Hypromellose (purity ≥ 99%) and paracetamol (purity ≥ 99%) were obtained from Sigma-Aldrich (Steinheim, Germany). Both hypromellose and paracetamol were used in this study as received.

Binary physical mixtures of API and excipient consisting of 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 95% of paracetamol were prepared by a gentle mixing of ingredients in a porcelain mortar using a plastic spatula and a pestle. An analytical balance model XA 105 Dual Range (Mettler Toledo, Schwerzenbach, Switzerland), was used to weigh the paracetamol and hypromellose. To achieve complete homogenization, API and excipient were thoroughly mixed over a period of 10 min.

DSC curves were recorded on a heat-flux DSC 822^e instrument (Mettler Toledo, Schwerzenbach, Switzerland), with a liquid nitrogen cooling system (Dewar vessel) and STAR^e 9.10 software. The samples, ~ 4 mg in weight, were accurately weighed (±0.01 mg) and placed in 40 μl flat-bottomed aluminum pans with crimped-on lids. Measurements in triplicate were performed over the temperature range of 25 to 300°C at a heating rate 10°C/min under nitrogen stream (purity 99.9997%, Air Products, Warsaw, Poland) at a flux rate of 70 ml/min.

A heating-cooling-heating test was carried out using the following temperature program: heating samples at a rate of 10°C/min from 25 to 250°C, isotherm for 2 min, cooling at a rate 40°C/min from 250 to −25°C, isotherm for 2 min and the second heating at a rate 10°C/min to 250°C.

Indium (In, purity 99.999%) and zinc (Zn, purity 99.998%) standards (Mettler Toledo, Schwerzenbach, Switzerland) were used to calibrate a DSC cell. Reference values for onset temperature and heat flow were as follows: 156.6°C and 28.45 J/g (In); 419.6°C and 107.5 J/g (Zn), and for those measured: 156.6°C and 28.80 J/g (In); 420.1°C and 110.7 J/g (Zn).

A HSM equipped with a BX41polarizing microscope (Olympus, Shinjuku, Japan) and a color video digital camera SC30 supported by Olympus CellA software was used to record imagines during temperature scans. A 1–5 mg of sample was placed between two glass cover slides and put on a hot stage (Semic, Bioelektronika, Krakow, Poland) equipped with an SR90 temperature regulator (Shimaden, Tokio, Japan) and Heating Desk Shimaden software. Measurements were performed over the temperature range of 25 to 300°C at a heating rate of 10°C/min.

FT-IR spectra were collected using a Nicolet 380 FT-IR spectrometer (Thermo Fischer Scientific, Madison, USA), with a DTGS KBr detector and OMNIC software. The samples analyzed were prepared as KBr pellets with the aid of a hydraulic press (Specac, Orpington, UK), each pellet being prepared from a 1-mg sample and 100 mg of spectroscopy-grade KBr (Merck, Darmstadt, Germany). Measurements in triplicate were performed in the 4,000–400 cm⁻¹ spectral region with a spectral resolution of 4 cm⁻¹. Prior to each measurement, background spectra was taken with an average of 16 scans.

Raman spectra were recorded on a DXR SmartRaman spectrometer (Thermo Fisher Scientific, Madison, USA). The instrument was equipped with a Raleigh filter, CCD detector and OMNIC software. Measurements were run in triplicate over a spectral range of 3413–99 cm⁻¹ with resolution of 2 cm⁻¹. A 15-mW DXR 780 nm laser with an aperture of 25 μm was deployed. Exposure time was 1 s (twice).

The solubility curve of crystalline API in an amorphous polymer was calculated in accordance with the procedure described in the literature (Tian et al., 2015; Baghel et al., 2016). If a polymer is considered as a solvent, the quantity of paracetamol soluble in hypromellose is expressed as the mole fraction (x_API) of the dissolved API in relation to the activity coefficient (γ_API), according to the equation:

where: ΔH_fus is the melting enthalpy of paracetamol, R the gas constant, T_m the melting temperature of paracetamol in Kelvin, and T the temperature in Kelvin of the two phases of paracetamol in equilibrium.

