Formulation, Development And Evaluation Of Enteric Coating Rifaximin Pellets For Small Intestinal Delivery

Table no. 1 Materials used

2. Instruments and Equipment

All instruments and equipment used during the study were calibrated and qualified prior to use, and equipment qualification records were maintained throughout. The complete list of instruments and equipment.

Table no.2 Instruments and Equipment

EXPERIMENTAL WORK

1. Organoleptic Properties

The organoleptic properties of rifaximin were evaluated to confirm the identity and physical characteristics of the drug sample. The drug was found to be an orange-red crystalline powder with a characteristic appearance and no objectionable odour. The observed properties were in agreement with the reported literature values.

Table no.3 Organoleptic Properties of Rifaximin

Solubility Study

The solubility of rifaximin was determined in various solvents. The drug exhibited poor aqueous solubility and was found to be practically insoluble in water, confirming its BCS Class IV nature.

Table no. 4 Solubility Profile of Rifaximin

The poor aqueous solubility of rifaximin justified the development of a specialized pellet-based delivery system for targeted intestinal drug release.

Melting Point Determination

The melting point of rifaximin was determined using the capillary method and compared with the reported literature value.

Table no.5 Melting Point of Rifaximin

The observed melting point was found to be within the reported range, indicating purity of the drug sample.

UV spectrophotometric analysis

The UV-Visible spectrophotometric analysis of Rifaximin was carried out using a Jasco Corporation, Japan, V-550 spectrophotometer with Spectra Manager software. Methanol was used as the solvent system for both the blank and the sample preparations. A 20 µg/mL solution of Rifaximin was scanned, and the wavelength of maximum absorption (max) was found to be 290 nm.

The UV absorption spectrum of Rifaximin was obtained using a Jasco Corporation, Japan, V-550 UV-Visible Spectrophotometer equipped with Spectra Manager software. All glassware used was thoroughly cleaned with double distilled water and dried before use. Analytical grade reagents were used throughout the study, and methanol was selected as the solvent due to the solubility of Rifaximin.

For the determination of the UV spectrum, 10 mg of Rifaximin was accurately weighed and dissolved in methanol, and the volume was made up to 10 mL to obtain a stock solution of 1000 µg/mL. From this stock solution, 0.2 mL was withdrawn and diluted to 10 mL with methanol to obtain a working solution of 20 µg/mL. The solution was scanned over the wavelength range of 200–400 nm to determine the wavelength of maximum absorbance (λmax). The λmax of Rifaximin was found at approximately 290 nm.

Figure no.1 UV spectrophotometric of Rifaximin drug

This figure presents the UV absorption spectrum of pure methanol, scanned over the relevant wavelength range and used as the reference blank for all subsequent spectrophotometric

measurements. The spectrum shows a flat baseline with negligible absorbance across the scanned range, confirming the absence of any interfering absorption from the solvent itself in the region of interest around 290 nm.

Figure no.2 UV spectrophotometric of Rifaximin drug + Excipients

Calibration Curve of Rifaximin in Methanol

A calibration curve of Rifaximin was prepared in methanol. A stock solution containing 1000 µg/mL was prepared by dissolving 10 mg of Rifaximin in 10 mL of methanol. From this stock solution, 1 mL was transferred into a 10 mL volumetric flask and diluted to volume with methanol to obtain Solution A (100 µg/mL).

Aliquots of 0.5, 1.0, 1.5, 2.0, and 2.5 mL were withdrawn from Solution A and diluted to 10 mL with methanol to obtain concentrations of 5, 10, 15, 20, and 25 µg/mL (ppm), respectively. The absorbance of each solution was measured at 290 nm against methanol as a blank. A calibration curve was plotted by taking concentration (µg/mL) on the X-axis and absorbance on the Y-axis. The calibration curve showed good linearity over the selected concentration range

Figure no.3 Calibration Curve of Rifaximin in Methanol

FTIR Analysis of Rifaximin

The Fourier Transform Infrared (FTIR) spectrum of Rifaximin was recorded using a Shimadzu IRAffinity-1 FTIR spectrophotometer. The spectrum was recorded using the potassium bromide (KBr) pellet method, with KBr used as the blank. The spectrum was scanned over the range of 4000–400 cm⁻¹ at a resolution of 4 cm⁻¹. The characteristic absorption peaks obtained for Rifaximin were compared with the reported reference spectrum to confirm the identity, purity, and integrity of the drug. The FTIR analysis was also performed to evaluate the compatibility of Rifaximin with the selected excipients used in the formulation of enteric-coated pellets by comparing the spectra of the pure drug, excipients, and their physical mixture.

