# Effect of Selected _Trichoderma_ Strains and Metabolites on Olive Drupes
## 2 Materials and Methods
### Phenolics' Isolation, Identification, and Quantification  
2.7.1. Metabolites' Analysis  
The analysis of metabolites was performed on an Agilent HP 1260 Infinity Series liquid chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with a DAD system (Agilent Technologies, Santa Clara, CA, USA) and a Q-TOF mass spectrometer model G6540B (Agilent Technologies, Santa Clara, CA, USA).  
Chromographic Conditions  
An InfinityLab Poroshell 120 EC-C18 column (2.1 mm \(\times\) 100 mm, 2.7 \(\upmu\)m) (Agilent Technologies, Santa Clara, CA, USA), at controlled temperature (25 \({}^{\circ}\)C) was used as stationary phase. Two elements, phase A (0.1% (\(v/v\)) formic acid in water) and phase B (0.1% (\(v/v\)) formic acid in acetonitrile), were employed as mobile phase. The gradient was set as follows: 0 min, 95% A; 4 min, 91% A; 7 min, 88% A; 8 min, 85% A; 9 min, 84% A; 14 min, 80% A; 15 min, 78% A; 18 min, 72% A; 19 min, 70% A; 20 min, 69% A; 21.50 min, 68% A; 23 min, 66% A; 24 min, 65% A; 25.5 min, 60% A; 27 min, 50% A; 30 min, 0% A; 35 min, 0% A; and 37 min, 95% A. Flow rate was 0.5 mL min\({}^{-\mathrm{w}}\).  
Spectroscopic and Spectrometric Conditions  
The UV spectra were recorded every 0.4 s, with a resolution of 2 nm, from 190 to 750 nm by DAD (Agilent Technologies, Santa Clara, CA, USA).  
The MS system was equipped with a dual ESI (electrospray ionization) source and operated in negative mode as reported by [PERSON] et al. [50]. All the parameters were controlled by the Agilent MassHunter Data Acquisition Software, version B.05.01. Mass spectra were recorded in the mass range 100-1600 \(m/z\) (3 scans per second). Hexakis (\({}^{1}\)H,\({}^{1}\)H, \({}^{3}\)H-tetrafluoropentoxy)-phosphazene (C\({}_{18}\)H\({}_{18}\)O\({}_{6}\)N\({}_{3}\)P\({}_{3}\)F\({}_{24}\) at \(m/z\) 922.009798, 2 umol L\({}^{-9}\)) (Sigma-Aldrich, St. Louis, MO, USA), and purine (C\({}_{5}\)H\({}_{4}\)N\({}_{4}\) at \(m/z\) 121.050873, 10 umol L\({}^{-1}\)) (Sigma-Aldrich, St. Louis, MO, USA) were injected in the source (0.060 mL min\({}^{-\rm e}\)) to perform the lock mass correction in real-time. The capillary was set at 4000 V, cone 1 (skimmer 1) at 45 V, fragmentor at 180 V. Gas temperature was 350 \({}^{\circ}\)C, and the nebulizer was at 45 psi. The injection volume was 5 \(\mu\)L. For each treatment, three biological samples were analyzed in triplicate.  
Mass Profiler Professional (Agilent Technologies, MPP v 13.1.1, Santa Clara, CA, USA) was used for molecular feature normalization, alignment, compound identification, and statistical analysis. MPP normalization and alignment parameters were: minimum number of ions (2); abundance filter (>5000 counts); intercept (0.4 min), alignment RT window, and slope (0%); intercept (2 mDa), alignment mass window, and slope (20 ppm). Only masses occurring in two of three samples were accepted. Masses found in blank runs from filtered masses were used to remove background noise. The ion chromatogram (EIC) was extracted with \(\pm\)20 ppm single ion expansion with MassHunter software v B.06.00 (Agilent Technologies, Santa Clara, CA, USA).