TABLE 2.

2.8.2 Operating conditions.

System	Dionex ultimate 3000 RS UPLC with Thermo Scientific Q Exactive Orbitrap Hybrid Tandem Mass Spectrometer
Column	Waters XBridge TM C18, 2.5 um, 2.1 mm i.d x 50 mm
Column temperature	40oC
Mobile phase	A = 0.1% formic acid in water
Separation gradient	Time (min)	%B
	0.0	10
	15.0	100
	20.0	100
Flow rate	0.4 ml/min
Injection volume	5 uL
m/z range	100–1,500
Ion source and polarity	ESI (+ve and -ve)
Resolution	70,000 (full MS scan) and 35,000 (ddMS2 scan)
Collision energy	25, 40, and 55
Software for data analysis	Compound discoverer 3.1
Workflow description	Natural product unknown ID w stats online and local database searches (untargeted food research ID workflow with statistics: Detect and identify unknown compounds with differential analysis). Perform retention time alignment, unknown compound detection, and compound grouping across all samples. Predict elemental compositions for all compounds and hide chemical background (using blank samples). Identify compounds using MZCloud (ddMS2 and/or DIA), ChemSpider (exact mass or formula), and local database searches against mass lists (exact mass with or without RT) and mzVault spectral libraries. Perform spectral similarity search against MZCloud for compounds with ddMS2. Apply mzLogic to rank order structure candidates from ChemSpider and mass list matches. Apply spectral distance scoring to ChemSpider and mass list matches and perform differential analysis on detected compounds