Monolithic Silica-Based Capillary Reversed-Phase Liquid Chromatography/Electrospray Mass Spectrometry for Plant Metabolomics

Abstract

Application of C18 monolithic silica capillary columns in HPLC coupled to ion trap mass spectrometry detection was studied for probing the metabolome of the model plant Arabidopsis thaliana. It could be shown that the use of a long capillary column is an easy and effective approach to reduce ionization suppression by enhanced chromatographic resolution. Several hundred peaks could be detected using a 90-cm capillary column for LC separation and a noise reduction and automatic peak alignment software, which outperformed manual inspection or commercially available mass spectral deconvolution software.

An introduction to liquid chromatography–mass spectrometry instrumentation applied in plant metabolomic analyses

“…The sample is introduced either by manual direct infusion (DI) or via HPLC or UHPLC followed by DI, following which the sample is delivered to the probe via a capillary; the probe functions to translocate the liquid sample from the capillary to the needle of the ion source. Two ionisation sources are generally employed in LC-MS based metabolomics: electrospray ionisation (ESI) is the most commonly employed (Tolstikov et al, 2003;Jander et al, 2004;von Roepenack-Lahaye et al, 2004;Vorst et al, 2005;Moco et al, 2006;Rischer et al, 2006;De Vos et al, 2007;Hanhineva et al, 2008) and is particularly well suited to the ionisation of a wide range of metabolites including, drug compounds (Chen et al, 2007), amino and organic acids Figure 3. The relationship between column stationary phase particle size, fl ow velocity and the height equivalent to the theoretical plate (HETP) value as an expression of chromatographic separation effi ciency.…”

“…chromatography combined with mass spectrometry (MS) or nuclear magnetic resonance (NMR) spectroscopy is capable of detecting all metabolites within a given biological sample Hall, 2006;Allwood et al, 2008). Over the past decade many methods for the high-throughput metabolomic analysis of plant derived samples have been established for gas chromatography combined with MS (GC-MS; Fiehn et al, 2000;Fernie et al, 2004;Lisec et al, 2006), direct injection mass spectrometry (DIMS; Goodacre et al, 2003;Catchpole et al, 2005;Allwood et al, 2006), liquid chromatography MS (LC-MS; Tolstikov et al, 2003;Jander et al, 2004;von Roepenack-Lahaye et al, 2004;Vorst et al, 2005;Moco et al, 2006;Rischer et al, 2006;De Vos et al, 2007), capillary electrophoresis MS (CE-MS; Sato et al, 2004), and 1 H-NMR (Ratcliff e and Shachar-Hill, 2001;Le Gall et al, 2003;Ward et al, 2003;Choi et al, 2004Choi et al, , 2006. 1 H-NMR has proven to be an appropriate tool for untargeted plant metabolomics, especially where studies focus upon samples that contain highly abundant bulk metabolite species, for example sugars in fruits (Biais et al, 2009).…”

“…Plant metabolism embodies a huge range of semi-polar compounds, including many key groups of secondary metabolites, which are best separated and detected by LC-MS approaches (Tolstikov et al, 2003;von Roepenack-Lahaye et al, 2004;Dixon et al, 2006;Hall, 2006;Moco et al, 2006;De Vos et al, 2007). MS-based techniques which are applicable to LC are also undergoing continual development, showing ever improving levels of mass resolution and precision and thus aiding metabolite detection and identification greatly (Kind and Fiehn, 2007;Draper et al, 2009).…”

“…separation on the basis of hydrophobicity) and thus they elute at later RTs (Snyder et al, 1997;Meyer, 2004). RP-LC has been and still is the most commonly employed method in metabolomics due to the high stability off ered by the silica-C 8 H 17 /C 18 H 37 stationary phase materials, robust analytical methodologies have also been developed by several plant research groups (Tolstikov et al, 2003;von Roepenack-Lahaye et al, 2004;Rischer et al, 2006;De Vos et al, 2007); therefore this review will focus upon the analysis of semi-polar plant metabolites with RP-LC.…”

“…2) (Kimura and Rodriguez-Amaya, 2002;Allwood et al, 2006). However, for the analysis of complex metabolomics samples there are many coeluting peaks which can prevent identifi cation and quantifi cation in this manner, thus MS is employed to perform these tasks (Tolstikov et al, 2003).…”