The solubility of 2-acetoxy benzene carboxylic acid in water is about 3 mg/cm³. According to USP solubility criteria, 2-acetoxy benzene carboxylic acid is slightly soluble in water. The samples were prepared according to the Huguchi and Connors method by adding a poorly soluble pharmaceutical active substance (ASA) in excess to aqueous cyclodextrin solutions of various concentrations. Phase solubility tests were performed in triplicate.

A constant amount of pure 2-acetoxy benzene carboxylic acid (0.15 g) was suspended in 10 mL of an aqueous solution containing successively higher concentrations of cyclodextrin (2 - 14 mmol). Well-closed vials were continuously stirred for 72 hours at room temperature (25˚C) and then the samples were filtered through 0.45 μm filter paper. Fresh samples were prepared and used to measure phase solubility on a UV-spectrophotometer with a previous dilution that corresponds to the interval of the calibration curve of 2-acetoxy benzene carboxylic acid in methanol, on the basis of which the concentration is measured (Figure 4).

• Phase solubility measurements were done on a UV-spectrophotometer Perkin Elmer Lambda 25 at 226 nm, on a previously constructed calibration curve. UV/VIS spectroscopy is used for analytical purposes to quantify various analytes, such as conjugated organic compounds, organic substances and biochemical macromolecules. Determination usually takes place in solutions. Organic substances, especially those with a high degree of conjugation, absorb light in the UV or visible region of the electromagnetic spectrum.

For preparation of calibration curve it is used pure 2-acetoxy benzene carboxylic acid as a standard. At the analytical balance was measured 5.00 mg of standard in volumetric flask of 10 ml and added ethanol to the mark. After dissolving, we pipetted 0.4, 0.5, 0.6, 0.7 and 0.8 ml of stock solution in volumetric flask of 10 ml and added distillated water to the mark, so we had five diluted standards with concentrations: 20, 25, 30, 35, 40 and 45 μg/ml. Groups of atoms that do not absorb radiation by themselves, but their presence in molecules causes the absorption maxima to shift to longer wavelengths is auxochromic groups. Shift of the absorption maximum (λ_max) towards longer wavelengths caused by the presence of auxochromes or changes in the solvent. It is a bathochromic shift or red shift. For example: introduction of groups like: -OH, -OCH₃, causes λ_max to shift to higher values.

• Qualitative interpretation of the complex was performed with FTIR-spectrophotometer Perkin Elmer 1000; Fourier transform spectrophotometers significantly improved the quality of the infrared spectrum, accelerated the analysis process and enabled comparison with the database. In this way, they enabled greater sensitivity and the use of minimal sample quantities. Infrared spectra of the samples were recorded on a resolution of 4 cm⁻¹ and an interval of 4000 - 400 cm⁻¹.

The sample is prepared by taking 10 mg of the sample which is well powdered and homogenized, with about 90 mg of pure and well dried KBr. It is very important that KBr is dry during the preparation of the sample, because otherwise bands originating from water appear on the spectrum. The resulting mixture is filled into a metal cuvette which is placed in an FTIR spectrometer and the IR spectrum of the powder component is recorded. The spectrum can be interpreted by visual expertise of the position, intensity and shape of the absorption bands: The position of the band is determined by its peak (maximum). The intensity of the band is proportional to the absorbance, its size is the length of the absorption band in the ordinate direction. The shape of the band depends on a number of factors such as concentration, type of solvent, resolution of the apparatus, etc.

Using β-CD as a natural cyclodextrin increases the solubility of 2-acetoxy benzene carboxylic acid in water. The diagram (Figure 5) shows the influence of different concentrations of β-cyclodextrin on the phase solubility of 2-acetoxy benzene carboxylic acid in an aqueous environment. According to the classification of phase solubility profiles according to Higuchi and Connors, this solubility belongs to the A_L type since the increase in cyclodextrin concentration results in a linear increase in the solubility of the pharmaceutical active compound. The most common type of cyclodextrin complex is the 1:1 pharmaceutical active compound/cyclodextrin (D/CD) complex, where one molecule of the pharmaceutical active compound forms a complex with one molecule of cyclodextrin.

