## 2 Materials and Methods
### Determination of Volatile Compounds
#### 2.4.8 Quantitation of Selected Volatile Compounds  
Determination of Selected Volatile Compounds by SAFE-GC-MS  
Nineteen selected key odorants with FDs \(\geq\) 64 based on AEDA and/or previously reported as important volatile compounds associated with blueberry flavor [8; 21] were quantitated by solvent extraction combined with SAFE-GC-MS, as described earlier. A solution of stable isotope-labeled internal standards (hexanal-d\({}_{12}\), 2-phenyl-d\({}_{5}\)-ethan-1,1,2,2-d\({}_{4}\)-ol, and linalool-d\({}_{3}\)) was added to the sample prior to extraction to quantify these target odorants using stable isotope dilution analysis (SIDA). This method is highly accurate due to the similarity in the physicochemical properties between the isotopically labeled internal standards and the target analytes. Based on this approach, losses of the analytes caused by analytical procedures, i.e., extraction, distillation, or degradation, are compensated for. This differs from the use of generic internal standards that usually require the determination and use of recovery factors. Therefore, in this study, recovery factors were not calculated for compounds determined using unlabeled generic internal standards. Thus, these concentrations are considered semi-quantitative.  
The target volatile compounds, isotopically labeled internal standards, selected MS ions, and response factors (R\(f\)) are indicated in Table S1.  
A total of 2 \(\upmu\)L of each blueberry volatile extract was analyzed using the aforementioned GC-MS-O system under the same analytical conditions, except that selective ion monitoring (SIM) (dwell time, 50 ms; ions monitored) was used for quantitation. The unique quantitation mass ion was chosen to ensure the highest response and lowest interference (Table S1).  
Determination of Selected Volatile Compounds by Headspace Solid-Phase Microextraction-Gas Chromatography-Mass Spectrometry (HS-SPME-GC-MS)  
HS-SPME-GC-MS was used to determine another four selected key aroma compounds, i.e., methyl-3-methylbutanoate, ethyl-3-methylbutanoate, eucalypt, and \(\alpha\)-terpineol, with the same criteria as those in the earlier section for better sensitivity than SAFE-GC-MS extraction, as previously described [8]. With SPME, volatiles in a sample are adsorbed onto a coated fiber and then desorbed into a hot GC inlet. A total of 30 g of frozen blueberries was powdered with liquid nitrogen by an IKA analytical mill. Then, 2 g of blueberry powder was weighed into a prechilled 20 mL vial and mixed with 5 mL of citrate buffer (0.2 M, pH 3.2, 1% NaF, saturated with NaCl). Then, appropriate isotope-labeled internal standards (d\({}_{3}\)-methyl 3-methylbutanoate, d\({}_{3}\)-ethyl 3-methylbutanoate, d\({}_{3}\)-eucalypt, and d\({}_{3}\)-\(\alpha\)-terpineol) were added, and the vial was sealed with a PTFE-lined septum cap. Triplicate analyses were performed on each sample.  
Analysis was performed with a 7890 GC-MS system (Agilent Technologies, Inc.) coupled to a time-of-flight mass spectrometer (TOF-MS) (Pegasus 4D; LECO(r); St. Joseph, MI, USA) with a CombiPal (Leap Technologies, Inc., Fort Lauderdale, FL, USA) automated injector. Prior to the run, the sample was incubated (45 \({}^{\circ}\)C, 250 rpm) for 10 min prior to sampling. Following this, an SPME fiber (Supelco, 2 cm, DVB/Carboxen/PDMS) was deployed into the sample vial headspace and incubated for 50 min at 45 \({}^{\circ}\)C prior to the fiber being removed from the sample vial and desorbed for 20 min into the 260 \({}^{\circ}\)C splitless inlet of the GC. Volatile separation was accomplished with a Stabilwax column (30 m \(\times\) 0.25 \(\times\) 0.25 \(\upmu\)m) manufactured by Restek (Bellefonte, PA, USA), with helium carrier gas at a flow rate of 1 mL/min. The oven temperature was ramped from 40 \({}^{\circ}\)C to 225 \({}^{\circ}\)C at a rate of 4 \({}^{\circ}\)C per minute. The MS interface was 230 \({}^{\circ}\)C, and the ionization energy was 70 eV, with a 35 to 300 amu mass range and a scan rate of 50 scans/s.