Metabolite profiling of plant extracts by ultra-high-pressure liquid chromatography at elevated temperature coupled to time-of-flight mass spectrometry.J Chromatogr A. 2009 Jul 24; 1216(30):5660-8.JC
Detailed metabolite profiling of crude plant extracts, mandatory for both quality control and metabolomics purposes, requires high-resolution separation and sensitive detection with a reasonable sample throughput. In this respect, the use of ultra-high-pressure liquid chromatography (UHPLC) working at high temperature (HT) and coupled to time-of-flight mass spectrometry (TOF-MS) was evaluated in the present study in terms of achievable peak capacities for given analysis times. Prior to the analysis of complex mixtures, the effects of TOF-MS detection on peak capacity were evaluated, and a loss of 15-30% compared to UV was observed due to the additional band broadening generated by this detector. Extracts from a model plant Arabidopsis thaliana and from a widely used phytochemical preparation Ginkgo biloba, as well as a standard mixture of representative natural products (NPs), have been analyzed. As expected from the theory, the increase in mobile phase temperature of up to 90 degrees C for the profiling of extracts containing metabolites spread over a large polarity range (e.g., Arabidopsis thaliana) generated similar peak capacities to those obtained at room temperature, but with a 2- to 3-fold reduction in analysis time, demonstrating the power of this approach for such applications. On the other hand, for the analysis of more polar extracts (e.g., Ginkgo biloba), the use of higher temperature was not beneficial, as it induced a significant decrease in retention, and thus resolving power, because of the increase in elution strength. The use of HT-UHPLC-TOF-MS raised the question of NP stability under high temperature conditions. This work demonstrated that no apparent degradation was evidenced at high temperature for a representative mixture of NPs and also for the different metabolites detected in the selected plant extracts.