Compass software

The high-resolution mass spectrometry (HRMS) analysis of the extracts was carried out on an Impact II QToF HR mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany) coupled to an Elute UHPLC system (Bruker Daltonik GmbH, Germany). The data acquisition was performed using Compass software (Bruker, Germany), while the data were processed by Metaboscape 5.0 software (Bruker, Germany). The lyophilized extracts were dissolved in 50% acetonitrile (LC-MS grade, Thermo Fisher, Pittsburgh, PA, USA) in water (LC-MS grade, Thermo Fisher, PA, USA) to achieve a concentration of about 0.2 mg/mL. The following chromatography program was applied: 0.1% formic acid in water as solvent A and 0.1% formic acid in acetonitrile as solvent B, with gradients as follows: 1% B for 2 min, 1% to 99% B in 15 min and 100% B for 3 min, using a flow rate of 0.25 mL min⁻¹. The separation was carried out on a Bruker Intensity Solo 2 C18, 2.1 × 100 mm RP column at a constant temperature of 35 °C. An injection volume of 5 µL was used for the analysis. The analysis was performed in positive and negative ESI modes sequentially. The ESI source was operated at a nebulizer pressure of 2.0 bar, and the dry gas rate was set to 8.0 L min⁻¹ at a temperature of 200 °C. The MS/MS spectra were recorded in Auto MS/MS mode, resulting in collision energy stepping from 20 to 50 eV.

The crude extract of the S. ecbatanensis was subjected to mass analysis and HRESIMS data were recorded on a maXis UHR-TOF mass spectrometer (Bruker, Billerica, MA, USA). Analytical RP HPLC was carried out with an Agilent 1200 (Santa Clara, CA, USA) HPLC system equipped with a diodearray UV detector (DAD) at 200–600 nm connected with the maXis ESI TOF mass spectrometer. HPLC conditions: Waters Acquity UPLC BEH C18 column 50 × 2.1 mm, 1.7 μm, solvent A: H2O, 0.1% formic acid; solvent B: acetonitrile, 0.1% formic acid; gradient system: 5% B for 1 min, increasing to 95% B in 20 min; flow rate 0.6 mL/min; column oven temperature 40 °C. The Data Analysis software (Compass-software, Bruker, USA) was used for analysis of the spectra and calculation of molecular formula including the isotopic pattern (Smart Formula algorithm). New compounds were detected by comparison of molecular weights and UV spectra with the characteristics of the known natural derived compounds in the databases of (Dictionary of Natural Products, CRC press) and SciFinder (Chemical Abstract Service, USA).

The dries extracts were dissolved in a solution of 50% (v/v) chromatographic grade acetonitrile (Tedia, Fairfield, OH, USA), 50% (v/v) deionized water, and 0.1% formic acid. The solutions were infused directly individually into the ESI source by means of a syringe pump (Harvard Apparatus) at a flow rate of 50 μL min⁻¹. ESI(+)-MS and tandem ESI(+)-MS/MS were acquired using a hybrid high-resolution and high accuracy (5 μg/L) microTof (Q-TOF) mass spectrometer (Bruker Scientific) under the following conditions: capillary and cone voltages were set to +3500 V and +40 V, respectively, with a desolvation temperature of 100°C. For ESI(+)-MS/MS, the energy for the collision induced dissociations (CID) was optimized for each component. Diagnostic ions in different fractions were identified by the comparison of their ESI(+)-MS/MS dissociation patterns with compounds identified in previous studies. For data acquisition and processing, compass software (Bruker Scientific) was used. The data were collected in the m/z range of 70–800 at the speed of two scans per second, providing the resolution of 50,000 (FWHM) at m/z 200. No important ions were observed below m/z 180 or above m/z 650; therefore ESI(+)-MS data is shown in the m/z 180−650 range.