Spectrophotometric Determination of Metronidazole by Prior Reduction and Subsequent Diazotisation and Coupling with N-( 1-naphthyl ) ethylenediamine – Application to Pharmaceutical Preparations

ABSTRACT A simple, rapid and sensitive spectrophotometric method for the determination of microgram amounts of metronidazole (MZ) has been proposed. The method is based on the reduction of metronidazole with iron metal and hydrochloric acid followed by diazotisation and coupling with N-(1-naphthyl)ethylenediamine in acidic medium to give a pinkish-red colored azo dye which has maximum absorption at 503 nm. Beers law is obeyed in the concentration range of 20-500 μg of metronidazole in a final volume of 25 ml, i.e., 0.8-20 ppm, with a molar absorptivity of 4.673×10 l.mol. cm and Sandell's sensitivity index of 0.0366 μg.cm , a relative error of – 1.73 to +1.58% and relative standard deviation of ±0.47to ±1.32 %, depending on the concentration level. The method has been applied successfully to the determination of metronidazole in pharmaceutical preparations.

Different methods have been reported for the determination of metronidazole and most of which have been applied to assay in dosage forms.These including: voltammetry (Manohara et al., 2010), spectrophotometry (Adegoke and Bourne, 2009), chromatography (Nora, 2007), polarography (Bartlett, 2005), gas chromatography (Safwan, 2010), and flow injection analysis.(Simone, 2006).The non aqueous titration method using perchloric acid as titrant and determining the end -point potentiometrically for the assay of metronidazole has also been described (British pharmacopoia, 2000).
Most of the spectrophotometric methods found in the literature for the determination of metronidazole are in the visible region and involve initial reduction by treatment with Zn powder and HCl (Manohara et al., 2010), followed by the diazotisation and coupling of the resulting amine with different coupling agents such as: 4-chloro-3-nitroaniline (Thulasamma and Venkateswarlu, 2009), 3-methylbenzothiazolin-2-one hydrazone (Nagaraja et al., 2002), p-benzoquinone (Atta et al., 2005), 8-quinolinol (Saffaj et al., 2004), and β-naphthol (Charrouf et al., 2004).Various chromatographic methods have been reported for the analysis of metronidazole in pharmaceutical dosage form and biological fluids (Mustapha et al., 2006).High performance liquid chromatography (HPLC) method was employed to measure metronidazole in human plasma and application to single dose pharmacokinetic and bioequivalence studies (Jaber et al., 2006).The objective of the investigation reported in this paper was to evaluate a simple spectrophotometric method for the determination of metronidazole; the method involves the diazotisation of reduced metronidazole followed by subsequent coupling with N-(1-naphthyl)ethylenediamine dihydrochloride (N-NED) to form a highly coloured dye.

Apparatus
CECIL CE7200 UV-Visible Recording Spectrophotometer UV-160 with 1.0 cm matched quartz cells were used for all absorption measurements.

Reagent
All chemicals used were of analytical-reagent grade.
Reduced metronidazole solution (500 µg /ml).A 0.05g of metronidazole (NDI,Iraq) is dissolved in about 10 ml distilled water followed by addition of 0.4 g of powdered iron and 5 ml of concentrated HCl then the solution is cooled, and filtered, the clear mixture is then transferred to a 100 -ml volumetric flask and is completed to the mark with distilled water .The solution is kept in a brown bottle, where it is stable for 2 days.
Reduced working metronidazole solution (200 µg /ml).A 40 ml of (500 ug/ml) is diluted with distilled water to the mark in a 100-ml volumetric flask.

Hydrochloric acid solution(1M
).This solution is prepared by diluting 8.47 ml of the concentrated acid (Fluka) to the mark with distilled water in a 100-ml volumetric flask.

Sodium nitrite solution( 1%
).This solution is prepared by dissolving 1g of sodium nitrite (BDH) in distilled water and the volume is completed to the mark in a 100-ml volumetric flask.

Sulphamic acid solution( 3%).
This solution is prepared by dissolving 3 g of sulphamic acid (Fluka) in distilled water and the volume is completed to the mark in a 100-ml volumetric flask.

N-(1-naphthyl)ethylenediamine dihydrochloride (N-NED) solution( 0.1%).
This solution is prepared by dissolving 0.1 g of the compound (Molekula, UK) in distilled water and the volume is completed to the mark in a 100-ml volumetric flask.
Reduced metronidazole tablets solution (500 µg /ml).Weigh and mix the contents of ten tablets(each one contains 500 mg MZ), then an accurately weighed amount of powder equivalent to 0.05 g MZ was dissolved in 10 ml of distilled water, then the procedure of reduction is followed as above.After filtration of the solution, a suitable aliquot of solution was taken and the recommended procedure was followed for analysis of the drug.Reduced metronidazole suspension solution(500 µg /ml).The suspension solution is shaken very well and 1.25 ml was taken (equivalent to 200 mg MZ) with 10 ml distilled water and the procedure for reduction was followed as above; a suitable aliquot of solution was taken and the recommended procedure was followed for analysis of the drug.
Reduced metronidazole injection solution (500 µg /ml).10 ml of the solution was taken (equivalent to 500 mg MZ) with 10 ml distilled water and the procedure for reduction was followed as above.a suitable aliquot of solution was taken and the recommended procedure was followed for analysis of the drug.

RESULTS AND DISCUSSION
For the subsequent experiments, 200 µg of metronidazole is taken in 25 ml final volumes and absorbance measurements are performed at 503 nm.

Principle of the method
The method included the following steps: Reduction of metronidazole (Denniston et al., 2004): Reduced metronidazole is reacted with excess nitrite in acidic medium to form the diazonium ion : The residual nitrite (as nitrous acid) which was undesirable due to its side reaction, such as, nitrosation of coupling agent (Bladyga et al., 1999), was be removed by sulphamic acid: The colored solution is formed by coupling diazotized MZ with N-NED in acidic medium.

