DEVELOPMENT AND VALIDATION OF METHODS FOR QUANTITATIVE DETERMINATION OF SODIUM HYALURONATE IN THE COMPOSITION OF COMBINED DENTAL GEL

Abstract

According to the EP monograph "Sodium hyaluronate" for identification they use the infrared transmission spectrum of the substance, quantification is carried out by spectrophotometry. The aim of the work was to develop a method for quantitative determination of hyaluronic acid in the gel in the presence of other substances and its validation. Materials and Methods. The object of the study were samples of combined dental gel of the following composition: metronidazole benzoate 16 mg/g, miramistin 5 mg / g, sodium hyaluronate 2 mg / g. Identification and quantification of sodium hyaluronate was performed by liquid chromatography (SPhU, 2.2.29, 2.2.46). The test solution and the reference solution were chromatographed, obtaining the number of parallel chromatograms not less than when checking the suitability of the chromatographic system. Chromatography is performed on a liquid chromatograph with a diode-matrix detector under the following conditions: chromatographic column PL-aquagel-OH, Agilent size 300 mm × 7.5 mm, with a particle size of sorbent 8 μm; mobile phase A: 0.1 M sodium sulfate solution; mobile phase B: acetonitrile for chromatography P; detection at a wavelength of 210 nm. Results. The retention time of sodium hyaluronate on the chromatogram of the test sample of the gel coincides with the peak and the retention time on the chromatogram of the comparison solution of the standard sample of the substance. The suitability of the chromatographic system for 3 parallel determinations was checked: the relative standard deviation (RSD) is equal to 0.25, the number of theoretical plates is 980, the symmetry coefficient is 1.293. The validation characteristics of the developed methodology meet the established eligibility criteria. The spectral purity coefficients (Fp) of the sodium hyaluronate peak on the chromatograms of the model solution are Fp1=997.665 and Fp2=997,802. The method is linear in the range of sodium concentration of hyaluronate 80–120 %, the calculated linear dependence of the reduced area of the chromatographic peak on the reduced concentration of sodium hyaluronate is |a|=1.9490≤Δa=2.56. The confidence interval of the unit value for the sample of relations is found / entered Δz=1.08, which corresponds to the condition Δz≤1.6 %. The value of the systematic error is equal to δ=0.12, which satisfies the condition δ≤0.51 %. Conclusions. The method of quantitative determination of sodium hyaluronate by the method of high-performance liquid chromatography has been developed and investigated. The method allows the identification and quantification of sodium hyaluronate in the composition of the dental gel, in the presence of metronidazole benzoate and miramistin. Validation of the methodology was performed and the main validation characteristics were determined. In terms of specificity, linearity, correctness, convergence of the method meets the eligibility criteria established by the SPhU.