Optimization of extraction conditions for LC-ToF-MS analysis of mevalonate pathway metabolites in engineered E. coli...

Isoprenoids give rise to many functional products used today such as flavours, fragrances and even pharmaceutical compounds. Mevalonate pathway metabolites are the key intermediates that affect the production yield of isoprenoids. With increasing demand and benefit of isoprenoids, the present study adopts Analytical Quality-by-Design (AQbD) approach to establish an efficacious extraction protocol prior to the determination of mevalonate pathway metabolites in an engineered Escherichia coli model. The statistical experimental design approach, described in this work, has successfully validated an optimised sample preparation method i.e., using acetonitrile: 50 mM ammonium formate (pH 9.5) (7:3) (ACN73) at -20 °C for 10 min without solvent evaporation to retain the targeted mevalonate metabolites in engineered E. coli strain. The study also demonstrates the use of liquid chromatography paired with a Time-of-Flight Mass Spectrometer (LC-ToF-MS) for the quantitative analysis of the mevalonate pathway metabolites in E. coli. The analytical method was validated in accordance with guidelines in Metabolomics Standards Initiative and ICH Q2 (R1) with analyte spike recoveries at 80% and above. In short, the present study overcomes the one-variable-at-a-time (OVAT) limitations in analytical development, minimises metabolite losses and gives better cost and time efficiencies by eliminating the solvent evaporation and swapping process. This work highlights the importance of analytical methods development in microbial metabolomics studies.