Elias Abebe Bekele

Multi-response optimization of the extraction and derivatization protocol of selected polar metabolites from apple fruit tissue for GC–MS analysis

Given the complexity of the plant metabolome exhibiting a wide spectrum of physico-chemical properties, finding the best compromise for GC-MS analysis is a challenging exercise. In this study, the GC-MS protocol for extracting and analyzing polar metabolites from apple tissue is optimized. We found pure methanol extraction to be slightly better as compared to the two phase methanol/chloroform/water extraction in terms of introducing less degradation of the extracted metabolites while being comparable in extraction efficiency and repeatability. The methanol extraction was superior to the single phase methanol/chloroform/water extraction in all performance measures. A multi-response optimization based on a desirability function was applied to optimize the derivatization. This procedure allows searching for optimal parameters while simultaneously considering overall detection enhancement of metabolites from various metabolic classes. A short oximation at a high temperature in combination with a low silylation temperature gave results similar to a longer oximation at low temperature in combination with a high silylation temperature. Increasing silylation time from 0.5 h to 2 h resulted in an improvement of the silylation reaction.