Dispersive liquid-liquid microextraction using natural deep eutectic solvents combined with LC-MS/MS for the determination of Epothilone(s) in cell culture media.

In the present communication, a sensitive and reliable analytical method was developed for the quantitative determination of epothilones A, B, and D from different culture media, namely GSM (glucose skim milk medium), EPM (enriched peptone medium), CYE (casitone yeast extract medium), and EPMS (enriched peptone medium with salt) using hydrophobic deep eutectic solvent (DES)-based extraction approach. Epothilones are naturally occurring macrolides originally isolated from myxobacteria that exhibit potent anticancer activity by stabilizing microtubules and having a superior efficacy, including activity against multidrug resistance cancer. The extraction employed a thymol-menthol DES at a 2:1 M ratio as the extraction solvent, with acetonitrile serving as the disperser solvent, followed by precise identification and quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The developed method enables effective monitoring of epothilone production in different culture media, namely GSM (glucose skim milk medium), EPM (enriched peptone medium), CYE (casitone yeast extract medium), and EPMS (enriched peptone medium with salt). Key extraction parameters, including DES volume, disperser solvent volume, and pH, were systematically optimized using a DoE approach. The method exhibited excellent linearity over the concentration range of 5-1000 ng/mL with correlation coefficient (r) greater than 0.99. The LOD and LOQ were determined and found to be 5 ng/mL and 10 ng/mL, respectively. High extraction efficiency was achieved, with recoveries ranging from 92.92% to 98.54% for epothilones A, B, and D, along with acceptable precision (intra-day RSD < 4% and inter-day RSD < 9%). The validated method was successfully applied to quantify and monitor epothilone production yields in various culture media. The highest production of epothilones was observed in GSM medium. Overall, the method offers a powerful and versatile tool for the simultaneous identification and quantification of epothilones and holds significant potential for applications in epothilone production monitoring, biological and environmental culture analysis, pharmaceutical research, and related fields.