The use of the HPLC method for the quantitative determination of cetylpyridinium hexafluorosilicate in a dental gel

Cetylpyridinium hexafluorosilicate has been proposed as a caries preventive and antibacterial agent for use as a gel for oral application, which requires development of quality control methods. Aim. To develop the method for the quantitative determination of cetylpyridinium hexafluorosilicate in a dosage form using HPLC. Materials and methods. The object of the study is the experimental dosage form – a gel proposed for use in dental practice. The research method is HPLC. Results and discussion. The method for the quantitative determination of the active component, which allows determining cetylpyridinium hexafluorosilicate in the gel in the presence of excipients of the dosage form, has been developed. It is recommended to carry out this determination by the HPLC method using the column of 150 × 4.6 mm in size filled with octadecyl silica gel for chromatography, the sorbent particle size of 3.5 μm, a mixture of perchlorate buffer solution (pH 3.0) and acetonitrile (30 : 70) as a mobile phase and UV detection at the wavelength of 254 nm. Conclusions. The conditions of the chromatographic study by the HPLC method proposed provide sufficient selectivity for the quantitative determination of cetylpyridinium hexafluorosilicate in the dosage form, and it has been confirmed by studying the validation characteristics of the method.

Ключевые слова: цетилпиридиния гексафторосиликат; гель; количественное определение; метод ВЭЖХ; валидация It is known that the element of fluorine is involved in the mineral metabolism in the bone tissue and teeth due to the caries-protective and osteoprotective activity. Unfortunately, overdose with fluorine can lead to binding of calcium ions to inert calcium fluoride and appearance of the hepatotoxic activity. It has been found that among various complex fluoride forms only hexafluorosilicates reduce the ability of fluorine to bind calcium ions and practically do not possess the hepatotoxic activity compared to sodium fluoride.
The biochemical studies have proven that the mechanism of caries preventive action of fluorine preparations is based on their ability to increase the mineralizing index and the level of non-specific immunity of the teeth pulp. Cetylpyridinium hexafluorosilicate synthesized at the premises of the Odessa National Medical University showed the highest activity in the dose of 15 mg/kg when used as a gel for oral application [1].
The composition and technology of the gel for oral application has been developed and experimentally proven. Its composition includes a gel former -hydroxyethyl cellulose, solubilizer -PEG-40, sorbitol introduced to provide moderate osmotic properties, and sodium benzoate proposed as a preservative [2].
The aim of the work is to use the high-performance liquid chromatography method for the quantitative determination of cetylpyridinium hexafluorosilicate in the gel composition.

Materials and methods
Cetylpyridinium hexafluorosilicate RS with the nominal content of 100.17 %; hydroxyethyl cellulose, PEG-40 (b. 40 manufactured by the JSC TOS plant "BARVA"), sorbitol (b. KV9523 manufactured by Cerestar), sodium benzoate, mint oil (b. L01-211D50, Pharmaceuticals and cosmetics, 07.11.2011), the class A glassware for measuring, reagents meeting the requirements of the State Pharmacopeia of Ukraine (SPhU), as well as an AB-204/A Mettler Toledo analytical balance were used in the study.
The chromatographic determination was carried out on a ProStar (Varian, USA) chromatograph under the following conditions: Test solution. Place approximately 2.5 g (accurate weight) of the gel in a 50 ml volumetric flask, add 30 ml of water R, sonicate for 15 min. Stir the solution using a laboratory mixer till the complete dissolution of the gel. Dilute the solution to the volume with the same solvent, mix thoroughly, and filter through a "red tape" paper filter. Filter the solution obtained through a 0.45 μm membrane filter.
Standard solution. Place approximately 50 mg (accurate weight) cetylpyridinium hexafluorosilicate RS in a 50 ml volumetric flask, dissolve in water R, dilute the solution to the volume with the same solvent and mix. Filter the solution obtained through a 0.45 μm membrane filter.
Column: Waters XBridge ТМ C18, 0.15 m × 4.5 mm with the precolumn filled with octadecyl silica gel for chromatography with the sorbent particle size of 3.5 μm.
The flow rate of the mobile phase: 1.5 ml/min. Detection: spectrophotometry at the wavelength of 254 nm.
The volume of injection: 20 μL. The run time: 20 min. The standard solution and the test solution were chromatographed alternately.
The quantitative content of cetylpyridinium hexafluorosilicate in 100 g of the drug (g) was calculated by the formula: where: S 1 -is the average value of the peak area of cetylpyridinium hexafluorosilicate calculated from the chromatograms of the test solution; S 0 -is the average value of the peak area of cetylpyridinium hexafluorosilicate calculated from the chromatograms of the standard solution; m 1 -is the sample weight of the drug, (g); m 0is the sample weight of cetylpyridinium hexafluorosilicate RS, (g); P -is the amount of active substance in cetylpyridinium hexafluorosilicate RS, (%).