Study of the optimum reaction conditions
The various parameters affecting the colour intensity of the dye have been studied and optimum conditions are selected.

Choice of coupling agent
Different coupling agents are used for the reaction with diazotized reduced metronidazole in acidic medium.The results in Table 1 show that N-NED gives the more sensitive reaction (ε = 4.986 ×10 3 l.mol - .cm - ) in acidic medium.

Effect of N-NED amount
The effect of different N-NED amount on the colour intensity of the dye has been studied ( Table 2).From the results, it can be observed that 5 ml of 0.1% N-NED solution is the more suitable amount which gives the highest value of formed azo dye absorbance and also it gives highest determination coefficient (R 2 ) values.

Effect of acids on the diazotization
The effect of the amount of different acids (weak and strong) used for the diazotisation of reduced MZ has been investigated.The results indicated that 2 ml of 1M HCl gives the highest colour intensity therefore, it has been selected in subsequent experiments (Table 3).

Effect of nitrite amount and time
The colored dye reached its maximum intensity when using 1 ml of 1% sodium nitrite solution after 5 minutes as a reaction standing time ( Table 4).

Effect of sulphamic acid amount and time
The presence of unreacted nitrite is undesirable in diazotisation reaction.Therefore, it should be removed by sulphamic acid which rapidly reacts with nitrite.The results indicated that 1 ml of 3% sulphamic acid solution with 3 minutes standing time are considered to be the most suitable (Table 5), and therefore are selected subsequently.

Effect of time and amount of MZ on absorbance
The effect of time on the development and stability period of the formed colored dye was investigated under optimum experimental conditions described before.The formation of colored dye being complete after mixing the components of the reaction and the absorbance of the colored species remained constant for at least 75 minutes (Table 6).

Effect of order of addition
The results indicated that the effect of order of addition (Table 7) of reagents is important: From the orders cited above, we can conclude that the reaction of nitrite with sulphamic acid is faster than the reaction of nitrite with reduced metronidazole and hence an absorbance decrease is observed.The last order reveals that nitrite will nitrosate N-NED to form the nitroso derivative which might be yellow to orange in colour, depending on the nature of the amine.

Final absorption spectra
Absorption spectra of the colored dye formed by coupling of diazotized reduced MZ with N-NED reagent in acidic medium was recorded against its corresponding reagent blank and show a maximum absorption at 503 nm in contrast to the N-NED reagent blank which shows no absorption in the visible region (Fig. 1).

Procedure and calibration graph :
To a series of 25-ml calibrated flasks, an aliquot of aqueous solution containing 20 -500 µg 0f reduced MZ are transferred, 1 ml of 1 M hydrochloric acid is added and the mixture is shaken, then 1 ml of 1% sodium nitrite solution is added and the mixture is allowed to stand for 5 minutes then 1 ml of 3% sulphamic acid solution is added with occasional shaking for 3 minutes.A 5 ml of 0.1% N-NED solution is added and the volumes are completed to the mark with distilled water, the absorbances are read at 503 nm against blank.The colour was stable for at least 1 hour.The calibration graph is linear over the range 0.8-20 ppm (Fig. 2).The apparent molar absorptivity, referred to MZ, has been found to be 4.673×10 3 l.mole - .cm - .

Accuracy and precision
To check the accuracy and precision of the calibration curve, MZ was determined at three different concentrations.The results illustrated in Table 8 indicate that the method is satisfactory.

Nature of the Dye
Job's method (Hargis, 1988) indicates that the azo dye has a composition of 1:1 [MZ] to N-NED reagent (Fig. 3).

Interference
The effect of some foreign compounds, which often accompanied pharmaceutical preparations, was studied by adding different amounts to 200 µg metronidazole in a final volume 25 ml (Table 9).The results in Table 9 indicated that the studied foreign compound do not interfere in the determination of metronidazole using the proposed method.

Effect of organic solvents
Different organic solvents have been examined to evaluate their effects on the spectrum of the resulting azo dye, the results are given in Table 10 and Fig It can be seen from the above table that water is the best solvent from both economic as well as analytical points of view.Therefore, water is still chosen in the subseqent experiments.

Application of the Method
The proposed method was successfully applied to determine metronidazole in its pharmaceutical preparations (Table 11).The performance of the proposed method was assessed by calculation of the t-test compared with the standard method (British Pharmacopeia, 2000) (non aqueous titration with perchloric acid) for 95% confidence level with four degrees of freedom.The results showed that the t-value was less than the critical value=2.306,indicated that there was no significant difference between the proposed and standard method for metronidazole (Table 12).

Comparison of Method
Table 13 shows the comparison between the analytical variable obtained from the present method with those of recent spectrophotometric method.

*
The application of the present method is of wider scope.
) has a bacterial action against anaerobic bacteria, its spectrum of activity includes the anaerobic gram negative bacilli which include most of Bacteroides such as Fusobacterium and Veillonella, anaerobic gram positive cocci such as Peptococcus and Peptidostreptococcus species and anaerobic gram positive bacilli which include Clostridium and Eubacterium species.(Block et al., 2004).methyl-5-nitroimidazole(Sweetman, 2009)

Fig. 1 :
Fig. 1: Absorption spectra of 200 µg MZ treated according to the recommended procedure and measured against (A) blank, (B) distilled water and (C) blank measured against distilled water

Fig. 5 :
Fig. 5: Effect of organic solvents on optical properties of azo dye

Table 1 : Selection of coupling agent.
Sλmax B , Where S= The dye , B=Blank **: no colour contrast

Table 5 : The effect of sulphamic acid amount and time on the dye absorbance.
* negative absorbance values.