GNPS - Si11 - standards mixture to test annotation of ISF features

A mixture of 11 compounds (2-methoxybenzoic acid, biochanin A, trans-ferulic acid, 3-indoleacetonitrile, indole-3-carboxaldehyd, kaempferol, kinetin, p-coumaric acid, L-(+)-alpha-phenylglycine, phloridzin dihydrate, rutin trihydrate) each at a concentration of 20 µM was prepared in a 1:1 mixture of acetonitrile and water.

1 µl of the Si11-mixture was analyzed by reversed phase ultrahigh-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry multiple times. The sample was separated using ultra high-performance liquid chromatography, performed on a Dionex Ultimate 3000 UPLC system (Thermo Fisher Scientific, Waltham, MA) using a 150 by 2.1 mm Kinetex C18 column with 1.7-mm particle size (Phenomenex, Aschaffenburg, Germany) column with a flow rate of 300 ml/min. Gradient elution with water with 0.1% (vol/vol) formic acid as eluent A and acetonitrile with 0.1% (vol/vol) formic acid as eluent B was run as follows: 1% B for t = 0 min to t = 2 min, linear gradient from 1% B to 100% B from t = 2 min to t = 20 min, hold 100% B until t = 25 min, and linear gradient from 100% B to 1% B from t = 25 min to t = 30 min. The sample was analyzed by positive mode electrospray ionization quadrupole time-of-flight mass spectrometry on a maXis HD QTOF (Bruker, Bremen, Germany) using data-dependent MS/MS by collision-induced dissociation of the three most abundant ions in each scan, making use of Bruker’s “Smart Exclusion” functionality to minimize multiple fragmentation of the same ion. Accurate masses were obtained by internal calibration using an ion cluster of sodium formate and lock mass calibration.