Quantitative determination of rhamnolipid using HPLC-UV through carboxyl labeling

Rhamnolipid, as a low-toxic, biodegradable and environmentally friendly biosurfactant, has broad application prospects in many industries. However, the quantitative determination of rhamnolipid is still a challenging task. Here, a new sensitive method for the quantitative analysis of rhamnolipid based on a simple derivatization reaction was developed. In this study, 3-[3'-(l-rhamnopyranosyloxy) decanoyloxy] decanoic acid (Rha-C10-C10) and 3-[3'-(2'-O-α-l-rhamnopyranosyloxy) decanoyloxy] decanoic acid (Rha-Rha-C10-C10) were utilized as the representative rhamnolipids. Liquid chromatography-mass spectrometry and high-performance liquid chromatography-ultra violet results showed that these two compounds were successfully labeled with 1 N1-(4-nitrophenyl)-1,2-ethylenediamine. There was an excellent linear relationship between rhamnolipid concentration and peak area of labeled rhamnolipid. The detection limits of the Rha-C10-C10 and Rha-Rha-C10-C10 were 0.018 mg/L (36 nmol/L) and 0.014 mg/L (22 nmol/L), respectively. The established amidation method was suitable for the accurate analysis of rhamnolipids in the biotechnological process. The method had good reproducibility with the relative standard deviation of 0.96% and 0.79%, respectively, and sufficient accuracy with a recovery of 96%-100%. This method was applied to quantitative analysis of 10 rhamnolipid homologs metabolized by Pseudomonas aeruginosa LJ-8. The single labeling method was used for the quantitative analysis of multiple components, which provided an effective method for the quality evaluation of other glycolipids with carboxyl groups.