AQbD-guided development of a green RP-HPLC method for dual drug quantification in lipid–polymer hybrid nanoparticles for endometriosis therapy

Abstract

Endometriosis (EM) is a gynaecological disorder for which drug combinations such as atorvastatin (ATV) and silybin (SB) hold therapeutic promise. However, their limited solubility and bioavailability necessitate advanced delivery systems like lipid–polymer hybrid nanoparticles (LPHNPs). But no validated method exists for their simultaneous quantification in such nanocarriers. In this study, a simple, novel, robust, and environmentally sustainable reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed utilizing the Analytical Quality by Design (AQbD) framework using Box-Behnken Design (BBD), ensuring consistent and reliable performance.

Highlights

Development of a novel and robust RP-HPLC method for simultaneously quantifying Atorvastatin and Silybin in LPHNPs.
Method development was designed employing Analytical Quality by Design framework with Box–Behnken Design optimization.
The validated method showed excellent linearity, precision, robustness, and ICH Q2(R2) compliance.
The developed methodeffectively determined the entrapment efficiency and in vitro release from dual-loaded LPHNPs.
Greenness assessment confirmed the environmental sustainability of the analytical approach.

The optimized chromatographic conditions employed an Agilent Eclipse XDB C18 column (150 mm × 4.6 mm, 5 μm), a mobile phase consisting of acetonitrile (ACN) and 0.1 % formic acid in water (20,80 v/v), a flow rate of 1.0 mL/min, and UV detection at 266 nm. Under these conditions, sharp and well-resolved peaks were obtained at retention times of 7.210 min (ATV) and 5.229 min (SB). The method exhibited excellent linearity (r² = 0.9999 for ATV; r² = 1.0000 for SB), high precision (%RSD < 2 %), robustness, and full compliance with ICH Q2(R2) guidelines.

Its applicability was successfully demonstrated in quantifying entrapment efficiency (EE) and in vitro release from dual drug-loaded LPHNPs. Furthermore, greenness evaluation using the AGREE tool validated its eco-friendly characteristics. Overall, this validated RP-HPLC method offers a reliable and regulatory-compliant tool for simultaneous quantification of ATV and SB in LPHNPs, thereby facilitating the advancement of eco-friendly nanotherapeutics for endometriosis management.

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Materials

Atorvastatin (off- white crystalline powder, purity >98 %; MW: 558.6 g/mol) was provided as a kind gift sample from Mankind Pharma, New Delhi, India, while Silybin (white crystalline powder, purity >98 %; 482.24 g/mol) was obtained from Cayman Chemicals, USA. HPLC-grade chemicals such as methanol (MeOH) and acetonitrile (ACN) were procured from Merck Specialities Pvt. Ltd., Mumbai, Maharashtra India. Soluplus was generously supplied as a gift sample by BASF, Mumbai, India. Soy lecithin and…

Sharanya Paramshetti, Mohit Angolkar, Darshan Patil, Asha Spandana K.M., Riyaz Ali M. Osmani, Hosahalli Veerbhadrappa Gangadharappa, Adel Al Fatease, Ali H. Alamri, Umme Hani, AQbD-guided development of a green RP-HPLC method for dual drug quantification in lipid–polymer hybrid nanoparticles for endometriosis therapy, Microchemical Journal, Volume 219, 2025, 116242, ISSN 0026-265X, https://doi.org/10.1016/j.microc.2025.116242.