Satpur, Nashik for providing gift samples of drugs for this work and R.C. Patel College of Pharmacy for providing necessary facilities.

REFERENCES
1. Moffat, Osselton and Widdop., Clarkes Analysis of Drugs and Poisons, 3rd Edn., vol 2, The Pharmaceutical press, Great Britain, 2004, 1172. 2. Goodman and Gilmans, The Pharmacological Basis of Therapeutics, 10th Edn., McGraw-Hill, New York, 2001, 1129, 1183. 3. Reynolds, J.E.F., In Martindale the extra pharmacopoeia, 30th Edn, The Pharmaceutical press, London, 1993, 523. 4. Sivasubramanian L., Kasi Sankar v., Sivaraman V., Senthil Kumar K., Muthukumaran A. and Raja T, K: Visible spectrophotometric determination of levofloxacin in Tablet dosage forms, Indian J Pharm Sci., Nov-Dec 2004, 66(6), 799-802. 5. Bottcher, S., Baum, H. V., Hoppe-Tichy, t., Benz, C., Sonntag, H. G: An HPLC assay and microbiological assay to determine levofloxacin in soft tissue, bone, bile, and serum., J Pharm Biomed Anal., 2001, 25(2), 197 -203. 6. Kasture V.S., Bhagat A.D., Puro N.C., More P. S., and Bhandari N.K:

Spectrophotometric method for simultaneous estimation of ofloxacin and ornidazole in tablet dosage form, Indian drugs, 2004, 41(1), 51-53. 7. Patel P, U, Suhagia B. N., Patel C. N., Patel M, M., Patel G. C. and Patel G, M: Simultaneous spectrophotometric estimation of gatifloxacin and ornidazole in mixture, Indian J Pharm Sci, 2005, 67(3), 356-357. 8. Beckett, A. H. and Stenlake, J. B., Practice Pharmaceutical Chemistry, 4th Edn., vol 2, CBS publisher, New Delhi, 1997, 285.

TABLE I Absorptivity Values (E 1%, 1cm) of LEV and ORN at 298 (isoabsorptive point) nm and 320 nm (Method 1)

Sr. No.

Absorptivity at 298.0 nm

Absorptivity at 320.0 nm

LEV 1 2 3 4 5 Mean S. D. 606.37 605.37 607 605 607.12 ax1 == 606.17 0.9539

ORN 303.18 302.68 303.50 303.50 303.56 ay1=303.08 0.3691 212.5 212 212.8 213.3

LEV 362.2 360.9 361.5 361.1 362

ORN

211.25 ax2= 212.17 0.7839

ay2=361.5 0.5594

Table II Tablet formulation analysis data for Method 1 and 2

Method 1 9

Method 2

*Amount Sample Label claim found (%Label claim) Brand-I LEV 250mg ORN 500mg Brand-II LEV 250mg 99.09 99.29 99.24 0.93 0.52 0.58 0.93 0.52 0.58 0.44 0.41 0.23 0.26 0.19 S.D. %R.S.D. S.E.

*Amount found (%Label claim) 98.45 98.79 98.89 98.36 0.61 0.77 0.68 0.98 0.62 0.77 0.69 1.0 0.27 0.34 0.30 0.44 S.D. %R.S.D. S.E.

ORN 500mg 99.10 0.44 *mean of five estimations

Table III Recovery Studies (For method 1 and 2) Sample No. Method 1 Quantity of Drug Added % (mcg/mL ) LEV ORN Recovery* % Method 2 Recovery*

S.D. LEV ORN

S.D. LEV ORN

10

Brand -I

6.4 8.0 9.2

12.8 16.0 19.2

100.37 0 .68 99.73 0.95 99.86 0.27

99.94 0.37 99.85 0.30 100.33 0.68

99.82 0.88 100.12 0.62 100.1 0.47

99.91 .28 99.83 0.47 100.03 0.21

Brand -II

6.4 8.0 9.2

12.8 16.0 19.2

99.91 0.25 98.27 0.97 98.65 1.20

100.02 0.68 98.89 0.40 98.72 0.49

99.82 0.88 100.12 0.62 100.1 0.47

99.91 0.28 99.83 0.47 100.03 0.21

*Mean of three estimations at each level

Table IV Repeatability and Ruggedness data Parameters Repeatability 0.76 (%RSD (n=5) 0.28 0.67 0.22 Method 1 LEV ORN Method 2 LEV ORN

Precision (%RSD) 0.21-0.072

11

Intra-day (n=3)

0.16-0.049

0.19-0.067

0.19-0.052

Inter-day (n=3) Ruggedness (%RSD)

0.15-0.072

0.15-0.045

0.13-0.070

0.17-0.057

Analyst 1 Analyst 2

1.53 1.26

0.62 0.75

1.35 1.21

0.66 0.79

Fig 1:

Overlain spectra of LEV and ORN I: Isoabsorptive point, LEV: Levofloxacin, ORN: Ornidazole

12

Fig. 2 Multicomponent Analysis of Mixed Standards

13

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