To Formulate and Evaluate Of Ternary Co-Amorphous System for Solubility Enhancement of Ritonavir

Table 1: Drug and Coformers

Equipments

Table 2: Equipments and Suppliers

Calibration curve of Ritonavir

A series of standard solutions of  at concentrations of 10, 20, 30, 40, 50, and 60 µg/mL were prepared in 0.1 N HCl, and the absorbance of each solution was measured at 240 nm using a UV-visible spectrophotometer.

Solvent evaporation method for preparation of co amorphous

The required molecular ratio of and selected co-formers was dissolved in ml methanol under continuous stirring for 20 min to obtain a homogeneous solution. The solution was then allowed to evaporate at room temperature, resulting in the formation of a free-flowing co-amorphous powder.

Table 3: Composition of Ternary Co-Amorphous Formulations

Saturation solubility study

An excess quantity of sample was introduced into 10 mL of 0.1 N hydrochloric acid to evaluate saturation solubility. The dispersion was continuously mixed on a Magnetic Stirring system at 300–500 rpm while maintaining the temperature at 37 ± 0.5°C for 24 h to establish equilibrium. After equilibration, the undissolved particles were allowed to sediment, and the supernatant was separated by filtration through a 0.45 μm membrane filter. The filtrate was appropriately diluted and quantified using UV-Visible Spectroscopy at 240 nm.

Fourier transform infrared spectroscopy

Fourier Transform Infrared spectroscopy analysis was carried out to characterize the co amorphous using a Bruker Alpha ? FTIR spectrometer.

Differential scanning calorimetry

Differential Scanning Calorimetry analysis of optimized formulation was carried out using a METTLER TOLEDO DSC1 STARe System.

Powder x-ray diffraction

The crystalline nature of the sample was evaluated by powder X-ray Diffraction employing an Ultima ?, Riga Corporation, Japan.

Scanning electron microscopy

Surface morphology of the sample was examined using Scanning Electron Microscopy.

Pre-compression study

Bulk density

Bulk density of a powder may vary considerably depending on factors such as crystallization method, milling process, and formulation characteristics. It was determined by carefully transferring the powder blend into a graduated cylinder through a wide funnel, followed by measurement of the occupied volume and sample weight. The bulk density was then calculated as the ratio of powder mass to bulk volume.

Tapped density

The tapped density was measured by putting the powder mixture into a graduated cylinder using a large funnel, then tapping the cylinder 50 times, and finally recording the volume and weight of the compacted powder.

Angle of repose

The angle of repose was determined by allowing the powder to flow through a funnel onto a flat surface, forming a cone, and then measuring the angle between the powder slope and the horizontal plane.

Hausner’s ratio

This parameter indicates powder flow properties and is calculated from the ratio of tapped density to bulk density.

Carr’s index

The compressibility index was determined using the measured bulk density and tapped density values.

Post compression study

Hardness

Tablet hardness refers to the force needed to break a tablet under diametric compression. It was measured using a Monsanto hardness tester.

Tablet size and thickness

The thickness of the tablets was measured using a Vernier caliper.

Friability

The friability test was carried out to assess the ability of tablets to resist abrasion during packaging, handling, and transportation. An initial weight of 20 tablets was recorded and placed in a friabilator operating at 25 rpm for 4 minutes. The weight loss after the test was calculated and expressed as a percentage. The acceptable friability range is preferably between 0.5% and 1.0%.

Weight variation test

Uniformity of tablet weight is an important quality parameter for a batch. Any deviation in weight must comply with the prescribed limits. A total of 20 tablets were randomly chosen and weighed precisely for analysis.

In-vitro dissolution studies

In vitro dissolution studies of pure Ritonavir and the optimized formulation were carried out using a USP type II dissolution apparatus with paddle assembly operated at 50 rpm. The dissolution test was performed in 900 mL of 0.1 N HCl, which served as the dissolution medium, while maintaining the temperature at 37 ± 0.5°C. At predetermined time intervals, 5 mL aliquots were withdrawn and replaced with an equal volume of fresh 0.1 N HCl to maintain sink conditions. The collected samples were analyzed using UV-Visible Spectroscopy, and the dissolution profile was evaluated accordingly.