Saturation Solubility of Rifaximin in Methanol, Water, 0.1 N HCl and Phosphate Buffer pH 6.8

The saturation solubility of Rifaximin in methanol, distilled water, 0.1 N hydrochloric acid (HCl), and phosphate buffer pH 6.8 was determined by the shake-flask method. An excess amount of Rifaximin (approximately 50–100 mg) was added separately into glass vials containing 10 mL of each solvent. The vials were tightly sealed, labelled, and placed in an orbital shaker maintained at 25 ± 0.5°C with continuous shaking at 100 rpm for 48 hours to achieve equilibrium.

After equilibrium was attained, 1 mL of the supernatant solution was withdrawn from the middle layer of each vial and centrifuged at 5000 rpm for 10 minutes. The clear supernatant was filtered through a 0.45 µm syringe filter (PTFE filter for methanol and Nylon/PES filter for aqueous media). The filtrates were suitably diluted, wherever required, and analyzed using the validated UV–Visible spectrophotometric method at 290 nm. The concentration of dissolved Rifaximin was calculated from the calibration curve, and the saturation solubility in each solvent was expressed as mg/mL.

Figure no. 4 FTIR Analysis of Rifaximin

FTIR spectroscopy was performed to investigate possible interactions between rifaximin and formulation excipients. The characteristic peaks of rifaximin were retained in the optimized formulation, indicating the absence of significant drug-excipient interactions

Figure no. 5 FTIR Analysis of Rifaximin + Excipients

FTIR spectra of pure rifaximin, physical mixtures of rifaximin with excipients, and the optimized enteric-coated pellet formulation were recorded using an FTIR spectrophotometer. The samples were prepared by the KBr pellet method and scanned over the spectral range of 4000–400 cm⁻¹. The obtained spectra were analysed for the presence of characteristic functional group peaks and any possible drug–excipient interactions.

Table no. 6 FTIR Analysis of Rifaximin + Excipients

RESULTS

FORMULATION DEVELOPMENT RESULTS

Trial Batch Observations

Nine trial batches (F1–F9) were prepared by varying the concentration of enteric coating polymer and coating composition. The prepared pellets were evaluated for acid resistance, drug release, assay, and physical appearance.

Table no.7 Trial Batch Observations

The results demonstrated that increasing the concentration of Eudragit® L30D-55 improved acid resistance and reduced premature drug release in acidic media.

Optimized Batch (F9)

Table no.8 Optimized Batch (F9)

F9 can be justified as the optimized batch because it shows:

• Highest assay and drug content
• Excellent acid resistance (>99%)
• Very low acid stage release (<10%)
• Desired intestinal release (>80%)
• Lowest friability
• Acceptable moisture content
• Uniform particle size distribution.

1. Drug loading result

Drug loading was successfully performed on sugar spheres using a fluidized bed processor equipped with a Wurster insert. The prepared drug-loaded pellets exhibited uniform appearance, smooth surface morphology, and satisfactory flow characteristics. No agglomeration or sticking was observed during the drug layering process. The percentage yield of drug-loaded pellets ranged from 91.25% to 99.12%, indicating efficient processing conditions.

Table no. 9 Drug loading result

The results confirmed successful deposition of rifaximin onto the sugar sphere surface with acceptable content uniformity.

2. Barrier Coating Results

Barrier coating was applied to prevent direct interaction between rifaximin and the enteric coating polymer. The HPMC E5-based barrier coating produced a smooth and uniform film around the drug-loaded pellets. No cracking, peeling, or agglomeration was observed during the coating process.

Table no.10 Barrier Coating Results

The barrier coating layer effectively separated the drug layer from the enteric coating and improved overall coating integrity.

3. Enteric Coating Results

Enteric coating was successfully applied using Eudragit® L30D-55. The coated pellets exhibited excellent appearance, smooth surface, and adequate coating integrity. Acid resistance improved with increasing enteric polymer concentration.

Table no. 11 Enteric Coating Results

The results demonstrated that increasing enteric coating level improved gastric resistance and minimized premature drug release in acidic media.