C + D ⇌ K 1 : 1 D/CD

Under such conditions, the A_L type of phase solubility diagram, with a slope less than one, has a stability constant K_1:1 of the complex that can be calculated from the slope and the intrinsic solubility of the biologically active compound So in the aqueous complexation medium.

Intrinsic solubility is the solubility of a substance when cyclodextrin is not present, i.e. the point where the phase solubility curve intersects the y-axis.

The stability constant of the complex was calculated from the Higuchi and Connors equation using the parameters obtained from the phase solubility diagram. The stability constant for samples of the complex 2-acetoxy benzene carboxylic acid-β-cyclodextrin is K = 45.31 M⁻¹ (Figure 5).

The stability of cyclodextrin complexes with organic molecules in water is most often attributed to the so-called hydrophobic hydration of the interior of the macrocycle, that is, the hydrophobic functional groups of the guest molecules. This term refers to the restructuring of water molecules around non-polar chemical compounds and in the cavity of cyclodextrin. Consequently, with the formation of a complex, the water molecules that participated in the solvation of the guest and the interior of the host can form hydrogen bonds like those in the solvent. Since it is hydrophobic interactions that form between the host and guest molecules, parts of the 2-acetoxy benzene carboxylic molecule that contain aromatic nuclei are involved. NMR studies have shown that in the complex the benzene ring of 2-acetoxy benzene carboxylic acid is located inside the cavity of the cyclodextrin molecule, while the ester and acetyl groups come out of the cone cavity [9] . The presence of hydrophilic functional groups on the edge of cyclodextrins can also significantly influence their complexation properties. 2-acetoxy benzene carboxylic acid with two carbonyl chromophores can form hydrogen bonds with the hydroxyl groups of cyclodextrin and thus enables better incorporation of the drug into the host cavity. Figure 6 shows a proposed model of the formation of a complex between one molecule of 2-acetoxy benzene carboxylic acid and one molecule of cyclodextrin by placing the aromatic benzene nucleus in the lipophilic cavity of cyclodextrin [10] .

The results of research by UV spectroscopy and the analysis of UV spectra of complexed 2-acetoxy benzene carboxylic acid with different concentrations of β-cyclodextrin are shown in Figure 7. They indicate that increasing the concentration of β-cyclodextrin leads to an increase in the absorbance of 2-acetoxy benzene carboxylic acid at a wavelength of 226 nm, which indicates an increasing inclusion of the pharmaceutical active compound in the cavity of the β-cyclodextrin molecule. The spectrum shows a bathochromic and hyperchromic shift due to the presence of auxochromic groups such as hydroxyl groups derived from cyclodextrin.

The formation of a complex of 2-acetoxy benzene carboxylic acid and β-cyclodextrin was also confirmed by FTIR characterization. The FTIR spectra of 2-acetoxy benzene carboxylic acid show two significant regions that characterize its structure. The first region refers to the existence of an intense peak at 1605 cm⁻¹ which corresponds to the benzene ring. Peaks in the area of 1680 and 1750 cm⁻¹ confirm the presence of two C = O groups. The second part of the IR spectrum of aspirin includes shorter and broader peaks in the region of 2500 - 3500 cm⁻¹ that originate from the vibrations of O-H and C-H bonds [11] .

The FTIR spectra of β-cyclodextrin show a broad band around 3400 cm⁻¹ which is the result of valence –OH vibrations. In the region of 2900 cm⁻¹ there is a band of asymmetric and symmetric valence C-H vibrations of the –CH₂ group, but also valence C-H vibrations from the CHOH group. If some hydrogen or Van der Waals interaction were to be established, the spectra of the inclusion complexes compared to the spectra of the starting compounds should be different in intensity, shape, position and number of bands.

The analysis of FTIR spectra (Figure 8) of prepared complexes 2-acetoxy benzene carboxylic acid complex and β-cyclodextrin shows that the broad hydroxyl band of pure β-cyclodextrin at 3281.2 cm⁻¹ is narrowed in the FTIR spectra of the complex, which is a good indicator of the formation of an inclusion complex. This is a common phenomenon observed by many researchers in the synthesis of an inclusion complex between a β-cyclodextrin (host) and a guest molecule. Increasing the concentration of β-cyclodextrin leads to a decrease in the frequency of valence ν(OH) vibrations, which favors complexation.