The activity coefficient (γ_API) of paracetamol can be calculated on the basis of Hansen solubility parameters (δ), the molar volume (V) of mixture ingredients (k), the mixture volume (V¯) and the molar volume of weighted Hansen solubility parameter (δ¯), according to the equation:

The mixture volume (V¯) and molar volume of weighted Hansen solubility parameter (δ¯), taking into account the character of interactions, i.e., dispersion forces (δ_d), polar interactions (δ_p) and hydrogen bonding (δ_h), can be calculated according to the following equations:

where ϕ is the volume fraction of API or polymer (k), x the mole fraction, M the molecular weight, and ρ the density of ingredients.

The miscibility curve of paracetamol with hypromellose can be predicted based on the Florry-Huggins theory and the Gibbs free energy (ΔG_mix) (Forster et al., 2001; Djuris et al., 2013; Maniruzzaman et al., 2014; Tian et al., 2015). The Gibbs free energy (ΔG_mix) of mixing both ingredients is described by the equation:

where n is the number of API lattice sites, defined as the volume of API molecule occupied by a polymer chain, and χ is the API-polymer interaction parameter.

The number of API lattice sites occupied by a polymer chain (n) and the API-polymer interaction parameter (χ) can be calculated by the following equations:

where v is the volume of paracetamol lattice site (volume of the API), and δ is the Hansen solubility parameter (δ).

Miscibility curve (spinodal curve) can be predicted by setting the second derivative of the Gibbs free energy to zero, as expressed by the equation:

The glass transition curve can be predicted using the relation between the glass transition temperature (T_g) of the mixture and the weight fraction of paracetamol (w_API) in the mixture, according to the Fox equation (Tian et al., 2015):

where T_g is the glass transition temperature of a mixture, T_gAPI and T_gpolymer are the glass transition temperatures of paracetamol and hypromellose, respectively.

The DSC curves of paracetamol, hypromellose and their mixtures prepared by carefully mixing ingredients with a plastic spatula in a porcelain mortar are shown in Figure 2A, those mixed using a pestle in Figure 2B. The DSC curve of paracetamol (curve a) displays a single endothermic peak due to the melting of polymorphic form I (monoclinic) at 169.4°C, which is consistent with the literature data (Klı̀mová and Leitner, 2012). The physical mixtures homogenized using a spatula (Figure 2A, curves b-f) revealed that the endothermic peak due to the melting of paracetamol appeared on the DSC curves over the entire concentration range and decreased as the content of active ingredient in the mixture decreased. By contrast, DSC curves for mixtures prepared using a pestle (Figure 2B, curves b-f) did not display the melting peak of paracetamol for mixtures containing < 20% of API. This suggests that the interaction between paracetamol and hypromellose resulted in a partial reduction in paracetamol crystallinity, probably due to intermolecular interaction with the formation of hydrogen bonding. As reported in the literature, other physical interactions are also possible, e.g., formation of eutectic mixtures (Bi et al., 2003), solid dispersions (Jahangiri et al., 2015), dissolution of paracetamol in hypromellose (Tian et al., 2015), reduction of paracetamol crystallinity in the presence of microcrystalline cellulose (de Oliveira et al., 2017) and amorphization or polymorphic transformation (Salunkhe et al., 2018). The risk of initiating interactions between ingredients is greater when homogenization of the mixtures is carried out using a pestle (Byard et al., 2005; Curtin et al., 2013).

The data compiled in Table 1 shows that the correlation coefficients between the enthalpy of paracetamol melting and the API content in mixtures with hypromellose are high. Detailed inspection of other regression parameters revealed high values of intercept (b) in calibration equations for mixtures mixed using either plastic spatula or pestle. However, the intercept value increased approximately 3-fold for mixtures mixed with a pestle as opposed to those mixed with a spatula. This may be indicative of the fact that the heat of fusion values are lower than those expected for real content of paracetamol in the mixtures. The implication is that the immediate cause of this situation may be a reduction in crystallinity of paracetamol due to its contact with hypromellose. The intensity of this process is enhanced by mixing the ingredients with a pestle.

