## 4 Materials and Methods
### Wine C\({}_{13}\)-Norisoprenoids (Bound-Form) Analysis  
Wines were hydrolyzed in acidic condition and analyzed for bound-form C\({}_{13}\)-norisoprenoids with HS-SPME-GC-MS based on the previous method with minor modifications [42]. Two-milliliter wine sample was diluted 5-fold with 0.2 M citrate buffer (pH 2.5) saturated with sodium chloride in a 20 mL vial. The vial was placed in a shaking water bath (99 \({}^{\circ}\)C, 1 h) for acid hydrolysis. After hydrolysis, the sample was placed in an ice bath for 5 min before the addition of internal standard ((\(\pm\))-theaspirane, 1 mg/L). Internal standard calibration curves were built with devolatilized Pinot noir wine. Devolatilized Pinot noir wine was generated by passing wine through a solid-phase extraction column with 2 g Lichroult EN (Merck, Darmstadt, Germany). Standard mixtures of C\({}_{13}\)-norisoprenoids were added and diluted to different gradients. HS extraction conditions and GC method were the same as for the analysis of major volatile compounds.  
Untargeted Analysis with Liquid Chromatography High-Resolution Tandem Mass Spectrometry (LC-HRMS/MS)  
Wine samples were diluted ten times with aqueous methanol 50% _v/v_ before injecting 3 \(\upmu\)L into the system. Untargeted analysis of wine was performed according to a previously reported fingerprinting method with some modifications [44]. LC-HRMS/MS analysis was conducted using a UHPLC system (Shimadzu Nexera, Shimadzu Corporation, Kyoto, Japan) coupled to an AB SCIEX Triple TOF(r) 5600 mass spectrometer (Framingham, MA, USA) operated in positive electrospray ionization (ESI) mode. Chromatographic separation was performed utilizing an Inertsil Phenyl-3 column (4.6 mm \(\times\) 150 mm, 100 A, five \(\upmu\)m; GL Sciences, Torrance, CA, USA). Mobile phase A consisted of water containing 0.1% _v/v_ formic acid, and mobile phase B, methanol containing 0.1% _v/v_ formic acid. The flow rate was 0.4 mL/min. The entire run was 30 min in a multistep gradient as follows: 0-1 min at 5% B, followed by 5 to 30% B from 1 to 10 min, then 30 to 100% B from 10 to 20 min, held at 100% B from 20 to 25 min and then returned to 5% B from 25 to 30 min. For each year, a quality control (QC) sample was prepared by mixing an equal volume of each wine sample. In addition, QC was injected every two hours to monitor system stability.  
Data-dependent acquisitions (DDAs) were conducted to obtain precursor and fragment ion information to aid in annotating compounds. The following settings were used: spray voltage was set to 4500 V, full scan with ion accumulation of 150 ms, followed by a dynamic MS/MS selection of the eight most intense ions with 100 ms accumulation; after two MS/MS acquisitions, the precursor (fragmented) ions were excluded for 30 s; collision energy 35 V with collision energy spread (CES) of 15 V ramped through each MS/MS scan using a range of \(m/z\) 100-1200. In addition, the instrument was equipped with an automatic calibrant delivery system performing calibration every two hours. Annotation of compounds was performed as previously reported with some modifications [44; 45; 46]. In brief, exact mass, isotopic pattern, and MS/MS spectra were compared with online libraries: MassBank of North America (MoNA), METLIN, and Human Metabolome Database (HMDB) (online versions, July 2021). For compound annotation, a confidence score equal to or higher than 50 was considered adequate for putative annotation. L2 annotationswere performed according to [PERSON] et al. [47]. Because this score was more rigorous than previous reports using Progeness QITM with a score \(>\) 31.6 [48], in the case of compounds previously reported in wine, the score selected was equal to or higher than 35. A high confidence score was typically reached when MS/MS similarity was \(>\)50%, the isotopic similarity was above 80%, and the exact mass error was better than 10 ppm.