RESULTS AND DISCUSSTION

Calibration curve of Ritonavir

Table 4: Concentration and Absorbance of Ritonavir in 0.1 N HCL

Figure 1: Calibration Curve of Ritonavir

The calibration curve of Ritonavir in 0.1 N HCl showed a linear increase in absorbance with increasing concentration in the range of 10–60 µg/mL. The absorbance values increased from 0.1372 at 10 µg/mL to 0.8106 at 60 µg/mL, indicating good compliance with the Beer–Lambert law. This confirms that the analytical method is suitable for quantitative estimation of the drug.

Saturation solubility study

Table 5: Saturation Solubility Study Co-Amorphous System

Among all formulated batches of Ritonavir, batch F8 (1:3:2) was identified as the optimized formulation due to its highest solubility and better dissolution performance. It showed a solubility of 0.096 mg/mL, representing a 19.2-fold increase over pure drug. Further studies such as Fourier-transform infrared spectroscopy, Differential scanning calorimetry, and X-ray diffraction are required for confirmation.

Infrared spectroscopy

Comparison of the Infrared spectroscopy spectra of the pure drug and co-amorphous system indicated hydrogen bond formation between the drug and coformer, suggesting the formation of a new supramolecular synthon.

IR spectrum of Ritonavir

Figure 2: IR Spectra of Ritonavir

Table 6: Characteristic peaks from Ritonavir

IR spectra of Ritonavir+L-Arginine+Nicotinamide

Figure 3: FT-IR spectrum of Ritonavir+L-Arginine+Nicotinamide

Table 7: FT-IR spectrum of Ritonavir+L-Arginine+Nicotinamide

Differential scanning calorimetry

Differential scanning calorimetry of Ritonavir

Figure 4: DSC of Ritonavir

Differential scanning calorimetry of Ritonavir+L-Arginine+Nicotinamide

Figure 5: DSC of Ritonavir+L-Arginine+Nicotinamide

The co-amorphous formulation exhibited a noticeable change in thermal behavior compared to the pure drug. The melting temperature shifted from 122.65°C for the pure drug to 114.97°C for the co-amorphous system. Additionally, the onset temperature increased from 97.43°C in the pure drug to 112.56°C in the co-amorphous formulation. These variations in thermal characteristics suggest the successful formation of a co-amorphous phase and indicate molecular interactions between the drug and the co-former.

Powder x-ray diffraction

Powder x-ray diffraction of Ritonavir

Figure 6: PXRD of Ritonavir

Powder x-ray diffraction of Ritonavir+L-Arginine+Nicotinamide

Figure 7: PXRD of Ritonavir+L-Arginine+Nicotinamide

The PXRD pattern of the co-amorphous formulation containing Ritonavir, L-Arginine, and Nicotinamide exhibited significant changes compared with that of pure Ritonavir. The characteristic sharp diffraction peaks of crystalline Ritonavir were markedly reduced in intensity or disappeared, resulting in a broad halo pattern. This reduction in crystallinity indicates the transformation of the drug into an amorphous state. The absence of distinct crystalline peaks and the appearance of diffuse scattering suggest the successful formation of a co-amorphous system through molecular interactions among Ritonavir, L-Arginine, and Nicotinamide. These findings confirm modifications in the solid-state properties of the drug and support the development of a stable co-amorphous phase.

Scanning electron microscopy

Scanning electron microscopy of Ritonavir

Figure 8: SEM of Ritonavir

Scanning electron microscopy Ritonavir+L-Arginine+Nicotinamide

Figure 9: SEM of Ritonavir+L-Arginine+Nicotinamide

The surface morphology of the Ritonavir–L-Arginine–Nicotinamide system was examined by SEM, and the micrographs are presented in Figure X. This formulation was selected for SEM analysis due to its improved solubility. The SEM images revealed distinct morphological changes compared with pure Ritonavir, showing irregular and non-uniform particles. These observations suggest modification of the original solid-state structure and support the formation of a new drug–co-former system.

Pre compression studies of pure drug and co-amorphous formulation

Table 8: Pre compression study of pure drug and co-amorphous system

(No. of samples, n = 3)

The pre-compression evaluation demonstrated that the Ritonavir–L-Arginine–Nicotinamide co-amorphous system possessed better flow and packing characteristics than pure Ritonavir. The observed improvement in flowability and compressibility may be attributed to the modification of particle properties following co-amorphization, indicating its suitability for further tablet formulation and manufacturing processes.

Post compression parameters of pure drug and co-amorphous tablets

Table 9: Post Compression parameters of tablets

(No. of samples, n = 3)

Dissolution studies

Figure 10: Cumulative % drug release of Ritonavir+L-Arginine+Nicotinamide co amorphous Tablet