INTRODUCTION:

Imidazoles are five membered ring structures containing two nitrogen atoms with a complex side chain attached to one of the nitrogen atoms. Metronidazole (MET) is an antiprotozoal and chemically it is 2-methyl-5- nitroimidazole-1-ethanol.¹ Metronidazole acts as a prodrug. It is converted in anaerobic organisms by the redox enzyme pyruvate-ferredoxin oxidoreductase. The nitro group of metronidazole is chemically reduced by ferredoxin (or a ferredoxin-linked metabolic process) and the products are responsible for disrupting the DNA helical structure, thus inhibiting nucleic acid synthesis.² Extensive literature survey reveals that various analytical methods have been reported for the estimation of metronidazole in single and combination form such as UV spectrophotometric,^3-6 HPLC,^7-10 amperometry,¹¹ Supercritical Fluid Chromatography,^12-13 HPTLC,¹⁴ chemiluminescence ,¹⁵ voltammetry,^16-17 polarography,^18-19 GC-FID.²⁰

Further review of literature revealed that metronidazole in combination for intravenous admixture has been estimated by spectrophotometric method with first derivative spectrophotometry,²¹ but no spectrophotometric method is yet reported for intravenous metronidazole alone with zero order spectrophotometry.

Since there is lack of method to directly estimate metronidazole in intravenous formulation, this paper describes simple, rapid, accurate, precise and economical methods for determination of metronidazole in intravenous formulation by zero order spectrophotometry. This is supported by the fact that after obtaining the spectra no interference was observed by the excepients.

MATERIALS AND METHOD:

Instruments:-

UV-visible double beam spectrophotometer, JASCO V-630 with spectral bandwidth of 1 nm, wavelength accuracy of ± 0.3 nm and a pair of 10 mm matched quartz cells was used. Shimadzu AY220 balance was used for weighing the samples. All the chemicals used were of AR grade. Distilled water was used throughout the experimental work.

Materials:

Pure metronidazole was kindly gifted by Aarti drugs, Boisar (Tarapur). The commercially available injection, Nirmet manufactured by nirlife Healthcare (Label claim: metronidazole- 500 mg/100ml, i.e. 0.5% w/v) was procured from local market. All the chemicals and reagents were of analytical grade.

Preparation of standard stock solution:

Accurately weighed 10 mg of metronidazole (MET) pure drug taken in separate 100mL volumetric flask and dissolved with 70 mL of distilled water and shaken for 15 min and then diluted with distilled water to get 100 μg/mL standard stock solutions.

Construction of calibration curve:

Aliquots of standard stock solution were pipetted out and suitably diluted with distilled water to get the final concentration of 5-30 μg/mL. The solution was scanned in the spectrum mode from 400 nm - 200 nm wavelength range. Calibration curve was constructed by plotting the absorbance against the concentration and regression equation was computed.

Fig 1:- Overlain spectra of pure MET and IV preparation.

Fig 2:-Calibration curve of Metronidazole [Linearity].

Analysis of formulation:

For the estimation of metronidazole from injection, a portion of the formulation was taken in separate 100 mL volumetric flask and subsequently diluted with distilled water to get the final concentration of 20 μg/mL. Evaluation was performed with double beam spectrophotometer for metronidazole at 320 nm.(Fig- 3)

Fig-3 Overlain spectra of metronidazole

Method Validation:

Accuracy

To check the accuracy of the proposed method, recovery studies were carried out 80, 100 and 120% of the test concentration as per ICH guidelines. The recovery study was performed three times at each level.

Table No. 1: Optical parameters and regression characteristic for metronidazole (MET)

Parameter	Data
Beer-Lambert’s Law range (Linearity)	5-30 μg/mL
λ_max(nm)	320 nm
Regression equation (Y=mx + c)	Y = 0.060x - 0.011
Slope (m)	0.060
Intercept (c)	0.011
R²	0.999

Repeatability:

To check the degree of repeatability of the methods, suitable statistical evaluation was carried out. Repeatability was performed six times with tablet formulation. The standard deviation, coefficient of variation and standard error was calculated.

