Beh shield rp18 column

Ocimum basilicum leaf extracts collected when UAE via a sonotrode was applied with different parameters was evaluated by an ACQUITY Ultra Performance LC system (Waters Corporation, Milford, MA, USA) associated with an electrospray ionization (ESI) source operating in negative mode and a time-of-flight (TOF) mass detector (Waters Corporation, Milford, MA, USA). All measurements were carried out in triplicate. The active substances were partitioned on an BEH Shield RP18 column (1.7 μm, 2.1 mm × 100 mm; Waters Corporation, Milford, MA, USA) at 40 °C using a gradient previously stated by Verni et al. 2020 [68 (link)] and using water containing 1% acetic acid as mobile phase A and acetonitrile as mobile phase B. The data were elaborated using MassLynx 4.1 software (Waters Corporation, Milford, MA, USA).

Chromatographic analysis was performed on a Waters ACQUITY H-Class UPLC^® system, equipped with a quaternary solvent manager, sample manager, flow-through needle, high temperature column heater with active preheating, and QDA detector. Chromatographic separation was carried out at 35°C on a BEH Shield RP18 column (2.1 mm × 100 mm, 1.7 μm) (Waters). The mobile phase consisted of 0.1% formic acetonitrile (A) and 0.1% formic acid water (B), using an gradient elution of 5–30% A at 0–3 min, 30–40% A at 3–5 min, 40–100% A at 5–15 min, and 100–5% A at 15–18 min. The sample volume injected was 2.5 μL, and the flow rate was 0.4 mL/min.
The conditions of the electrospray ionization (ESI) source were as follows: ESI in positive mode; capillary voltage, 800 V; fragmentor, 15 V; sampling frequency, 5 Hz; Probe temperature 500°C. Ginsenoside Rg1 was detected in SIM 823.48 Da [M+Na]⁺ mode at 0–5.5 min; AG IV and AG III were detected in SIM 808.00 Da [M+Na]⁺ mode at 5.5–8 min; AG II and iAG II were detected in SIM 849.50 Da [M+Na]⁺mode at 0–8 min; AG I was detected in SIM 869.50 Da [M+H]⁺ mode at 0–8 min; iAG I was detected in SIM 891.50 Da [M+Na]⁺ mode at 0–8 min.

UPLC analysis was undertaken on an ACQUITY UPLC BEH Shield RP18 column (2.1 mm × 100 mm, 1.7 μm) at 40 °C by using a Waters ACQUITY UPLC system. The mobile phase, 0.1% formic acid solution (A), and acetonitrile (B) were eluted in a gradient program of 5–30% B in 0–4 min and 30–35% B in 4–5 min at a flow rate of 0.3 mL/min. During the stability study of this experiment, the gradient elution implemented was listed below: 5–35% B in 0–2 min, 35–45% B in 2–3 min, and 45–55% B in 3–3.5 min. The detection wavelength was monitored at 237 nm. Injection volume was 2 μL.

Lyophilized cells (2–3 mg) were suspended in 500 μL of methanol:acetone (5:5 [v/v]) and fractured using 0.5 mm glass beads in a multi-bead shocker MB1001C(S) as described for lipid analysis. The samples were centrifuged at 10,000×g for 2 min at 4 °C, and the supernatant was transferred to a new microtube. The extraction procedure was repeated four times to obtain 2 mL of supernatant. The supernatant (330 μL) was dried in a vacuum using an evaporator CEV-3100 (EYELA, Tokyo, Japan), resuspended in 500 μL of chloroform:acetonitrile (2:8 [v/v]) containing 1 μM trans-β-apo-8-carotenal as an internal standard, and filtered using a 0.22 µm Cosmospin Filter G (Nacalai Tesque). Pigments were identified and quantified using an ACQUITY ultra liquid chromatography (UPLC) system equipped with a photodiode array detector and a BEH Shield RP18 column (1.7 μm, 2.1 mm × 100 mm; Waters, Milford, MA, USA) [25 (link), 47 (link)].

The analysis of Morus alba leaf extracts was performed by HPLC-ESI-TOF-MS as previously reported by Verni et al., 2020 [22 (link)]. Three replicates of each sample were processed. The equipment consists of a UPLC system ACQUITY (Waters Corporation, Milford, MA, USA) coupled to a time-of-flight analyzer (TOF) (Waters Corporation, Milford, MA, USA). The phenolic compounds were separated using a BEH Shield RP18 column (1.7 µm, 2.1 mm × 100 mm; Waters Corporation, Milford, MA, USA). The analysis was carried out at 40 °C and the data were processed using MassLynx 4.1 software (Waters Corporation, Milford, MA, USA).

Calibration curves of hypothemycin (1) and (5Z)-7-oxozeaenol (2) were developed using pure standards isolated from fungal strains MSX78495 and MSX63935, respectively. Ten standard solutions were prepared in CH₃CN at a concentration range of 3–1536 ng ml⁻¹. HRESIMS data were collected in triplicate via a UPLC-HRESIMS (Thermo LTQ Orbitrap XL) system using the area under the curve (AUC) to generate the calibration curves. A BEH Shield RP18 column (Waters, 1.7 µm; 50 × 2.1 mm) heated to 40 °C was utilized with a flow rate of the mobile phase of 0.3 ml min⁻¹ and a gradient system of 15:85 to 100:0 of CH₃CN-H₂O (0.1% formic acid) over 10 min. MS data were collected from m/z 150 to 2000 in the positive mode. The linearity of each calibration curve, relative error (RE), and limit of quantitation (LOQ), which is the lowest amount of analyte that can be quantitatively determined with suitable precision and accuracy, were calculated and summarized (Fig. S7 and Table S3). Extracts from the fungal strains MSX78495, MSX63935, and MSX45109 were analyzed in triplicate, the areas were averaged, and the concentrations of 1 and 2 were extrapolated from the corresponding calibration curve. Statistical analysis was carried out using GraphPad Prism (GraphPad Software, La Jolla, CA), and comparisons were made using one-way ANOVA followed by Tukey post hoc test.