Microtof q hybrid quadrupole time of flight mass spectrometer

For LC–MS, the LaChromUltra (Hitachi Europe Ltd., UK) HPLC system coupled to a microTOF-Q hybrid quadrupole/time-of-flight mass spectrometer (Bruker Daltonics, Bremen, Germany) was used, equipped with an electrospray ionization (ESI) source.
2 μL of the sample was separated with the SeQuant^® ZIC^®-pHILIC 5 µm Polymeric column (150 × 2.1 mm) (Merck, Darmstadt, Germany). Eluent A (20 mM NH₄HCO₃, pH 9.3, adjusted with aqueous ammonia solution) and eluent B (acetonitrile) were applied at a flow rate of 0.2 mL min⁻¹ by use of following gradient: 0 min B: 90%, 30 min B: 25%, 37.5 min B: 25%, 40.0 min B: 80%.
The ESI source was operated in negative ionization mode. The temperature of the dry gas and the capillary was set to 180 °C. The scan range of the MS was set to 200–1000 m/z.

Samples of air-dried and ground plant material (0.5 g leaves) were extracted with ethanol–water (50:50, v/v) in a water bath at 60–70 °C. The extract was purified using a C16 Diapack cartridge and dissolved in 70% ethanol. Mass spectrometric analysis was carried out at the Novosibirsk Institute of Organic Chemistry SB RAS (Novosibirsk, Russia). HPLC-MS analysis was performed using an Agilent 1200 liquid chromatograph (Agilent Technologies, USA) and a micrOTOF-Q hybrid quadrupole-time-of-flight mass spectrometer (Bruker, Germany) with API-ES. Positive ions were identified in the range of 100–3000 m/z. Chromatographic separation was carried out at 30 °C using a Zorbax SB-C18 column (2.1 mm × 150 mm, inner diameter 3.5 µm) with a ZorbaxSB-C8 guard column (2.1 mm × 12.5 mm, inner diameter 5 µm). The composition of the mobile phase changed in a linear gradient from 15:85 (v/v) methanol (phase A) and 2% formic acid in water (phase B) to 100:0 (v/v) in 30 min, then in isocratic mode from 30 to 45 min. The volume of the injected sample was 10 µL. UV detection was carried out at four wavelengths/bandwidths: 255/16, 270/16, 320/16, 340/32 nm. Mass detection operating parameters were as follows: dryer gas flow (nitrogen) 8 L/min, nitrogen temperature 230 °C, nebulizer pressure 1.6 bar.