Table No. 2: Analysis of formulation

Drug concentration taken (μg/mL)	Absorbance reading (at 320 nm)	% Amount found
20	1.1998	100.49
20	1.1954	100.12
20	1.1897	99.64
20	1.1971	100.26
20	1.1924	99.87
20	1.1933	99.94
Average		100.053
S.D		0.3015
COV		0.3013
S.E		0.1231

S.D: Standard Deviation, COV: Coefficient of variation, S.E: Standard Error.

Intermediate precision (Interday and Intraday):

The interday and intraday precision by the assay of the sample solution on the same day and on different days at different time intervals was carried out respectively

Table No. 3: Result of recovery study

Sr No.	% Level of standard drug added	% Recovery	% Mean recovery ± S.D	% RSD
1	80%	99.79±0.81	99.85±1.00	1.001
		100.25±1.23
		99.51±0.96
2	100%	99.46±0.39	100.11±0.68	0.679
		101.02±0.76
		98.87±0.91
3	120%	99.48±1.01	99.60±0.89	0.893
		100.31±0.77
		99.01±0.89

S.D: Standard Deviation, % RSD: Relative Standard Deviation

Table No. 4: Validation data for precision study

Drug concentration taken (μg/mL)	Intraday precision		Interday precision
Drug concentration taken (μg/mL)	% Content*	% RSD	% Content*	% RSD
20	99.81	0.891	99.68	0.925

*Average of six determinations, % RSD: Relative Standard Deviation

RESULT AND DISCUSSION:

The proposed method for estimation of metronidazole (MET) in intravenous prepration was found to be simple, rapid, accurate, precise and economical. The λ_maxof metronidazole was found at 320.0 nm (Table-1). Drug follows Beer-Lambert’s law over the concentration range of 5-30 μg/mL giving the linear equation as 0.060x-0.006 and R²= 0.999 for the proposed method (Fig. 2). The values of standard deviation were found satisfactory and the recovery studies were close to 100%. Thus being a rapid method it can be applied in routine analysis of metronidazole in intravenous preparation.

The proposed method is based upon direct estimation of metronidazole in injectable preparation at 320 nm. The mean percentage content of drug was found to be within the limit which is determined by taking average of six readings (Table- 2). The developed method was validated as per ICH guidelines for repeatability, intermediate precision and recovery studies (Table-3). The precision of the method was checked in terms of Inter-day and Intra-day, where methods were repeated on six different days and also repeated on six different time periods in same day (Table-4). The accuracy of the method was proved by performing recovery studies in the commercially available formulations. Moreover there is no interference from the excipients present in the formulations.

CONCLUSION:

In the above developed method, there was no additional extraction or separation procedure to extract the active ingredient from the formulation and this can be explained from the fact that the spectra obtained can be overlain without much interference (Fig 1). The error in quantifications can be decreased by the elimination of this procedure. Hence, the developed method is simple, rapid, accurate, precise and economical for the routine estimation of metronidazole in its pharmaceutical dosage form (intravenous injection).

ACKNOWLEDGEMENT:

The authors are greatly thankful to P.D.V.V.P.F’s College of Pharmacy, Ahmednagar, India for providing access to facilities and necessary infrastructure to carry out research work. We are also thankful to Aarti drugs, Boisar (Tarapur) for providing us the free gift sample of Metronidazole.

REFERENCES:

1. British Pharmacopoeia, monograph, 2007, 675.

2. Tripathi K.D., Essentials of Pharmacology: Anti-amoebic and other antiprotozoal drugs, Jaypee brothers publication, New Delhi, 2008, pp 750-753.

3. Jadhav G.P., More H.N., Mahadik K.R. Simultaneous UV-Spectrophotometric estimation of Nalidixic acid and Metronidazole. Indian Journal of Pharmaceutical sciences, 60; 1998: 246-248.

4. Maliwal D., Jain A., Maheswari R.K. Simultaneous UV-Spectrophotometric estimation of Norfloxacin and Metronidazole., Asian Journal of Pharmaceutics, 2008.

