Targeted Primary Compound Analysis and Method Validation

The extraction of primary metabolites was carried out according to Fiehn et al. (2008) with some modifications. Briefly, 0.1 g of fresh leaf powder was subjected to extraction by adding 1 mL of cool (-20°C) extraction solvent, composed of isopropanol/acetonitrile/water (3:3:2 v/v/v). A 20 μL aliquot of a solution containing palmitic-D3, nicotinic-D4, and glucose-D7 (1000 mg/L) was added as an internal standard. The extraction mixture was vortexed for 10 s, shaken at 4°C for 5 min and centrifuged at 12,000 g for 2 min at 5°C. A second round of extraction was carried out following the same procedure. The two supernatants were merged and re-suspended in a final volume of 5 mL using the extraction solvent. A total of 250 μL of supernatant was placed in a 1.5 mL Eppendorf tube and evaporated to dryness under N₂. The residue was re-suspended in 500 μL of acetonitrile/water (50:50 v/v), vortexed for 10 s, sonicated and centrifuged at 12,000 g for 2 min. The supernatant was then transferred into a 1.5 mL Eppendorf tube and dried out under N₂. The dried extract was subject to derivatization, first by adding 20 μL of methoxamine hydrochloride in pyridine (20 mg/mL) to inhibit cyclization of reducing sugars and shaken at 30°C for 1 h; then by adding 80 μL of N-methyl-N-trimethylsilyl-trifluoroacetamide with 1% trimethylchlorosilane for trimethylsilylation of acidic protons and shaken at 37°C for 30 min. Finally, 5 μL of a solution containing decane and heptadecane (1000 mg/L) were added in order to monitor the chromatographic analysis and the instrumental conditions. The derivatized extract was then transferred into vials for analysis. One microliter of derivatized extract was injected for GC/MS analysis.

Analyses were performed using a Trace GC Ultra combined with a mass spectrometer TSQ Quantum GC and an autosampler Triplus (Thermo Electron Corporation, Waltham, MA, United States). A RXI-5-Sil MS w/Integra-Guard^®(fused silica) (30 m × 0.25 mm × 0.25 μm) column was used for compound separation. Helium was used as the carrier gas at 1.2 mL/min and the injector split ratio was set to 1:10. The injector, transfer line and source temperature were set to 250°C. The initial oven temperature was kept at 65°C for 2 min, increased by 5.2°C/min to 270°C and held at 270°C for 4 min. These conditions were shown to represent a good compromise in order to obtain a not excessively long chromatographic run, a high number of compounds and good peak separation. The mass spectrometer was operated in electron ionization mode. Data acquisition was performed in full scan mode from 50 to 700 m/z. Data processing was performed using XCALIBUR^TM 2.2 SOFTWARE.

The method for primary metabolites was validated according to the currently accepted US Food and Drug Administration (FDA) bio-analytical method validation guide (US Department of Health and Human Services, 2001). Validation assays were established on calibration standards and quality control (QC) samples prepared as a pool of grape samples, extracted and derivatized according to the procedure described above. QC samples were used to evaluate the recovery of each compound and the stability of sample, intra- and inter-day variability, and to evaluate the efficiency of the extraction procedure. The standard mix was used to determine the limit of detection (LOD), limit of quantification (LOQ), and linearity range for each compound. Matrix calibration curves built using QC samples were compared with solvent calibration curves. Matrix effect (ME) values were determined using the slope ratios: ME% = 100 × (1 - slope solvent calibration curve/slope matrix calibration curve) (Kwon et al., 2012). LOQ and LOD were evaluated at the concentration in which the quantifier transition presented a signal-to-noise (S/N) ratio of >10 and >3, respectively. Intra- and inter-day variability were evaluated using the coefficient of variation (CV%) of QC samples injected 10 times on 1 day and then for 5 consecutive days. The recovery test was estimated on 10 spiked grape samples and calculated as the average of the “measured value/expected value” ratio (%). Each compound was identified and quantified against the standard, using one, or in the case of a few compounds, two specific m/z characteristics for the individual metabolite (extracted ion monitoring) and excluding saturated fragments. The fragments used for quantitation and the linear retention index (RI) are reported in Supplementary Table S1. Compounds were expressed as mg/kg of fresh leaves.