Publication Ref No.

: IJPRD/2010/PUB/ARTI/VOV-2/ISSUE-6/AUG/004

ISSN 0974 9446

DEVELOPMENT AND VALIDATION OF UV SPECTROPHOTOMETRIC METHOD FOR THE DETERMINATION OF METRONIDAZOLE IN TABLET FORMULATION Arun K. Mishra1,2*, Rajdhar Yadava1, Amrita Mishra1, Anurag Verma1, Pronobesh Chattopadhyay1 Central Facility of Instrumentation, Department of Pharmacy, IFTM, Lodipur- Rajput, Moradabad 244001, India 2 Institute of Pharmacy, Bhagwant University, Ajmer 305004, India Email: arun_azam@rediffmail.com
1

Arun K. Mishra

ABSTRACT The present research work discusses the development of a UV spectrophotometric method for Metronidazole. Simple, accurate and cost efficient spectrophotometric method has been developed for the estimation of Metronidazole (MND) in Tablet dosage form. The optimum conditions for the analysis of the drug were established. The maximum wavelength ( max) was found to be 277nm. The percentage recovery of Metronidazole was in the 98.20.129. Beers law was obeyed in the concentration range of 1-10g/ml. Calibration curves shows a linear relationship between the absorbance and concentration. The line equation y=0.0283x+0.0407 with r2 of 0.9902 was obtained. Validation was performed as ICH guidelines for Linearity, accuracy, precision, LOD and LOQ. The sample solution was stable up to 36 hours. The proposed method may be suitable for the analysis of MND in tablet formulation for quality control purposes.

Key Words: Metronidazole (MND), UV determination, Tablet dosage form. INTRODUCTION Metronidazole [2-(2-methyl-5-nitro1H- imidazol-1-yl) ethanol]1 is an amebicide antiprotozoal and antibiotic effective against anaerobic bacteria and certain parasites. It is the drug of choice for first episodes of mild-tomoderate Clostridium difficile infection.2 Metronidazole exerts rapid bactericidal effects against anaerobic bacteria, with a killing rate proportional to the drug concentration. Concentration-dependent killing has also been

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observed with Entamoeba histolytica and Trichomonas vaginalis. Metronidazole kills Bacteroides fragilis and Clostridium perfringens more rapidly than clindamycin.3,4

Metronidazole (Gift sample by RPG Life sciences. Ltd. Mumbai), Methanol, Potassium dihydrogen phosphate, Hydrochloric acid, Sodium hydroxides were purchased from CDH (P) Ltd. New Delhi. STANDARD SOLUTION OF METRONIDAZOLE Standard Metronidazole (10 mg) was accurately weighed and transferred to 10 ml volumetric flask. It was dissolved properly and diluted up to the mark with phosphate buffer (pH 7.4) to obtain concentration of 1 mg/ml. This solution was used as working standard solution. From this solution, by suitably dilution, 20 g/ml concentrations was obtained and used as standard solution. For calibration curve, dilution was made from 20g/mL. MEASUREMENT OF ABSORBANCE AND CALIBRATION CURVE The absorbance of the solutions containing MND at 10 g/ml was determined in the UV range 200-800nm using an appropriate blank. The max was found to be 277nm. At these wavelength maxima, calibration curve was drawn by plotting graph between absorbance and concentrations. (Fig 1) VALIDATION OF METHOD PARAMETERS10,11 LINEARITY The aliquots of concentration ranging 1-20 g/mL were prepared in triplicate, but linearity was found to be between 1-10g/ml concentrations. The linearity was calculated by the least square regression method (Table 2). PRECISION The precision of the assay was determined by repeatability (intraday) and intermediate precision (inter-day) and reported as RSD %. For this, 5g/ml and 6g/ml concentration solution was measured three times in day and

Metronidazole . Review of Literature for MND analysis revealed that several existing methods including different technique such as HPLC with UV detection5, GC-FID6, HPLC PDA/MS7 ,UPLC-MS8 assay for its quantification in plasma and gastric juice fluids9 have been reported for assay of metronidazole. However there is no simple and accurate method reported for the detection of MND in pharmaceutical formulation by UV spectrophotometry. OBJECTIVE The aim of present work is to find out a simple, sensitive, specific, spectrophotometric method for the detection of MND in pharmaceutical tablet formulation. MATERIALS AND METHODS INSTRUMENTS UV-Visible double beam spectrophotometer (UV-1800, SHIMADZU Limited, Japan) with 1cm matched quartz cells, Micropipette of Variable volume 10-1000 L (Gene Pete Co.) and Digital balance (Citizen Co.) CHEMICALS AND REAGENTS

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Publication Ref No.: IJPRD/2010/PUB/ARTI/VOV-2/ISSUE-6/AUG/004

ISSN 0974 9446

same was measured in next three days. The RSD% was calculated. (Table 3) ACCURACY (RECOVERY) The accuracy of the method was evaluated through standard addition method. In this, known amount of standard Metronidazole was added in pre-analyzed sample. This was done for 5 g/ml, 6g/ml and in triplicate. (Table 3) STABILITY The standard stock solutions of Metronidazole (Concentration 10g/ml was subjected to heat treatment on 500C, 600C and absorbance were measured. The absorbance for 500c for 1hr was same while for 600c, the absorbance was decreasing which was indicative that Metronidazole is stable at 500C and but at 600c MND solutions unstable. DETERMINATION OF ACTIVE INGREDIENTS IN TABLETS: The proposed method was applied to analyze commercially available Metronidazole tablets. The tablet was having content of Metronidazole equivalent to 200mg. (Ten tablets were weighed and weight equivalent to 100mg was dissolved in phosphate buffer (pH 7.4). By Frequent shaking volume was made up to mark with phosphate buffer (pH 7.4. The solution was then filtered through Whattman filter paper #41.This filtrate was diluted suitably with solvent to get the solution of 5g/ml concentration. The absorbance was measured against solution blank. Amount of Metronidazole was calculated from the calibration curve. The readings were taken in triplicate by performing the same experimentation in three times. RESULT AND DISCUSSION: In the start of the method development for this drug, different solvents were tested such as water, methanol, 0.1N Hcl, 0.1N NaOH and