To investigate potential interactions between ingredients, HSM was also used. This technique is a combination of microscopy and thermal analysis to enable the investigation of solid state changes in substances of pharmaceutical interest under heating or cooling (Steger et al., 2012). It allows changes in appearance of paracetamol crystals, particles of amorphous hypromellose and their mixtures to be observed, along with the determination of characteristic temperatures.

The appearance of paracetamol, hypromellose and their mixture at a 1:1 mass ratio is shown in Figures 3A–C, respectively (ambient temperature), and Figures 3D,E (after melting of paracetamol) and Figure 3F (after liquidation of hypromellose). Image a reflects the fact that between ambient and sublimation temperature API crystals were characterized by regular shape but various sizes. Under heating, paracetamol sublimed above 150°C, and subsequently, its small crystals melted at ~ 168°C. Vapors of API sublimation solidified to form needle-like crystals on the cool surface. FT-IR used to investigate these crystals confirmed the existence of form I of paracetamol. DSC analyses revealed that the needle-like crystals melted at 168°C, which corroborates the melting of form I of paracetamol (Klı̀mová and Leitner, 2012). Complete melting of API and its intensive evaporation were observed above 172°C (image d). Under cooling to ambient temperature, the sample crystallized. On the other hand, image c presents particles of amorphous hypromellose at various sizes. Under heating, liquidation and browning of the polymer were observed at ~ 260°C (Figure 3F).

A HSM study revealed that at the initial stage of the heating process, API crystals and particles of amorphous polymer were easily visible (Figure 3B) in mixtures containing 10, 30, 50, 70, and 90% of paracetamol. Next, as with paracetamol heating, the sublimation of mixtures commenced at ~ 150°C. Afterwards, the melting of the finest crystals began at temperatures lower than that of paracetamol, i.e., ~ 162, 161, 159, 157, and 156°C, respectively, for mixtures with decreasing content of API. Figure 3E presents the 1:1 mass ratio mixture heated to 159°C and shows hypromellose particles and melted paracetamol. In brief, neither new crystals nor amorphous particles were found when the mixtures were heated.

FT-IR and Raman spectroscopy were used to investigate whether any intermolecular interactions or chemical reactions may have occurred between paracetamol and hypromellose. Figure 4 shows the FT-IR spectra of paracetamol, hypromellose, and their mixtures. In interpreting these data, special attention was paid to the characteristic bands of chemical groups capable of forming hydrogen bonding. Characteristic bands for monoclinic paracetamol (spectrum a) were found at 808 cm⁻¹ (amid group deformation), 968 and 1,259 cm⁻¹ (C–N stretching vibrations), 1,655 cm⁻¹ (C = O stretching and C–NH deformation), 3,034 cm⁻¹ (N–H stretching) and 3,161 and 3,325 cm⁻¹ (O–H stretching) (Burgina et al., 2004; An et al., 2008). This confirms the results of DSC analysis which indicates that form I of paracetamol is the API under investigation (Burgina et al., 2004; Łuczak et al., 2013). Detailed inspection of the FT-IR data for mixtures (spectra b–f) revealed that characteristic absorption bands of paracetamol were found in all the spectra. Their intensity decreases proportionate to decreasing paracetamol content in the mixtures. Moreover, the lack of shifting in the characteristic absorption bands of both ingredients in conjunction with a lack of new bands suggests that neither physical interaction nor chemical reaction occurred between ingredients. It concurs with the literature data which revealed that hydrogen bonds have not been formed between ingredients in the physical mixtures, but they can be formed during hot-melt extrusion, e.g., nimodipine with hydroxypropyl methylcellulose succinate acetate (Zhang et al., 2018). It has also been confirmed that shifting of NH band at 3,300 cm⁻¹ to 3,357 cm⁻¹ in the spectrum of nimodipine extrudate with polymer strongly evidenced the formation of hydrogen bonds between the amine and carbonyl groups of API and polymer, respectively. The hydrogen bonds can also be created in the presence of solvent, e.g., using aqueous polymer solutions (Wen et al., 2005) or during electrospray-drying (Liu et al., 2018).

