Solutions for studying the character of the amoxicillin UV spectra in different solvent media were prepared: control sample – 0.001% solution of amoxicillin in 0.1 M HCl; test samples – 0.001% solution of amoxicillin with addition of Ca^{2 +}and Mg^{2 +}salts aliquot. Weights of salts were taken in the stoichiometric ratio of salt to antibiotic 1: 2.The analysis was carried out on spectrophotometer “Evolution 60S”(Thermo Scientific, USA) at a wavelength of 200–400 nm.

For analysis of possible complex formation, Job’s method of continuous variations was used (Job 1928). For that purpose initial solutions of amoxicillin and salts with equal molar ratios (1*10–3m/l) were prepared and further diluted with 0.1 M HCl to get final ratios of the antibiotic and salt 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1.The absorbance of obtained solutions was measured in maxima 203nm, 230nm and 272 nm.

To determine the possible interaction of amoxicillin with calcium and magnesium salts, an effect on the release of amoxicillin from tablets in a medium of 0.1 M hydrochloric acid solution with the addition of a mineral waters portion was conducted and “Dissolution” test was performed(United States Pharmacopeia2017; State Pharmacopoeia of Ukraine 2014a, 2014b, 2015).

The subjects of the study were amoxicillin tablets AMOXIL 500 mg, No. 20, Corporation Arterium, Ukraine.

An analysis of the mineral water market was conducted, as well as an analysis of their composition. To study the interaction, the following trademarks of mineral water, containing the largest number of cations, were selected:

– “Borjomi”, manufacturer: IDS Borjomi, Georgia.

– “Karpatska dzherelna”, manufacturer: “The Carpathian mineral waters”, Ukraine.

– “Yessentuki №17”, manufacturer: “Essentuki Mineral Water Factory”, Russian Federation.

– “Truskavetska”, manufacturer: “Аkva-ЕКО”, Ukraine.

Despite the benefits of mineral water, patients do not always take them with medicines. Therefore, for research, it was also taken tap urban water. Also, mineral waters from local Kharkiv sources often used by citizens were selected for research: “Kharkiv city spring water #1” and “Kharkiv city spring water #2”.

Since the content of mineral substances is regulated in a certain range of permissible concentrations, the quantitative content of calcium and magnesium cations in the selected samples of mineral waters was determined by complexometric titration: Calcium cation. 25 ml of pre-degassed mineral water are placed in a conical flask for titration, 5 ml of a solution of sodium hydroxide diluted, and 25 mg of the indicator mixture of murexide are added. Titrate with 0.01 M disodium edetate solution until the color changes from pink to violet (Lytvynenko YeYu 2017; Georgiyants VA et al. 2018).

25 ml of pre-degassed mineral water are placed in a conical flask, 5 ml of ammonia buffer solution is added, 25 mg of the eriochrome black T indicator mixture is added and titrated with 0.01 M disodium edetate solution until the color of the solution changes from violet to blue.

To calculate the content of magnesium cation, we use the difference in the volume of titrant titrated by the number of cations of metals, and the volume of titrant spent on titration of calcium cation.

To simulate the interaction of amoxicillin with cations of metals, the “Dissolution” test was carried out in a medium of 0.1 M hydrochloric acid solution, which pH is closest to the pH of the stomach, in a volume of 700 ml with the addition of 100 ml of the test mineral water degassed on ultrasound bath.

For a control experiment as a dissolution medium, a 0.1 M solution of hydrochloric acid in a volume of 800 ml was used.

An analytical balance Mettler Toledo AB-204 / A and grade A volumetric flasks were used during the study. All reagents and test specimens meet the pharmacopoeia requirements. PharmaTest PT-DT70 (Germany) is used as test equipment for dissolution test, apparatus 2 (baskets). The rotation speed of the baskets was 150 rpm. Test time – 45 min. The samples were taken after 5, 10, 15, 30, 45 minutes from the beginning of the study. The number of repetitions for each of the studies was six.

Samples were taken at specified intervals of time, using a 10 ml pipette, from the middle region between the surface of the dissolving medium and the top of the rotating basket. The aliquot for analysis was filtered through a laboratory “blue ribbon” paper filter and not compensated for an equal volume of dissolution medium, but was taken into account in the final formula for calculating the amount of released amoxicillin.

After finishing the test, preparation of the test solutions was performed: 5 ml of the test solution filtrate was placed in a 20 ml volumetric flask and the volume of the solution was completed to the mark with a 0.1 M solution of hydrochloric acid and mixed.

