Agilent 6530b qtof spectrometer

To determine total phenolic and flavonoid contents of F. clypeata extracts, colorimetric methods were used based on our previous work [13 (link)]. In this regard, the results were finally expressed as namely gallic acid equivalents (GAE) for total phenolics and rutin equivalents (RE) for total flavonoids.
Thereafter, the phytochemical analysis of each plant extract was carried out using Agilent 1200 Infinity HPLC and Agilent 6530B QTOF spectrometer (Agilent Technologies, Santa Clara, CA, USA). The conditions of the analyses were described previously [14 (link)]. The identification was based on the obtained fragmentation patterns, which were compared to the data available in the scientific literature and the Metlin database (https://metlin.scripps.edu).

Extracted metabolites were dissolved in acetonitrile–methanol–isopropanol (1:1:2 v/v/v) and filtered through 0.22 μm PTFE syringe filters. The chromatographic and mass spectrometry conditions were as described previously (Sieniawska et al., 2021 (link)). Metabolites were separated on Gemini® chromatographic column (3 μm i.d. C18 with TMS end-capping, 110 Å, 100 × 2 mm) supported by a guard column (Phenomenex Inc, Torrance, CA, USA), using Agilent 1200 Infinity HPLC chromatograph (Agilent Technologies, Santa Clara, CA, USA). The solvents were pumped at the flow rate of 0.3 ml/min to perform the gradient of phase A [water with 0.1% formic acid (v/v)] and phase B [0.1% formic acid in acetonitrile (v/v)]: 5 min, 0% B; 20 min, 66% B; 35 min, 95% B. Then, metabolites were detected on Agilent 6530B QTOF spectrometer equipped with a Dual Agilent Jet Stream spray source (ESI) (Agilent Technologies, Santa Clara, CA, USA). The operating conditions were as follows: sheath gas temp: 400°C, sheath gas flow: 12 L/min; drying gas temp: 350°C, drying gas flow: 12 L/min; nebulizer pressure: 40 psig, capillary V (+): 4000 V, skimmer 65 V. The acquisition was performed in a positive ion mode taking 2 spectra/s in a scan range from 100 to 3,000 m/z.