The Raman spectra of paracetamol, hypromellose and their mixtures are shown in Figure 5. Characteristic bands for paracetamol (spectrum a) were found at 463 cm⁻¹ (out-of-plane ring deformation), 1,235 cm⁻¹(C–N stretching vibration), 1,560 cm⁻¹(N–H and C = O stretching), and 1,646 cm⁻¹ (C = O stretching) (Burgina et al., 2004). Raman shift at 463 cm⁻¹ is particularly characteristic of the polymorphic form I of paracetamol in contrast to that at 454 cm⁻¹ which is characteristic of form II (An et al., 2008). Thus, similar to the DSC and FT-IR data, form I of paracetamol has also been identified by Raman spectroscopy. Detailed inspection of the data for all API mixtures (spectra b-f) confirmed that the existence of characteristic Raman shifts is related to paracetamol. The intensity of these bands decreases in line with decreased paracetamol content in the mixtures. Under no circumstances did bands shift or new bands appear. Therefore, neither physical interactions nor chemical reactions between ingredients were to be expected.

The classical phase diagram is constructed using the melting temperatures of two ingredients existing in a crystal form. Hypromellose is an amorphous polymer that does not melt, but is characterized by glass transition. Preparation of the phase diagram for a mixture of crystalline API and amorphous polymer entails the calculation of three curves which predict the solubility of API in polymer, mutual miscibility of ingredients and temperature of glass transition. The phase diagram reflects an equilibrium state predicted from the calculations and enables the conclusions to be drawn relating to the quantity of API (that may be dissolved in polymer at a given temperature) and about the composition of the mixture characterized by complete miscibility at a given temperature.

The literature data used for calculations of the solubility, miscibility, and glass transition curves in equations 1, 11, and 12 (Calculations section) are listed in Table 2 (Espeau et al., 2005; Chan et al., 2006; Sibik et al., 2014; Adhikari et al., 2015), while the calculated values predicting the solubility of paracetamol in polymer, mutual miscibility of ingredients and the glass transition of mixture are compiled in Tables 3, 4. These data were used to prepare the phase diagram for physical mixture of API with hypromellose shown in Figure 6.

The glass transition curve reflects the predicted temperature at which the mixture undergoes glass transition at a given composition. Glass transition was also confirmed with the aid of the heating-cooling-heating test. DSC curves reflecting this test for mixtures containing 90, 70, 50, 30, and 10% of API are illustrated in Figure 7. Detailed inspection of these curves revealed that melted paracetamol underwent amorphization during the fast cooling cycle (Figure 7B, curve a), a fact confirmed by an inflection on the DSC curve due to paracetamol glass transition and the absence of exothermic peak due to the crystallization of API. The performance of all the mixtures under study was the same as paracetamol behavior (Figure 7B, curves b–f).

In the second cycle of heating, the DSC curve of paracetamol (Figure 7C, curve a) showed inflection at ~23°C caused by the glass transition. A further exothermic peak was found due to paracetamol recrystallization to polymorphic form II which melted at ~ 160°C. For all mixtures, the DSC curves of the second heating cycle (Figure 7C, curves b–f) revealed an inflection due to glass transition between ~ 24 and ~ 112°C, respectively, for decreasing quantity of paracetamol in mixture. These temperatures are compiled in Table 4 along with those calculated using a suitable Equation (12). Neither an exothermic peak due to paracetamol crystallization nor an endothermic peak caused by its melting was found on the DSC curves of the second heating cycle. Thus, the melting of paracetamol with hypromellose lead to amorphization of API with inhibition of its crystallization.

The phase diagram permits a prediction of the thermodynamic stability of the system containing API and hypromellose in relation to temperature (Djuris et al., 2013; Tian et al., 2015). Figure 6 shows that the area above the solubility curve is the thermodynamically stable region, while the area between the miscibility and solubility curves represents the thermodynamically unstable region in which spontaneous phase separation may occur. In addition, the area on the right-side of the miscibility curve indicates the region in which ingredients are separated. The glass transition curve represents the glass transition temperatures for paracetamol-hypromellose mixtures.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.