Development of the spectrophotometric method for quantitative determination of metronidazole in capsules

Pathological conditions of the digestive system are quite common nowadays and rank first among other human diseases. Increasingly, both newly-diagnosed and recurrent cases of stomach ulcers, as well as duodenal ulcers are recorded. Among solid drug dosage forms capsules are considered to be the most rational since the drug substance exhibits a low degree of dispersion, which leads to better absorption, resists additional negative impact in the production process, as well as possesses a lower damaging action on the mucous membranes of the digestive tract, etc. Thereby capsules are more and more often recommended for use by preference. Therefore, developing a feasible method of quantitative determination of metronidazole in capsules – the method of spectrophotometry in the ultraviolet region of the spectrum has become particularly urgent since high performance liquid chromatography recommended by the BP and USP is not used widely in Ukraine. The object of research was metronidazole capsules. The research has found that the excipients have little effect on the absorption spectrum of metronidazole obtained by extraction with 0.1 M hydrochloric acid solution from the content of capsules. The test solution has been proven to be complied with the Beer-LambertBouguer law within the concentration range of 0.5·10-3% – 3.0·10-3%. The certification of the method developed on the model mixtures and a production sample has confirmed its correctness. The average results uncertainty ε in the former case was 0.11%, whereas in the latter it constituted 0.70%.

Pathological conditions of the digestive system are quite common nowadays and rank first among other human diseases. Thus, the most common of these is ulcer of the stomach and duodenum [5,7,10,12]. As recommended by the Maastricht consensus two lines of therapy containing metronidazole are used for treatment of this disease [14,[16][17][18]. Among solid drug dosage forms capsules are considered to be the most rational since they have several significant advantages, including high bioavailability, increased therapeutic effect, reduction of the damaging action on the mucous membranes of the gastrointestinal tract, prolongation of the action to reduce a negative impact on the drug substance in the production process, etc. [8,16].
First of all, the necessary condition for safe use of medicines is the existence of quality control methods available in each particular region. The State Pharmacopoeia of Ukraine (SPhU) recommends to determine the quantitative content of metronidazole capsules by using the liquid chromatography method [3]. However, it should be noted that for Ukraine this method is not widely available because of the high cost of the analysis and impossibility of providing the drug quality control laboratories with chromatographs on a large scale [9].
The aim of our paper was to develop more available method for determining the quantitative content of metronidazole in the form of capsules by spectrophotometry in the ultraviolet region of the spectrum.

Materials and Methods
The substance of metronidazole (manufacturer: Luotian Hongyuan Biochemical Co., LTD, China, batch 08111803) and "TRIKACIDE" capsules containing 500 mg of metronidazole (manufacturer: Pharmascience Inc., 6111, Royalmaunt Avenue, Montreal, Quebec, Canada, batch 6452653) meeting the requirements of the SPhU were chosen as the objects of study.
Such analytical equipment as an "Evolution 60S" Spectrophotometer (USA), AB 204 S/A METTLER TOLEDO analytical balance, as well as glassware for measuring, class A (first class), and reagents meeting the requirements of the SPhU was used in the study.
The method of study was absorption spectrophotometry in the ultraviolet and visible regions by the method of standard.

Results and Discussion
As a solvent 0.1 M hydrochloric acid solution was chosen, which the world's leading Pharmacopoeias [3,11,15,20] recommend to use for the assay of metronidazole in the substance and the dosage forms.
First of all, the influence of excipients on the absorption spectrum of metronidazole was determined. In order to do that, the absorption spectra of the metronidazole standard solution, the extract from the content of capsules and placebo solution were compared. The UV absorption spectrum of the extract of the content of metronidazole capsules almost coincides with the UVspectrum of the metronidazole standard sample solution. The influence of optical absorption of the placebo solution on absorption of the extract solution from the capsules content is very insignificant at the analytical wavelength of 277 nm in the medium of 0.1 M hydrochloric acid solution (Fig. 1).
It has been found that the graph of dependence of the absorbance of metronidazole solution on its concentration is linear at the wavelength of λ = 277 nm (Fig. 2). The compliance of the test solutions with the Beer-Lambert-Bouguer law is observed within the concentrations from 0.5·10 -3 % to 3.0×10 -3 %. The average value of the specific absorbance in the experiment was 363.
The method for quantitative determination of metronidazole in capsules proposed was assessed on model mixtures. The results are presented in Tables 1 and 2. The uncertainty of the average result ε = 0.11% meeting the requirements of the SPhU.
The results of quantitative determination of metronidazole in capsules on the production sample of the drug and metrological characteristics obtained from the statistical processing of the measurement results, are presented in Tables 3 and 4 [1 -3, 4, 6]. In this case, the uncertainty of the average result ε meets the SPhU and is 0.70%.    Table 2 Metrological characteristics of the average results of quantitative determination of metronidazole in capsules.  Table 3 The results of quantitative determination of metronidazole in capsules on the production sample  Table 4 Metrological characteristics of the average results of quantitative determination of metronidazole in capsules.

Testing on model mixtures
Testing on the production sample