Syncronis c18 column

The separation of phenolic acids of the ME was performed with an HPLC (HITACHI L-6200, Hitachi High Technologies Com., Tokyo, Japan) system with a Syncronis C18 column (5 μm, 4.6 mm × 250 mm; Thermo Fisher Scientific Inc., Waltham, MA, USA). The mobile phase (1.0 mL min⁻¹) consisted of A: 0.1% formic acid (FA; Thermo Fisher Scientific Inc., Rockford, IL, USA); and B: 100% methanol. The gradient conditions (A:B) were programmed as 0–10 min, 5–25% A and 95–75% B; 10–20 min, 25–30% A and 75–70% B; 20–40 min, 30–60% A and 70–40% B. The column was maintained at 25 °C, and the eluents were detected by UV280. Instrument control and data analysis were conducted using ECL2000 software (Analab Corp., Taipei, Taiwan).
The functional phenyl compound 2,4-dimethoxy-6-methylbenzene-1,3-diol (DMMB) was purified by preparative HPLC (Primeline^TM Gradient pump 500G; Analytical Scientific Ins., El Sobrante, CA, USA). The DMMB was separated on a preparative Inspire^TM C18 column (21.2 mm × 250 mm; Dikma Technologies Inc., Lake Forest, CA, USA) with 40% methanol (10.4 mL min⁻¹). The fraction containing DMMB was detected by UV280 and collected using an Advantec SF2120 fraction collector (Advantec Toyo Kaisha, Ltd., Tokyo, Japan) simultaneously. The collected fractions were concentrated using a rotary evaporator and a pressured nitrogen gas-blowing concentrator.

Solutions of LSB, MLB, and ZLB were filtered through a 0.22 μm polyvinylidene difluoride (PVDF) membrane filter (Pall Corporation, Glen Cove, NY, USA) and subjected to HPLC analysis. The HPLC system coupled to a model 600E photodiode array detector (Waters Corporation, Milford, MA, USA) was performed using a Syncronis C18 column (4.6 × 250 mm inner diameter, 5 µm, Thermo Scientific, Waltham, MA, USA) with an eluting gradient as follows: 5% acetonitrile for 0–5 min; linear gradient from 5% to 70% acetonitrile for 5–35 min; 70% acetonitrile for 35–45 min. The acetonitrile solution used in the eluting gradient contained 0.5% acetic acid. The ultraviolet (UV) absorbance was detected at 280 nm. Mass spectrometric analysis was performed on a linear trap quadrupole tandem mass spectrometer (Thermo Electron, San Jose, CA, USA) equipped with an electrospray ionization interface and connected to a Surveyor LC system (Thermo Electron, Waltham, MA, USA) with a 5 μL sample loop. The analytes were separated under the same condition used for HPLC analysis. Negative ESI mode was firstly scanned ranging from m/z 150 to 1500. The other scans were set as the data-dependent MSⁿ scan using the high-purity helium (>99.99%) as the collision gas and the relative collision energy of 30%.

Phenolics in the methanol extract of N. rtanjensis were chromatographically separated on a Syncronis C18 column (100 × 2.1 mm) with 1.7 μm particle size (ThermoFisher Scientific, USA), thermostated at 40 °C. The mobile phase consisted of (A) 0.1% acetic acid in water and (B) acetonitrile (MS grade, FisherScientific, UK), which were applied in the following gradient elutions: 5% B in the first minute, 5–95% B from 1.0 to 16.0 min, from 95% to 5% B for 16.0–16.2 min, and 5% B until the 20th min. The flow rate was set to 0.3 mL min⁻¹, and the injection volume was 5 μL. Settings of the TSQ Quantum Access Max QQQ mass spectrometer for the time-selected reaction monitoring (tSRM) experiment were as previously described in Čolić et al. [33 (link)]. Xcalibur software (version 2.2) was used for instrument control, data acquisition, and analysis. The total amount of compounds in samples was calculated based on the calibration curve of pure compounds and expressed as μg 100 mg⁻¹ dry extract.