4. Dissolution Results

The dissolution study was performed in two stages consisting of an acid stage (0.1 N HCl for 2 hours) followed by a buffer stage (pH 6.8 phosphate buffer). The enteric-coated pellets exhibited excellent resistance to acidic conditions and released the drug predominantly in intestinal pH

Table no.12. Dissolution Results

In-Vitro Dissolution Study

The in vitro dissolution study of rifaximin enteric-coated pellets was carried out using the USP Dissolution Apparatus Type II (Paddle) to evaluate the drug release behavior under simulated gastrointestinal conditions.

A quantity of pellets equivalent to the required dose of rifaximin was placed in the dissolution vessel containing 900 mL of 0.1 N hydrochloric acid (pH 1.2) maintained at 37 ± 0.5°C and stirred at 50 rpm. The pellets were exposed to the acidic medium for 2 hours to assess the integrity of the enteric coating and acid resistance.

Table no. 13 In-Vitro Dissolution Study

Overall Performance of Optimized Batch (F9)

Ased on all evaluation parameters, formulation F9 was identified as the optimized batch. The formulation demonstrated excellent processability, superior acid resistance, acceptable assay, satisfactory dissolution profile, and desired intestinal targeting characteristics.

QUALITY BY DESIGN (QBD)

Quality by Design (QbD) is a systematic, science-based, and risk-based approach to pharmaceutical product development introduced by the International Council for Harmonisation (ICH Q8 guideline). QbD emphasizes building quality into the product during development rather than relying solely on end-product testing. The approach begins with predefined objectives and focuses on understanding the relationship between formulation variables, manufacturing processes, and product performance.

The fundamental principle of QbD is that product quality should be designed and developed into the formulation from the beginning. It involves identification of Quality Target Product Profile (QTPP), Critical Quality Attributes (CQAs), Critical Material Attributes (CMAs), Critical Process Parameters (CPPs), risk assessment, design of experiments (DoE), establishment of design space, and implementation of a control strategy.

Application of QbD in the development of enteric-coated rifaximin pellets helps in achieving robust formulation performance, consistent product quality, improved process understanding, reduced variability, and regulatory flexibility. The systematic optimization of formulation and process variables ensures targeted drug release in the small intestine and reproducible manufacturing performance

1. Quality Target Product Profile (QTPP)

The Quality Target Product Profile defines the desired quality characteristics of the final product that are necessary to ensure safety and efficacy.

Table no. 14. Quality Target Product Profile (QTPP)

2. Critical Quality Attributes (CQAs)

Critical Quality Attributes are the physical, chemical, biological, or microbiological properties that must remain within predefined limits to ensure product quality

Tablet no. 15 Critical Quality Attributes (CQAs)

3. Risk Assessment

Risk assessment was performed to identify formulation and process variables that could significantly affect the quality of enteric-coated rifaximin pellets. The assessment was carried out using Ishikawa (Fishbone) analysis.

The major sources of variability were categorized into:

• Material
• Method
• Machine
• Man
• Environment
• Measurement

4. Critical Material Attributes (CMAs)

Table no. 16 Critical Material Attributes (CMAs)

5 Critical Process Parameters (CPPs)

Table no.17 Critical Process Parameters (CPPs)

DESIGN OF EXPERIMENT (DOE)

Design of Experiment was employed to evaluate the influence of critical formulation and process variables on product performance and to establish an optimized formulation

1. Independent Variables

Table no.18 Independent Variables

2. Dependent Variables (Responses)

Y2 = MOISTURE CONTENT

3. Design Space

Design space represents the multidimensional combination of formulation variables and process parameters that consistently provide the desired product quality.

The optimized design space was established based on response surface analysis and contour plot interpretation. Operating within the design space ensures consistent quality, robust process performance, and regulatory flexibility

Table no.19 Design Space

A control strategy was developed to ensure consistent manufacturing and maintenance of product quality throughout the production process.

4. Control Strategy

Table no.20 Control Strategy

The established control strategy ensures reproducible pellet quality, effective enteric coating performance, desired acid resistance, and targeted intestinal drug release.

Stability Studies

Evaluation of the optimized formulation (F9) under stress conditions to ensure shelf-life and efficacy.

ICH Q1A (R2) GUIDELINES The stability study was conducted to evaluate the physical and chemical integrity of the optimized enteric-coated Rifaximin pellets.

Batch: Optimized Formulation (F9)

Conditions: 40°C ± 2°C / 75% RH ± 5% RH

Table no.21 Stability study