Development, Validation, and Greenness Assessment of Eco-Friendly Analytical Methods for the Determination of Abiraterone Acetate in Pure Form and Pharmaceutical Formulations.

This study describes the development and validation of two environmentally sustainable analytical methods for the quantification of Abiraterone Acetate (AA) in its pure form and in commercial tablet formulations. 

A green High-Performance Liquid Chromatography (HPLC) method was optimized using an Agilent Extend C18 column (250 mm × 4.6 mm, 5 µm) maintained at 25 °C. The chromatographic separation was achieved under isocratic conditions with a mobile phase composed of ethanol and formic acid, delivered at a flow rate of 1.0 mL min⁻¹, and detection was carried out at 253 nm.

In parallel, a spectrophotometric method was established following a comprehensive assessment of AA’s absorption behavior in various solvents, identifying ultrapure water as the most suitable medium for quantitative analysis at 253 nm. 

Both analytical procedures were validated according to ICH guidelines, demonstrating excellent selectivity, linearity, precision, accuracy, robustness, and sensitivity. 

Calibration curves were linear over the concentration range of 5–30 µg mL⁻¹, with correlation coefficients (R²) greater than 0.999.

Statistical comparison of the two methods using Student’s t-test and Fisher’s F-test revealed no significant differences between their performance parameters. 

The environmental sustainability of the developed methods was evaluated using AGREE and GAPI assessment tools, confirming their green analytical profiles. Overall, the proposed methods provide accurate, precise, and eco-friendly alternatives for routine determination of Abiraterone Acetate in pharmaceutical formulations, supporting safer and more sustainable practices in pharmaceutical quality control.

 Materials and Methods

 Instruments

Chromatographic analyses were carried out using an Agilent 1260 Infinity HPLC system (Agilent Technologies, Palo Alto, CA, USA.
The system comprised a quaternary pump, vacuum degasser, column oven, autosampler, and UV–Vis detector. Data acquisition and processing were performed using ChemStation Software (version B.04.03, November 2010).

Chromatographic separations were achieved on an Agilent Extend C18 column (250 mm × 4.6 mm, 5 µm) maintained at 25 °C.

Spectrophotometric measurements were conducted with a Shimadzu UV-1800 double-beam spectrophotometer (Shimadzu Corporation, Kyoto, Japan), equipped with 1.0 cm quartz cuvettes and UV-Probe Software (version 2.70).

The pH of the solutions was measured with a Mettler-Toledo digital pH meter (Mettler Toledo GmbH, Greifensee, Switzerland
) using a glass electrode.

Ultrapure water was produced using a Millipore Milli-Q purification system (MilliporeSigma, Bedford, MA, USA).

Reagents, Solvents, and Materials

Abiraterone acetate (AA, purity ≥99.5%) reference standard was obtained from the United States Pharmacopeia (USP, Rockville, MD, USA).
Commercial Zytiga® tablets (250 mg) were purchased from a registered pharmacy in Afyonkarahisar, Turkey.

All solvents were of HPLC-grade purity and included acetonitrile (≥99.9%), methanol (≥99.0%), ethanol (≥99%), and formic acid (≥99.0%), sourced from Sigma-Aldrich Chemie GmbH (Istanbul, Turkey).

Mobile phases were freshly prepared daily, filtered through 0.20 µm nylon membrane filters (Whatman™, Cytiva Life Sciences, Maidstone, UK) and degassed in an ultrasonic bath (Elmasonic S, Elma Schmidbauer GmbH, Singen, Germany).

All glassware was rinsed with deionized and ultrapure water and dried before use.

Preparation of Pharmaceutical Solutions

• A stock standard solution of AA (500 µg·mL⁻¹) was prepared by accurately weighing 25 mg of the USP reference standard and transferring it into a 50 mL volumetric flask.
• Approximately 20 mL of ultrapure water was added, and the mixture was sonicated for 6 minutes to ensure complete dissolution. The volume was then adjusted to 50 mL with ultrapure water.
• Working standards (5–30 µg·mL⁻¹) were prepared by serial dilution of the stock solution using ultrapure water.
• For the sample solution, ten Zytiga® tablets (250 mg each) were weighed, and the average tablet mass was determined. 
• Tablets were crushed into a fine powder in a dry mortar. 
• A portion equivalent to 50 mg AA was transferred to a 100 mL volumetric flask, followed by 40 mL ultrapure water. 
• The mixture was shaken for 30 minutes on a rotary shaker, sonicated for 10 minutes, and diluted to volume with water. 
• The solution was then filtered through a 0.45 µm membrane filter to yield a 25 µg·mL⁻¹ stock sample solution.

Determination of λmax

Standard solutions (5–30 µg·mL⁻¹) were analyzed using the Shimadzu UV-1800 spectrophotometer over the wavelength range 200–400 nm.
A solvent blank (ultrapure water) was used for baseline correction. The maximum absorbance (λmax) for Abiraterone Acetate was found at 253 nm.

 All measurements were conducted at room temperature in triplicate for reproducibility.

Method Development and Optimization

Chromatographic conditions were optimized to achieve sharp, symmetrical peaks with minimal tailing and short retention times.

 Various mobile phases (water/methanol, water/acetonitrile, and water/ethanol) and buffer systems were tested. Optimal performance was achieved using ethanol–formic acid (isocratic mode), with a flow rate of 1.0 mL·min⁻¹ at 25 °C, and UV detection at 253 nm.
 

The analyte exhibited stable retention and resolution under these eco-friendly chromatographic conditions.

Spectrophotometric analysis confirmed ultrapure water as the most suitable solvent, providing consistent spectral characteristics and maximal absorbance at 253 nm.

Method Validation

Validation was conducted following ICH Q2(R1) guidelines to confirm selectivity, linearity, precision, accuracy, sensitivity, robustness, and system suitability.

• Linearity: 5–30 µg·mL⁻¹, with correlation coefficients (R²) > 0.999.
• Selectivity: No interference observed from excipients or solvents.
• Precision: Intra- and inter-day %RSD < 2%.
• Accuracy: Recoveries between 98.0–102.0%.
• Sensitivity: LOD and LOQ calculated using σ/slope method.
• Robustness: Assessed by minor changes in wavelength (±2 nm), flow rate (±0.1 mL·min⁻¹), and mobile phase composition (±2%).

Application to Commercial Formulations

The validated HPLC and spectrophotometric methods were applied to quantify AA in Zytiga® tablets (250 mg).
Both techniques showed excellent agreement with labeled claims, confirming their suitability for routine quality control of Abiraterone Acetate in pharmaceutical formulations.

Greenness Evaluation

The environmental impact of both analytical methods was evaluated using the Analytical GREEnness (AGREE) tool and the Green Analytical Procedure Index (GAPI).

 Results confirmed the eco-friendly nature of the developed procedures, due to the elimination of toxic solvents, minimal waste generation, and reduced energy consumption—aligning with the principles of Green Analytical Chemistry (GAC).