A novel ¹H NMR-based quantitative method for the direct quantification of mono-rhamnolipids and di-rhamnolipids

Rhamnolipids are environmentally friendly biosurfactants with a wide range of applications; however, their structural complexity and the limited availability of high-purity standards pose significant analytical challenges. In this study, we developed a ¹H NMR-based method for the direct quantification of both mono-rhamnolipids and di-rhamnolipids within complex mixtures. By identifying characteristic proton signals and optimizing a mixed deuterated solvent system (DMSO‑d₆/CD₃OD, 4:1 v/v), accurate quantification was achieved using sodium p-hydroxybenzoate as an internal standard. The method demonstrated strong anti-interference capability, excellent reproducibility (RSD <2 %), high accuracy (relative error <5 %), a relative abundance detection limit of 5 %, and satisfactory spike recovery rates, thereby addressing the limitations of conventional analytical techniques. This approach eliminates the need for high-purity reference standards and simplifies sample preparation, providing a reliable and efficient tool for both rhamnolipids research and industrial quality control.