Phosphate buffer (pH7.4). Due to greater solubility and reproducible readings of maximum absorbance, phosphate buffer was taken under consideration for further work. By serial dilution 20g/ml, the different dilutions of standard drug having concentration 1,2,3,4,5,6,7,8,9,10g/ml were prepared and calibration curve was prepared by plotting graph between absorbance and concentration (g/ml) (Fig1). The results of linearity are presented in table 2. The data was statistically validated by means of least square regression method. The detection and quantization limits as LOD (k=3.3) and LOQ (k=10) were calculated and these were found to be 1.07g/ml and 3.35g/ml respectively. The precision (measurements of intraday and interday) results showed good reproducibility with percent relative standard deviation (% RSD) is below 2.0. This indicated that method is highly precised. The evaluation of accuracy of the method was performed by standard addition method and for this, in 5g/ml and 6g/ml concentration solution, 100% addition was done and it was found to be 98.2% and 98.8% respectively. This indicated accuracy of proposed method. The proposed method was also applied for the assay of Metronidazole in tablet formulation (in triplicate) and the results as tabulated in Table 4. The results obtained were good agreement with the label claims. CONCLUSION: The proposed methods can be successfully applied for MND assay in tablet dosage forms without any interference in quality control. Analysis of the tablets by this method were reproducible reliable and in good agreement with labeled claim of the drug. ACKNOWLEDGEMENT: The authors would like to acknowledge Managing Director, IFTM, Moradabad, India, for providing facilities for conducting research.

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Publication Ref No.: IJPRD/2010/PUB/ARTI/VOV-2/ISSUE-6/AUG/004

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REFERENCES: 1. The Merck Index, 11th Edn., Merck and co. Inc., Rahway, USA 6181,(2001). 2. Heisterberg L, Branebjerg P E, Journal of Perinatal Medicine 11(2), 1983, 114-118 3.Marchioretto M.A.M., Ecclissato C., Cassiano N.M., Mendona S., Bernasconi G.C.R. Brazilian J. Medical and Biological Research 38, 2005, 437-444 4. The Martindale 35th ed. The complete drug reference, 2006, published pharmaceutical press, Lambeta high street, Londan SEI 75M, UK. 5. Kaye C M, Sankey M J, Thomas L A, Br J Clin Pharmacology 9(5), 1980, 528529. 6. Ashour S, Kattan N, International Journal of biomedical science 6(1), 2010, 13-19 7. Wang P, Li-Jie, Zheng H, Chinese Journal of Chromatography 5, 2007, 5-27 8. Liu H, Li F, Yang R, Wang L, Ma Y, Chinese journal of chromatography 27(1), 2009, 50-53 TABLES AND FIGURES:

9.Mustapha K B, Odunola M T, Garba M, Obodozie O. African Journal of Biotechnology 5 (13), 2006, 1188-1190 10. International Conference on Harmonization, Draft Guideline on Validation Procedure, Definition and Terminology Federal Register, 1995, 60, 11260 11. ICH, Q2A validation of analytical procedure, Methodology International Conference on Harmonization, Geneva, October 1994

Table No.1 - Calibration Curve Parameter Conc. (g/Ml) 1 2 3 4 5 6 7 8 9 10 Absorbance 0.09 0.109 0.133 0.161 0.191 0.215 0.231 0.262 0.291 0.319

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Publication Ref No.: IJPRD/2010/PUB/ARTI/VOV-2/ISSUE-6/AUG/004

ISSN 0974 9446

Figure 1: Calibration Curve Of Standard Metronidazole

Chart Title A s rb n e bo a c 0.4 0.3 0.2 0.1 0 0 5 10 15 Concentration Linear (Absorbance ) y = 0.0283x + 0.0407 2 R = 0.9902 Absorbance

Table.No. 2 - Validation Parameters S.No 1. 2. 3. 4. 5. 6. 7. Parameter Absorption Maxima(Nm) Linearity Range (G/Ml) Standard Regression Equation Correlation Coefficient (R2 ) Accuracy(% Recovery Sd) Lod (G/Ml) Loq (G/Ml) Result 277 1-10 Y = 0.0283x + 0.0407 0.9902 98. 20.129 1.07 3.35

Table 3: Results Of Recovery And Precision Ingredient Amount Amount % %Recovery Precision Precision Amount Of Added Recovered (Inter (Intra Drug From Addition Day)* (G/Ml) (G/Ml) Day)* Formulation 5 6 100 100 5 6 4.91 5.93 98.2 98.8 1.01 0.78 0.88 0.51

Metronidazole

* Percentage Rsd Of Three Samples Table No. 4 Determinations Of Active Ingredients In Tablets Sample Label Claimed 200mg Amount Found Mg /Tab. 198.120.126 % Labeled Claim * 99.06

Metronidazole

(* Average Of Three Determinations)

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