The increasing environmental concerns and regulatory restrictions on toxic conventional solvents have driven the search for sustainable alternatives. Dimethyl isosorbide (DMI), a bio-renewable solvent, has shown potential as a replacement for short-chain glycol ethers, although its use as solvent in liquid chromatography (LC) is underexplored. This study presents a physicochemical characterization of DMI with a particular focus on its application as an innovative solvent in LC analyses. The partition coefficient (log P = −0.44) was determined using the OECD 107 method, and viscosity measurements for DMI and its mixtures with water and ethanol were conducted at 25 °C, 40 °C, and 60 °C. Viscosity ranged from 1.28 mPa·s at 60 °C to 2.62 mPa·s at 40 °C. The Central Composite Face 2³ experimental design for studying the chromatographic behavior of DMI confirmed that 50% (v/v) DMI can be effectively utilized in the mobile phases, at a column temperature of 40 °C, with backpressures ranging from 160 to 300 bar and a UV cut-off at 240 nm. Its effectiveness as an eluent in LC was demonstrated for the quantification of methylparaben and propylparaben in pharmaceutical formulations. This study highlights DMI’s promise as a sustainable bio-renewable alternative to conventional organic solvents used as eluents in LC, supporting eco-friendly practices in pharmaceutical analysis.

Introduction: Pharmaceutical quality control techniques frequently rely on hazardous organic solvents, raising environmental and regulatory concerns. In response, environmentally friendly solvents and highly aqueous chromatographic approaches have become increasingly important. One such solvent is Dimethyl Isosorbide (DMI), which has emerged as a potential alternative to conventional organic solvents. This study aimed to evaluate the potential use of DMI as a green mobile-phase component in a Per-Aqueous Liquid Chromatography (PALC) method for the analysis of cefalexin in capsule dosage forms and to validate the method in accordance with ICH Q2(R2) guidelines. Additionally, a detailed forced degradation study was performed to assess the stability-indicating properties of the developed method. Methods: Chromatographic separation was performed on a silica column using a highly aqueous mobile phase consisting of acetic acid and DMI in a 90:10 (v/v) ratio. The chromatographic parameters, including mobile phase composition, temperature, and flow rate, were optimized. The method was validated. Results: The newly developed PALC method using a highly aqueous mobile phase was shown to provide efficient separation, good peak symmetry, and high reproducibility. Validation confirmed its specificity, linearity, accuracy, precision, robustness, and stability-indicating properties, including the separation of cefalexin from its related impurities and degradation products under all stress conditions. The AGREE tool used for environmental assessment yielded a high green score of 0.8, thus confirming the method’s reduced environmental impact without compromising its analytical reliability. Discussion: The results confirm that the integration of DMI as a bio-renewable solvent under PALC conditions offers a reliable and environmentally sustainable alternative to conventional HPLC methods for cefalexin analysis. However, this study is limited to capsule formulations, and further work is needed to extend its assessment to other dosage forms, evaluate transferability across different instruments and columns, and perform a more comprehensive sustainability evaluation beyond the scope of AGREE. Conclusion: This study demonstrates that bio-renewable DMI can effectively replace conventional organic modifiers (ACN/MeOH) in the PALC method without compromising analytical performance for the quantification of cefalexin.