2-Hydroxypropyl-β-cyclodextrin (HPβCD), a chemically modified β-cyclodextrin, has higher water solubility and lower parenteral toxicity compared to its parent compound β-cyclodextrin. Also, is a potential ingredient in pharmaceutical formulations. The phase solubility diagram for complexes of 2-acetoxy benzene carboxylic acid and aqueous solutions of 2-hydroxypropyl-β-cyclodextrin show that the solubility of 2-acetoxy benzene carboxylic acid increases linearly with increasing cyclodextrin concentration, so it can be classified as an A_L type diagram according to Higuchi and Connors with the possibility of forming a 1:1 complex and a 1:2 complex with cyclodextrin (Figure 9). The stability constant obtained from the slope of the phase solubility diagram is 53.57 M⁻¹.

UV spectra (Figure 10) of aqueous solutions of 2-acetoxy benzene carboxylic acid with 2-hydroxypropyl cyclodextrin show that the absorption maximum has shifted towards lower wavelengths. UV spectra show a hypsochromic shift of the absorption maximum of 2-acetoxy benzene carboxylic acid, which may be a consequence of the influence of the auxochromic group of cyclodextrin on the carbonyl group of 2-acetoxy benzene carboxylic acid. An increase in the concentration of 2-hydroxypropyl cyclodextrin is accompanied by an increase in absorbance intensity with a bathochromic shift, which is a confirmation of complexation.

The FTIR spectrum (Figure 11) of 2-acetoxy benzene carboxylic acid shows absorption maxima at 2696.78 cm⁻¹, which originate from the stretching vibrations of the carbonyl –OH group, at 1749.764 cm⁻¹ corresponding to the carbonyl group of the ester and at 1679.79 cm⁻¹ which corresponds to the acetate carbonyl group (Table 1). When forming a complex with 2-hydroxypropyl-β-cyclodextrin, the carbonyl band has a slight shift to a lower wavenumber, due to the host-guest interaction. This suggests the possibility of hydrogen bonding between the hydroxyl group of the host cavity and the carbonyl group of 2 acetoxy-benzene carboxylic acids. A slight shift of the absorption band for carbonyl groups to lower frequencies can be attributed to the breaking of hydrogen bonds between drug molecules and the formation of intermolecular hydrogen bonds of the drug with cyclodextrin.

Modified methyl-β-cyclodextrin (mβCD) offers a significant advantage as a host molecule over β-cyclodextrin (β-CD) because its solubility in aqueous solution at room temperature (>2000 mg/mL) is significantly higher. Therefore, it is expected that the higher solubility of methyl-β-cyclodextrin in the aqueous medium will contribute to the higher solubility of the pharmaceutically active compound when it is in the complex state.

Figure 12 shows the effect of different concentrations of methyl-β-cyclodextrin on the phase solubility of 2-acetoxy benzene carboxylic acid in an aqueous medium. We can see that by increasing the concentration of methyl-β-cyclodextrin there was an increase in the measured concentration of 2-acetoxy benzene carboxylic acid and practically doubles it. We can conclude that, according to the phase profile, it belongs to the AL type of phase solubility.

The stability constant obtained from the phase solubility diagram is 76.55 M⁻¹. Compared to the results obtained for β-cyclodextrin and 2-hydroxypropyl-β cyclodextrin, we can say that methyl-β-cyclodextrin binds drug molecules more strongly. The reason for this may be that the methyl group obviously increases the non-polar part of the cyclodextrin cavity, effectively increasing the depth of the cavity itself, which affects the better incorporation of the benzene ring of the “guest” molecule and thus its solubilization.

Confirmation of complexation is also shown by the UV spectra (Figure 13) of the samples, where we see that the increase in the concentration of methyl-β-cyclodextrin is accompanied by a higher intensity of the absorption maximum and a slight shift towards longer wavelengths.

FTIR characterization of the complex of 2-acetoxy benzene carboxylic acid and methyl-β-cyclodextrin confirms the formation of the complex. By comparing the spectra of pure 2-acetoxy benzene carboxylic acid and pure methyl-β-cyclodextrin with the spectra of the complex, we see that there are differences in the position of some bands in the complex compared to the starting compounds (Figure 14).