Development of ultraviolet spectroscopic method for the estimation of metronidazole benzoate from pharmaceutical formulation

Abstract

Background:

The present study was undertaken with an objective to develop a simple, accurate, cost-effective and reproducible ultraviolet spectrophotometric method for the estimation of metronidazole benzoate (MB) from pharmaceutical formulations.

Materials and Methods:

The analysis was performed on λ_max 268 nm by using 0.1 NHCl as diluents. The proposed method was validated on International Conference Harmonization guideline including the parameters viz., accuracy, linearity, precision, specificity and reproducibility. The proposed method was also used to access the content of MB in two commercial brands of Indian market.

Results:

Beer's law was obeyed in the concentration range of 1-10 μg/ml having regression equation y = 0.078 x-0.012. The accuracy value for 4 μg/ml and 5 μg/ml concentration of MB was found to be 99.37% and 98.9% respectively. The relative standard deviation of interday and intraday was lesser than 1%. The developed method was applied on two different marketed brands and contents of MB were found to be 98.62% and 98.59% incompliance with labeled claim. The results were under the limit of acceptance statistically.

Conclusion:

It was concluded that the proposed method can be used for routine analysis of MB in bulk and commercial formulations.

INTRODUCTION

Chemicallymetronidazole benzoate (MB) is 2-(2-methyl-5-nitro-1H-imidazole-1-yl) ethylbenzoate [Figure 1], belonging to the category of antiprotozoal drug, used to treat various infections including amoebicdysentery, trichomoniasis, periodontitis, giardiasis and many other anaerobic infections.[1] Asaprophylactic treatment of infection, MB is used before surgery aswellas of tenusedinoral liquid dosage for msdueto its known bitter taste.[2]

Figure 1.

Chemical structure of metronidazole benzoate

Earlier reported method includes kinetic sp ectrophotometry,[3] high performance liquid chro matography (HPLC),[4] ultra-performance liquid chro matography,[5] LC-mass spectrometry (MS)-MS,[6] gas liquid chromatographic (GLC)[7] and differential pulse polarographic method[8] for quantification of MB from plasma, urine and milk samples. The use of HPLC, GLC and LC-MS-MS involves the use of sophisticated instruments, which requires expensive instrumentation and more time consuming steps in sample preparations. The long run time of the se methods limits their applicability for the large number of samples. The existing spectroscopic method involves tedious derivatization step, which not only increase the complexity of method, but also increases the cost and time of analysis.[9]

Upon the literature survey, it was observed that no simple, cost-effective and reproducible methods area available for the estimation of MB from bulk dosage form as well as pharmaceutical dosage form. Keeping in view this fact, focus was laid to develop a simple, cost-effective and reproducible ultraviolet (UV) spectroscopic method for the estimation of MB and validated as per International Conference Harmonization (ICH) guideline.[10] There covery of MB by the proposed method was high, which accounts for the accuracy, novelty and reliability of the proposed method. The method was applied to determine the content of MB in two different marketed products in India.

MATERIALS AND METHODS

Instruments and materials

Shimadzu 1800 double-beam UV/V is spectrophotometer with1 cm matched quartz cells, digital balance (Citizen Co., Mumbai, India) and micropipette (The Modern Scientific Industries, Meerut, India) were used in the present study. MB was obtained as a gift sample from TTK Pharma, Vapi-Gujrat. The other chemicals and reagents were used of analytical grade.

Standard stock solution

Standard stock solution of MB was prepared by dissolving 25 mg of MB in 10 ml of 0.1 NHCl in a 25 ml volumetric flask, shaken well and finally volume was adjusted to get final concentration of 1 mg/ml. This prepared solution was used as standard stock solution.

Calibration curve

A total volume of 5 ml of 1 mg/ml aliquot solutions was further diluted up to 50 ml by 0.1 NHCl in a 100 ml volumetric flask and finally volume was adjusted up to the mark by 0.1 NHCl. Spectrophotometric scan was performed for the prepared diluted sample to find out λ_max in the wavelength region 200-800 nm. The λ_max was found to be 268 nm against blank solution [Figure 2]. From 1 mg/ml stock solution, the serial dilution pattern was followed to obtain aliquots of 1-10 μg/ml. The calibration curve was plotted between the absorbance and concentration [Figure 3]. The optical characteristics are presented in Table 1.

