Analysis of sesquiterpene hydrocarbons in grape berry exocarp (Vitis vinifera L.) using in vivo-labeling and comprehensive two-dimensional gas chromatography–mass spectrometry (GC×GC–MS)

Sesquiterpenes are structurally diverse, potent flavoring substances that significantly influence the aroma profile of grapes (Vitis vinifera L.) at the time of physiological ripening. To investigate these natural compounds, freshly harvested, ripe berries of the red wine variety Lemberger (Vitis vinifera subsp. vinifera L.) were analyzed using comprehensive two-dimensional gas chromatography (GC×GC) coupled to a time-of-flight mass spectrometer (TOF–MS) after headspace-solid phase microextraction (HS-SPME). The identification of structurally complex natural compounds, such as sesquiterpenes from fruits and vegetables, is often reported as “tentative”, as authentic standards are not commercially available for most of the analytes. For this reason, feeding experiments (in vivo labeling) were carried out using the stable isotope-labeled precursors [5,5-(2)H(2)]-1-deoxy-ᴅ-xylulose (d(2)-DOX) and [6,6,6-(2)H(3)]-(±)-mevalonolactone (d(3)-MVL) to clearly identify the volatiles. Based on the recorded mass spectra of the unlabeled and deuterated compounds, mechanisms for sesquiterpene formation in V. vinifera could be proposed and already known pathways could be confirmed or disproved. For example, the HS-SPME–GC×GC–TOF–MS measurements of fed sample material showed that the tricyclic sesquiterpene hydrocarbons α-copaene, β-copaene, α-cubebene, β-cubebene and the bicyclic δ-cadinene were biosynthesized via (S)-(−)-germacrene D rather than via (R)-(+)-germacrene D as intermediate.