Application Note # LCMS-79 Metabolic Profiling of Different Coffee Types on the Bruker compact™ QTOF System Introduction Metabolomics studies have gained major importance in food applications. Quality control of food samples can be established on a large scale via state-of-the-art metabolic profiling based on statistical data evaluation. However, the metabolomics workflow puts high demands on an analytical system in terms of its robustness, dynamic range and identification capabilities. The Bruker compact QTOF System combines the robustness of Bruker orthogonalTime-of-Flight instruments with new 10-bit digitizer technology that allows for compound detection and identification across an unrivalled dynamic range. After water and black tea, coffee represents the third most important beverage with a worldwide consumption of 4.5 million tons per year [1]. Analytically, it constitutes a very complex mixture of small molecules which differ in composition and quantities based on the different coffee cultivars, cultivation regions and processing procedures. As a proof of concept study for the compact, we analyzed 13 different types of coffee capsule extracts assigned by their manufacturer to different intensity categories. The major analytical task was to correlate high resolution LC-MS data to the manufacturer’s description via a non-targeted metabolomics approach. Authors Dr. Verena Tellström, Dr. Alexander Harder, Klaus Meyer, Dr. Aiko Barsch Bruker Daltonik GmbH, Bremen, Germany Structure Elucidation FragmentationExplorer ProfileAnalysis

Capsules of 13 different types of coffee were extracted using 35 ml of water on a standard coffee capsule machine (Krups XN 301T Nespresso Pixie). Two replicates of each type were prepared. A QC sample was generated by pooling equal volumes of all coffee samples profiled in this study. Extracts were diluted 1:50 in water prior to analyzing 5µl of 3 replicates for each extract by UHPLC-MS. Chromatographic separation was carried out using an RSLC system (Dionex) with a 50 x 2.1 mm BEH C18, 1.7 um column (Waters), at a flow rate of 0.45 mL/min, with Solvent A: Water + 0.1% HCOOH and Solvent B: methanol...

Robust instrument performance for large metabolomics data sets Intens. x106 QC_GA2_01_310.d: BPC +All MS, Masses excluded QC_GA2_01_321.d: BPC +All MS, Masses excluded Figure 1: A: Overlay of Base Peak Chromatograms of interspersed quality control (QC) samples run during the acquisition of a coffee metabolomics sample batch demonstrates the stability of the Compact QTOF for analysing large sets of complex samples. B: A PCA scores plot of coffee metabolomics (green) and QC samples (red) reveals tight clustering of QC samples as an additional proof for reproducibility during data acquisition. 5...

Coffee samples cluster according to the assigned coffee intensity For a detailed evaluation of the different coffee types analysed in this study, the QC samples were removed from the calculated PCA model. In total, 13 different coffee extracts, comprising espresso and lungo varieties from different blends and geographical regions were analysed. The two “biological” and three technical replicates for each sample type (highlighted by using the same colour and symbol) formed clusters in the PCA scores plot as seen in Figure 3 A. In order to avoid the dominance of the caffeine content on the clustering...

Identification of trigonelline and nicotinic acid Compound identities were elucidated using the MS/MS capabilities of the Compact QTOF instrument. SmartFormula3D readily provides the correct molecular formula by combining accurate mass and isotopic pattern information for MS and MS/MS spectra (Fig. 4 A). Furthermore fragment formulae are easily accessed and can be used for gaining structural information. For compound X a unique elemental composition of C6H6NO2 ([M+H]+) was generated. Via a direct link to MetFrag (Fig. 4 C), an open source tool for in-silico fragmentation, the molecular formula...

Conclusion Many compounds that contribute to the typical flavour of coffee as a beverage are only formed during the high temperature treatment of the roasting process. Trigonelline is one of the major analytes in unprocessed coffee and is transformed during the roasting process mainly to pyridine and nicotinic acid [3, 5]. The obtained results perfectly match this prior knowledge. As seen in figure 5, trigonelline (compound Y) has a higher content in coffees classified with strength 3 suggesting a weaker roasting. In contrast, the trigonelline degradation product nicotinic acid (compound X) is...