Charlotte Legrum

• Alkyl methoxypyrazines (MPs) have been identified in foodstuffs of plant origin as potent flavor compounds, contributing significantly to the characteristic vegetative, so-called green sensory impression of products made thereof. Of particular interest are 2-isopropyl-3-methoxypyrazin (IPMP), 2-methoxy-3-sec-butylpyrazin (SBMP) and 2-isobutyl-3-methoxypyrazin (IBMP) as they are often dominating in flavor due to their low odor thresholds. Biogenesis of MPs takes place in various species, resulting in variing concentration levels and distributions (ppt levels in Vitis vinifera wine berries, ppb levels in vegetables). The before mentioned and further MPs are also found in body fluids of insects such as ladybugs, e. g. in the species Coccinella septempunctata and Harmonia axyridis. Ladybugs may play a role in the generation of off-flavors in wine in case of their incorporation during harvest and wine making. Furthermore, MPs are products from the metabolism of certain microorganisms and could lead to off-flavors indirectly via contact materials like cork stoppers for wine. The last step in the proposed biosynthetic pathway(s) was clarified als O-methylation of alykl hydroxypyrazines, whereas the initial steps considering naturally occurring amino acids as starting materials are not yet fully explored. In case of SBMP, the alkyl side chain may thus derive from L-isoleucine, resulting in the same, namely (S)-configuration of the stereo center in SBMP. Considering analytical approaches, MPs in high concentration levels as in vegetables are accessible using classical extraction techniques, simple separation and detection techniques. On the other hand, for lower concentration ranges near the odor thresholds of MPs in wine and for the highly complex matrix of wine most analytical methods do not yield the necessary levels of detections. In this work a trace-level method for routine analysis and quantitation of MPs in the concentration range of the odor thresholds in white wine of 1 to 2 ng/L was developed. The extraction of the analytes was based on automated headspace solid phase micro extraction (HS-SPME). The separation was done either by heart-cut multidimensional GC (H/C MDGC) or comprehensive two-dimensional GC (GCxGC). MPs were detected using mass spectrometry (MS) in the selected ion monitoring (SIM) mode, especially for higher concentrations. To better clarify co-elution situations and for lower MP concentrations, tandem MS (MS/MS) was used in the selected reaction monitoring (SRM) mode. For a more reliable quatitation of trace levels, the stable isotope dilution assay (SIDA) was applied. The optimized method using HS-SPME H/C MDGC-MS/MS had levels of detection at concentrations below the odor thresholds of MPs in wine and allowed for the analysis of further MPs (isomers of dimethyl methoxypyrazine). As the core piece of enantiodifferentiation, one of the analytical columns in MDGC or GCxGC was replaced by a column with chiral selectors on the stationary phase, enabling the evaluation of the enantiomeric composition of SBMP in various samples. The quantitation of MPs in vegetable samples in this work revealed levels in the range of a few ng/kg up to µg/kg, which is in accordance with results from literature. Quantitative studies of wine (Sauvignon blanc and Cabernet blanc) indicated influences of oenological and viticultural processes on MP levels. The evaluation of the enantiomeric composition of SBMP resulted in the exclusive detection of (S)-SBMP in all analyzed samples, various vegetables, wine and ladybug species. The congruency of the configuration of the stereo center in the side chain of (S)-SBMP and the side chain of L-Isoleucin supports the hypothesis that natural amino acids serve as starting material in the biosynthesis of MPs. Extending the optimized H/C MDGC method, a successful separation of the isomers 3-methoxy-2,5-dimethylpyrazin (DMMP) and 2-methoxy-3,5-dimethylpyrazin (MDMP) was achieved. Of these isomers only MDMP was detected in cork and wine with an atypical cork off-flavor and it was identified for the first time in two ladybug species, Harmonia axyridis and Coccinella septempunctata. The methods developed in this work allowed for the quantitation of MPs in the range of a few ng/L. This can be used for further studies on influences on endogenic MP levels in Vitis vinifera or on influences on MP levels in wine by oenological processes or by contamination from different sources. For further clarification on the biogenesis of MPs, studies using labeled precursors or intermediates have to be developed. For the analysis of the resulting compounds from these fundamental studies, the quantitative (and enantioselective) analytical methods described here are essential.