5. Vega E, Sola N. Quantitative analysis of metronidazole in intravenous admixture with ciprofloxacin by first derivative spectrophotometry. Journal of Pharmaceutical and Biomedical Analysis, 25; 2001: 523-530.

6. Talwar N, Jain NK. High-Performance Liquid-Chromatographic Determination of Metronidazole in Tablets and Biological Samples. Indian Drugs. 29; 1991: 78-81.

7. Ravishankar S., Vasudevan M., Nanjan M.J., RPHPLC method for estimation of Metronidazole. 35; 1998: 359- 363.

8. Krishnan. Reddy S.R., Satyanarayan P.R.B. Estimation of Metronidazole in pharm. Dosage form. Acta Indica Chemistry. 28; 2002: 27-30.

9. Talwar N, Jain N K. HPLC Determination of Metronidazole in Pharmaceutical Dosage forms. Indian Drugs. 29; 1991: 78-81.

10. Fang ZJ, Sun HQ, Wang YJ, Sui ZY. HPLC Determination of Metronidazole Effervescent Tablets. Yaowu Fenxi Zazhi. 18; 1998: 335-336.

11. Palomeque M, García Bautista JA, García Mateo JV, Martinez Calatayud Flow-Injection Biamperometric Determination of Metronidazole with Online Photodegradation. Journal of Analytical Chemistry Acta. 401; 1999: 229-236.

12. Bari VR, Dhorda UJ, Sundaresan M. Simultaneous Estimation of Nalidixic Acid and Metronidazole in Dosage Forms Using Packed Column Supercritical Fluid Chromatography. Analytical Chemistry Acta. 376; 1998: 221-225.

13. Bhoir IC, Raman B, Sundaresan M, Bhagwat AM. Separation and Estimation of Diloxanide Furoate and Metronidazole in Solid Dosage Form Using Packed Column Supercritical Fluid Chromatography. Analytical Chemistry Acta. 354; 1997: 123-128.

14. Argekar AP, Raj SV, Kapadia SU. Determination of Ofloxacin., Norfloxacin and Lomefloxacin, Ofloxacin, Perfloxacin and Ciprofloxacin by RP-HPLC Indian Drugs. 33; 1996: 167-170.

15. Fu ZF, Chen H, Zhang ZJ. Determination of Metronidazole in Preparations by Flow Injection Chemiluminescence Analysis. Fenxi Shiyanshi. 23; 2004: 1-4.

16. Lu SF, Wu KB, Dang XP, Hu SS. Electrochemical Reduction and Voltammetric Determination of Metronidazole at a Nanomaterial Thin Film Coated Glassy Carbon Electrode. Talanta. 63; 2004: 653-657.

17. Ozkan O, Ozkan Y, Senturk Z. Electrochemical Reduction of Metronidazole at Activated Glassy Carbon Electrode and Its Determination in Pharmaceutical Dosage Forms. Journal of Pharmaceutical and Biomedical Analysis. 17; 1998: 299-305.

18. El-Sayed GO. Polarographic Determination of Metronidazole in Pharmaceutical Formulations and Urine. Journal of Microchemistry. 55; 1997: 110-114.

19. Sankar PS, Reddy SJ. Polarographic Determination of Metronidazole. Asian Journal of Chemistry. 2; 1990: 245-248.

20. Safwan A., Nuha K. Simultaneous Determination of Miconazole Nitrate and Metronidazole in Different Pharmaceutical Dosage Forms by Gas Chromatography and Flame Ionization Detector (GC-FID), International Journal of Biomedical Science. 6,(1); 2010: 13-18.

21. Vega E, Sola N. Quantitative analysis of metronidazole in intravenous admixture with ciprofloxacin by first derivative spectrophotometry. Journal of Pharmaceutical and Biomedical Analysis, 25; 2001: 523-530.

Received on 25.01.2012 Modified on 13.02.2012

Research J. Pharm. and Tech. 5(4): April 2012; Page 494-496