The system suitability
The standard solution is used to verify the suitability of the chromatographic system. The chromatographic system is considered to be suitable if the following requirements of the system suitability test are met: • the efficiency of the chromatographic column calculated for the main peak is not less than 5000 theoretical plates; • the symmetry coefficient calculated for the main peak is not less than 2.0; • the relative standard deviation (RSD) calculated for the areas of the main peaks of the repeated chromatograms must not exceed the values given in Tab. 1: In order to meet the requirements of the chromatographic system suitability the changes in the chromatographic conditions within the limits are permitted by the SPhU, 2.2.46, "Methods of chromatographic separation".
Preparation of the buffer solution with pH 3.0. Dissolve 2.0 g of sodium perchlorate R in 300 ml of water R, add 0.5 ml of triethylamine R and mix. Dilute the pH of the solution obtained to 3.0 ± 0.1 with diluted phosphoric acid R. Filter the resulting solution through a 0.45 μm membrane filter.

Results and discussion
The physicochemical properties of cetylpyridinium hexafluorosilicate described in the literature were used when developing the method for the quantitative determination of the active pharmaceutical ingredient in the composition of the gel [3].
The existing redox method of assay for cetylpyridinium hexafluorosilicate in the substance [4] in this case did not give a positive result because of the presence of excipients in the dosage form. To develop a method for the quantitative determination of the gel active component according to the requirements of the SPhU [5] it was necessary to choose the conditions under which enable the complete separation of the active substance and excipients was possible within a reasonable time. Various combinations of the mobile phase and the pH values of the medium were tested when developing this method.
Based on the experimental data it was found that as the mobile phase it was expedient to use the mixture of perchlorate buffer solution with pH 3.0 and acetonitrile in the ratio of 30 : 70. Separation of the active component and excipients that were not met under these conditions was achieved with isocratic elution. Water for chromatography was chosen as a solvent for preparation of the model, test and reference solutions.
The gel composition contains hydroxyethyl cellulose and sorbitol, which complicated the sample preparation and required additional filtering through a paper filter.
The conditions of the chromatographic determination proposed using the method of high performance liquid chromatography (HPLC) provide sufficient selectivity. The solution of the standard sample of cetylpyridinium hexafluorosilicate, the test solution of the drug Table 1 The relative standard deviation (RSD) calculated for the areas of the main peaks of the repeated chromatograms and the placebo solution were prepared and analyzed to verify selectivity of the method. The chromatograms obtained in the study of solutions are shown in Fig. 1-3.
As can be seen in the chromatogram of the placebo solution (Fig. 3), there are no peaks that can interfere with the determination of cetylpyridinium hexafluorosilicate, and it may indicate the reproducibility of the method.
The studies have shown that the retention time of the cetylpyridinium hexafluorosilicate peak in the test solution coincides with the retention time of the main peak in the reference solution with an accuracy of ± 2 %. A number of validation characteristics such as specificity, linearity, convergence, precision and accuracy were studied for the method proposed.  To estimate the error of the sample preparation of the test solution and standard solution the theoretical values for analytic operation uncertainty were calculated, it was Δsp = 0.89 % ≤ В • 0.32 = 1.6 %. Therefore, the estimated uncertainty of the sample preparation of the method of analysis in general should provide sufficient accuracy of measurements.
To prove the specificity of the method it is sufficient to meet all requirements regarding the criteria of linearity, correctness and precision since for the quantitative determination of cetylpyridinium hexafluorosilicate in the gel the HPLC method, which is specific, is used.
The assessment of linearity was performed in the entire range of the method application using the standard method. The study of the character of dependence of the peak area on the concentration was carried out using 9 model solutions with accurate weight of concentra-   Table 3 The results of analysis of the model solutions and their statistical processing  80,85,90,95,100,105,110,115, and 120 %) for analysis. For each of these nine solutions of the sample the average values of the peak areas (Sі) were calculated. Plotting of the calibration curve was carried out in the normalized coordinates (Fig. 4) after processing of the results obtained using the least squares method [5].
The statistical values b, Sb, a, Sa, Sr (residual standard deviation) and r (correlation coefficient) calculated are shown in Fig. 4 and Tab. 2. Requirements for linear dependence parameters are performed in the entire range of application of the method proposed (80-120 %).
To measure and calculate the metrological evaluation of convergence and correctness of the method three values of the peak areas for the standard solution and 27 values of the peak areas for model solutions were obtained. The actual values (Х і ), the ratio of the average values of the peak areas for each of 27 solutions to the average value of the peak area of the standard solution were calculated, the values Xi = (C i /C st ) · 100 %, Y i = (S i /S st ) · 100 %, as well as the value Z i = (Y i /X i ) · 100 % were obtained (Tab. 3).
The results of determination of the quantitative content of cetylpyridinium hexafluorosilicate in the dental gel studied are given in Tab. 4.
The analysis of the data has shown that the content of the active ingredient in the gel is 0.1611 g/100 g (normally 0.1660 g/100 g), the relative uncertainty of the result of the individual determination is 0.81 %. CONCLUSIONS 1. The method of the quantitative determination of cetylpyridinium hexafluorosilicate developed using the HPLC method is suitable for the quality control of the active substance in the dental gel. It has been proven that the content of the active component of the dental gel is within the permissible limits, and the metrological characteristics of the method allow recommending it for standardization of the product.
2. The validation characteristics of the method do not exceed the critical value of the error (1.6 %) and are characterized by qualitative analytical indicators.