4.6. Instrumentation and Experimental Conditions

Samples were applied in the 8 mm band using a Hamilton syringe (100 μL) (Hamilton, Switzerland) on the precoated silica gel plate 60 F₂₅₄ (size 20 × 10 cm × 0.2 mm thickness and particle size 5–6 μm) (Merck, Darmstadt, Germany) using the TLC applicator Linomat 5 (Camag, Switzerland) with vision CATS software (version 3.0.20196.1). The application positions (X and Y) were 20 and 8 mm, respectively, with a development of 80 mm in length. The solvent system used ethyl acetate: methanol: water (9:2:1.5 ) v/v/v) followed by TLC development using a 20 × 10 cm twin trough chamber. The optimized chamber saturation time for the solvent system was 20 min, at the temperature 25 ± 2 °C. The plates were dried and were analyzed at 254 and 366 nm and white light before and after derivatization with the NP-PEG reagent in TLC visualizer 2 (Camag, Switzerland).³³

Chromatography was achieved using a Shimadzu N-Series XS Al-PDA HPLC system. Chromatographic separation was carried out on a Phenomenex Luna, 5 μm C18 (2) 100 Å column (150 × 4.6 mm × 5 μm) at 30 °C. The mobile phase consisted of 0.1% formic acid in water (A) and acetonitrile (B). The gradient condition was 0.01–5.00 min, 20–20% B; 5.00–13.00 min, 20–80% B; 13.00–18.00 min, 80–80% B; and 18.00–21.00 min, 80–20% B. The run time was 25 min, followed by a flow rate of 1.0 mL/min and an injection volume of 10 μL, and the detection wavelengths are 250 nm for (1), 320 nm for (7), and 440 nm for (2–6).

Qualitative analysis was performed on Shimadzu Nexera X2 (Shimadzu Tech., Kyoto, Japan) consisting of a quaternary pump (LC-30AD), autosampler (SIL-30AC), column oven (CTO-20 AC), and diode array detector (SPD-M20A) and coupled with LCMS-8045 (Shimadzu Tech., Kyoto, Japan). The TQ-MS/MS was equipped with a thermally assisted ESI source with an outlet of the PDA detector connected to a splitter (Split; ratio 4:1) with the same chromatographic conditions with a precolumn (Phenomenex security guard ULTRA with C18 cartridge). The autosampler temperature was set at 15 °C with a flow rate of 1.0 mL/min and an injection volume of 10 μL for total ion chromatogram (TIC) acquisition. Mass analyses were performed for precursor ion scan in both positive- and negative-ion modes at a scan speed of 2000 u/sec with an interface temperature of 300 °C. The desolvation line and heat block temperatures were set to 250 and 300 °C, respectively. The gas flows for nebulizing, heating, and drying gas were kept at flow 10, 3, and 10 L/min, respectively. The sample (CSE) was analyzed in the Q1 scan mode, where the scan range was kept at 100–2000 m/z. The MS/MS fragmentation of compounds (1–7) was carried out in an ESI interface using the MRM mode. The mass resolution was set at 0.05 full width at half-maximum with resolving power Rp (1000–3000). The quadruple setting was set as voltage Q1 RF gain: 4998 Q1 RF offset 4990 and Q1 post-rod bias: −5.0 V CID CELL exit lens: −4.0 V. Datawere analyzed using Lab Solution software (Version 6.80).^{28,34,41−43}