Preparation of the comparison solution: 62 mg of amoxicillin was placed in a 100 ml volumetric flask, dissolved in a small amount of a 0.1 M solution of hydrochloric acid, and the volume of the solution was completed to the mark with the same solvent. After this, 5 ml of the resulting solution was placed in a 20 ml volumetric flask; the volume of the solution was brought to the mark with the same solvent and mixed.

The quantitative content of the active substance was determined by spectrophotometry in the ultraviolet region of the spectrum. Measurement of the test solution and the comparison solution absorbance was carried out on a spectrophotometer “Evolution 60S” at a wavelength of 250–300 nm. As a blank solution, a 0.1 M solution of hydrochloric acid was used.

A similarity factor was calculated to determine the similarity of the dissolution profiles (European Medicines Agency: Guideline on the investigation of bioequivalence 2010):

${\mathrm{f}}_2=50·\log \left[\frac{100}{\sqrt{1+\frac{1}{\mathrm{n}}·{\sum }_{\mathrm{t}=1}^{\mathrm{n}}{\left({\mathrm{R}}_{\left(\mathrm{t}\right)}-{\mathrm{T}}_{\left(\mathrm{t}\right)}\right)}^2}}\right]$,

where:

n – number of time points;

R_(t) – the mean percent reference sample dissolved at time t after initiation of the study (sample without adding mineral water), %;

T(t) – is the mean percent test sample dissolved at time t after initiation of the study (samples with mineral water), %.

When added Ca^{2 +}, Mg^{2 +} to the solution of amoxicillin in the medium of 0.1 M solution of HCl, visible changes were not observed.

Results of UV-spectrophotometry study of the simultaneous presence of amoxicillin and chosen metal salts in a medium of 0.1 M HCl are shown in Figure 2. We observe an increase in the absorbance of amoxicillin with the addition of Ca²⁺ as well as Mg²⁺ salts in a medium of 0.1 M hydrochloric acid at a maximum wavelength 230 ± 1, in comparison with the control sample.

Figure 2. 

UV spectra of amoxicillin with the addition of Ca²⁺, Mg²⁺ in a medium of 0.1 M hydrochloric acid.

The UV-spectrum of amoxicillin in 0.1M hydrochloric acid has three maxima at wavelengths 203nm, 230±1nm and 272±1 nm. Herewith, first maxima fluctuate while adding different salts from 202 to 208 nm. To control, we took into account changes in absorbance at all maxima. In the experiment with salts of calcium, proportional increase in the absorbance in all three maxima was observed, respectively as the concentration of the antibiotic increased. This could mean that no interaction occurs with calcium salts (Fig. 3).

Figure 3. 

Job’s plot for amoxicillin and Ca in 0.1 M HCl at wavelength 230 nm, 272 nm.

While adding magnesium salts maxima in absorbance at 273 nm was observed on Job’s plot for the solution with ratio 5:5. Absorbance reached the peak at this point and then remain steady – next solutions with ratios 6:4 and subsequent show approximately the same absorbance as 5:5 solution (Figs 4, 5). This may possibly indicate the formation of several complex compounds.

Figure 4. 

Job’s plot for amoxicillin and Mg in 0.1 M HCl at wavelength 239 nm, 272 nm.

Figure 5. 

Job’s plot for amoxicillin and Mg in 0.1 M HCl at 273 nm.

The results of determining the dissolution of amoxicillin from tablets in a 0.1 M HCl medium with addition of mineral waters are given in Table 3 and Figure 6.

As it can be seen in Fig. 6 there is a significant increase in the concentration of amoxicillin in a medium of 0.1 M HCl solution with addition of mineral water “Truskavetska” in comparison with the control sample. Concentration also increased slightly in samples with the addition of Kharkiv city spring water #1, “Yessentuki-17” and “Borjomi”. The increase in concentration can be explained by an increase in absorbance during the interaction of amoxicillin with metal salts.

Figure 6. 

Amoxicillin tablets dissolution profile in a 0.1M HCl medium with added mineral waters.

For each control point, the relative standard deviation of the average result (RSD_x¯) was calculated, which should be less than 20% for the first control point, and less than 10% for the remaining points (European Medicines Agency (2010) Guideline on the investigation of bioequivalence). All values meet the requirements (Table 3).

Similarity factors calculated for study samples are shown in Table 4.

Two dissolution profiles are considered to be similar if the similarity factor (f2) is in the range of 50% to 100%. This indicates that the bioavailabilities of the test sample and the comparison sample are considered to be equivalent.

The similarity factors determined show that the dissolution profiles of amoxicillin tablets in a medium of 0.1 M HCl with the addition of almost all, except one, mineral waters are similar. Similarity factor of “Karpatska dzherelna” mineral water is 47.77, which doesn’t correspond to the acceptance criteria.