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Purpose <p>Azelastine hydrochloride (AZE) and fluorometholone (FLU) are commonly used in combination therapy for the treatment of allergic conjunctivitis. Accurate and reliable quantification of these drugs in nano-micellar in situ gel formulations requires a robust, stability-indicating, and environmentally sustainable analytical method. Therefore, the present study aimed to develop and validate an Analytical Quality by Design (AQbD)-based RP-HPLC method for the simultaneous estimation of AZE and FLU.</p> Methods <p>Risk assessment was performed to identify critical method parameters (CMPs). Initial screening was conducted using a Taguchi L8 orthogonal array, followed by optimization through a Box–Behnken design. Chromatographic separation was achieved on a C18 column using a mobile phase consisting of acetonitrile and 0.1% orthophosphoric acid (50:50, v/v) at a flow rate of 0.8 mL min⁻¹, with detection at 230&#xa0;nm. The method was validated in accordance with ICH Q2(R1/R2) guidelines. Forced degradation studies were performed under different stress conditions to evaluate the stability-indicating capability of the method. Additionally, the environmental impact of the developed method was assessed using AGREE, AGREEprep, ComplexGAPI, Eco-Scale, and BAGI greenness assessment tools.</p> Results <p>The developed method exhibited excellent linearity in the concentration range of 2–12&#xa0;µg mL⁻¹ for both AZE and FLU (R² &gt; 0.999). The method showed high precision (%RSD &lt; 2%) and satisfactory accuracy with recoveries ranging from 98.09% to 101.64%. Forced degradation studies confirmed that degradation products were well separated from the analytes, demonstrating the stability-indicating nature of the method. No interference from formulation excipients was observed, and high recovery of both drugs was achieved from the nano-micellar in situ gel matrix. Greenness evaluation indicated that the method possesses good environmental sustainability.</p> Conclusion <p>The proposed AQbD-based RP-HPLC method is robust, reliable, stability-indicating, and environmentally friendly. It can be effectively applied for routine quality control analysis of AZE and FLU in advanced ocular nano-micellar in situ gel drug delivery systems.</p>

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Analytical Quality by Design-Driven Green RP-HPLC Method for Simultaneous Quantification of Azelastine Hydrochloride and Fluorometholone in Nano-Micellar In Situ Gel for Ocular Delivery

  • 期刊论文

  • 发表日期：

    2026年6月15日

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  • Swapnil S. Harale,
  • M. B. Patil,
  • Kishori P. Sutar,
  • Abhinandan A. Alman

摘要

Purpose

Azelastine hydrochloride (AZE) and fluorometholone (FLU) are commonly used in combination therapy for the treatment of allergic conjunctivitis. Accurate and reliable quantification of these drugs in nano-micellar in situ gel formulations requires a robust, stability-indicating, and environmentally sustainable analytical method. Therefore, the present study aimed to develop and validate an Analytical Quality by Design (AQbD)-based RP-HPLC method for the simultaneous estimation of AZE and FLU.

Methods

Risk assessment was performed to identify critical method parameters (CMPs). Initial screening was conducted using a Taguchi L8 orthogonal array, followed by optimization through a Box–Behnken design. Chromatographic separation was achieved on a C18 column using a mobile phase consisting of acetonitrile and 0.1% orthophosphoric acid (50:50, v/v) at a flow rate of 0.8 mL min⁻¹, with detection at 230 nm. The method was validated in accordance with ICH Q2(R1/R2) guidelines. Forced degradation studies were performed under different stress conditions to evaluate the stability-indicating capability of the method. Additionally, the environmental impact of the developed method was assessed using AGREE, AGREEprep, ComplexGAPI, Eco-Scale, and BAGI greenness assessment tools.

Results

The developed method exhibited excellent linearity in the concentration range of 2–12 µg mL⁻¹ for both AZE and FLU (R² > 0.999). The method showed high precision (%RSD < 2%) and satisfactory accuracy with recoveries ranging from 98.09% to 101.64%. Forced degradation studies confirmed that degradation products were well separated from the analytes, demonstrating the stability-indicating nature of the method. No interference from formulation excipients was observed, and high recovery of both drugs was achieved from the nano-micellar in situ gel matrix. Greenness evaluation indicated that the method possesses good environmental sustainability.

Conclusion

The proposed AQbD-based RP-HPLC method is robust, reliable, stability-indicating, and environmentally friendly. It can be effectively applied for routine quality control analysis of AZE and FLU in advanced ocular nano-micellar in situ gel drug delivery systems.