Figure 2.

Scanned spectra of metronidazole benzoate

Figure 3.

Calibration curve of metronidazole benzoate in 0.1N HCl

Table 1.

Calibration curve data of metronidazole benzoate in 0.1N HCl

Sample solution preparations

The proposed method was applied to analyze three commercially available brands of MB in suspension formulations. Six bottles of MB suspensions from two different manufactures were purchased from local market of Moradabad, India. Before measuring the suspension, content of all the six bottles from two different manufacturers were mixed separately and labeled as MB1 and MB2 for the formulation of two different manufacturers. The suspension of MB equivalent to 100 mg of the drug was measured and transferred to 100 ml volumetric flask and dissolved in 20 ml 0.1 N HCl. Solution was shaken for 20 min. The resulting solution was further diluted to 100 ml with 0.1 N HCl and filtered through what man filter paper no. 41.1 ml of the above solution was pipetted out into 100 ml volumetric flask and made up to mark with 0.1 N HCl. Absorbance was measured at 268 nm against the blank solution. Finally, employing the linear equation of the calibration curve, the concentration was calculated. Percentage addition method was employed to calculate the accuracy. Amount of active drug in the different marketed brands were calculated and presented in Table 2.

Table 2.

Determination of accuracy (by standard addition method) and precision

RESULTS AND DISCUSSION

Linearity

The linearity of the drug was obtained for 1-10 μg/ml concentration range of MB. The calibration curve was plotted between absorbance versus concentration and the regression analysis was performed for line equation. The linear equation was found to be y = 0.078 x - 0.012 and correlation coefficient (r²) 0.997 [Table 3]. The calibration curve was found to be linear in the above stated concentration.

Table 3.

Stability study

Accuracy (recovery)

Standard addition method was employed to access the recovery of MB in the proposed system. To get the accuracy, known amount of standard MB was added to pre-analyzed solution of MB.[11] This was carried out for 4 μg/ml and 5 μg/ml concentration in triplicate and were found to be 99.37% and 98.9% respectively [Table 2].

Precision

The precision of an analytical procedure express the closeness of agreement between a series of measurement obtained from multiple sampling of the same homogenous sample under the prescribed conditions.[12] The precision of the assay was determined by repeatability (intraday) and intermediate precision (interday) and reported as relative standard deviation (RSD)% for a statistically significant number of replicate measurements. To determine this, 4 μg/ml and 5 μg/ml concentration solutions were measured in triplicate in a day and the same was measured in the next 3 days. The % RSD values were measured and presented in Table 2. In all cases, the RSD was always less than 2 indicating the precision of the method.

Robustness

In order to determine robustness, analysis under different temperature condition was performed, i.e., at room temperature and 22°C temperature. The absorbance of 5 μg/ml concentration was measured in triplicate at three different temperature conditions and the result is presented in % RSD [Table 4].

Table 4.

Robustness analysis

Ruggedness

Ruggedness of the proposed method was assessed by carrying out the analysis by different analyst as well as by changing the instrument. The result is presented in % RSD [Table 5].

Table 5.

Ruggedness analysis

Limit of detection and limit of quantification

LOD and LOQ of MB were determined by using standard deviation of the response and slope [Table 6].

Table 6.

Validation parameters

Stability

The stability of MB in 0.1N HCl was studied by the proposed method. The sample solution of concentration 5 μg/ml was prepared and separately heated in order to maintain 50°C and 60°C for 60 min. Outcome of the study revealed the information about the stability of method [Table 3].

Determination of active ingredients in suspensions

This validated method was used to analyze two commercially available different brands of MB suspensions. Finding of the present amount requirement y revealed that both the marketed brands are incompliance with the % amount requirement (98-102%) with respect to labeled claim [Table 7].

Table 7.

Determination of active ingredients (%)

CONCLUSION

The developed UV spectrophotometric method was found to be simple, cost-effective, rapid, accurate and reproducible, with high accuracy and precision values. The validation parameters were evaluated as per ICH guidelines. The satisfactory finding of this work indicates that method may be applied for quantitativeestimation of MB from pharmaceutical dosage forms. This method may be employed in routine quality control aspects.