Abstract
In metabolomics studies, large sample sets have to be analyzed to allow statistical differentiation of sample types. Obviously, repeatability of the whole analytical workfl ow, including sample preparation, sample introduction, separation and detection, is hereby of the utmost importance. In this respect, automation of the sample preparation is very useful in order to reduce the analytical variability.
In a series of articles, we describe the use of the Gerstel MPS WorkStation for automated sample preparation applied to metabolomics studies. In a first part, an automated ultrasonic assisted extraction and fi ltration method was discussed. In this second part, an automated fractionation of lipid classes using solid phase extraction (SPE) is presented. The SPE fractions are concentrated using an mVAP evaporation station and re-dissolved in small amounts of solvent, followed by LC-QTOF analysis.
Introduction
Metabolomics focuses on the analysis of small molecules (MW<2000) in biological matrices. Hereby relatively large sets of samples are processed to allow differentiation between sample types. In this respect, analytical variability should be much lower than biological variability and automation of sample preparation can signifi cantly contribute to improved repeatability of the total analytical procedure. In a typical metabolomics workfl ow, extraction of the sample is followed by fractionation or clean-up, if needed derivatization, concentration, and fi nally GC or LC separation and MS detection. In a series of articles, we describe a number of automated methods that are currently applied in our laboratories. In a fi rst article, automated ultrasonic assisted liquid extraction and fi ltration using the Gerstel MPS Workstation were discussed [1]. In this second article, an automatic fractionation procedure based on solid phase extraction (SPE) is described. This method was used in a lipidomics study, focusing on the characterization of plant material based on the relative composition of different classes of lipids, including neutral lipids (triglycerides, sterols), free fatty acids and polar lipids. Due to the fact that these classes are present in the plant material at substantially different concentration levels, it was observed that fractionation and selective enrichment of lipid classes prior to LC-MS analysis resulted in a much better coverage of lipids [2]. After liquid-liquid extraction, based on the Folch method [3], a concentrated lipid fraction was obtained. Next, fractionation was performed in a “normal phase LC” mode on an aminopropyl SPE cartridge. Three fractions of increasing polarity were obtained and the extracts were concentrated using an mVAP evaporation station installed on the MPS Workstation. Finally, the concentrated extracts were analysed by LC-QTOF.
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