Green chiral HPLC method on a polysaccharide column under hydro-organic conditions for the determination of Levobupivacaine, Ropivacaine, and their enantiomeric impurities

A novel, rapid, green, and enantioselective HPLC method was developed and validated using a polysaccharide-based chiral column for the individual determination of Levobupivacaine and S (–) Ropivacaine together with their respective chiral impurities (R (+) Bupivacaine and R (+) Ropivacaine). The separation was optimized on a Chiralpak AD column under hydro-organic conditions using an isocratic mobile phase of isopropanol and water (85:15, v/v) at flow rate 0.5 mL/min, with UV detection at 220 nm. The method was validated according to ICH guidelines and assessed for environmental impact using NEMI, Analytical Eco-Scale, GAPI, and AGREE tools. Baseline separation of each enantiomer pair was achieved within 8 min. Good linearity (R 2 > 0.999) was exhibited over 20–200 μg/mL for the S-enantiomers and 2 – 20 μg/mL for the R-enantiomers. Precision (RSD < 1.36%) and accuracy (recoveries 99.4–100.7%) were satisfactory. Detection and quantitation limits for the chiral impurities were < 0.7 μg/mL and < 2.1 μg/mL, respectively. Thermodynamic studies indicated an enthalpy-driven enantioseparation involving hydrogen bonding, π–π interactions, and steric hindrance. The greenness assessment confirmed a superior environmental profile compared to literature methods, with an Eco-Scale score of 92 and AGREE score of 0.70. To our knowledge, the developed method is the first application of hydro-organic conditions on a polysaccharide column for these local anesthetics, offering a rapid, selective, and sustainable alternative to traditional modes used in chiral LC methods. Its simplicity and robustness make it well suited for routine quality control applications.