Ultra performance liquid chromatography (uplc)

The polyphenol profile of LPPE was analyzed with an ultra-high-pressure liquid chromatograph (UPLC, Agilent, Santa Clara, CA, USA) and a triple quadrupole mass spectrometer (6460QqQ-MS, Agilent, Santa Clara, CA, USA) equipped with an electrospray ionization source (ESI). A ZORBAX Eclipse Plus C18 column (100 mm × 2.1 mm i.d. 1.8 µm, Agilent, Waldbronn, Germany) was used. The mobile phase consisted of 0.1% aqueous formic acid (A) and methanol with 0.1% formic acid (B) at a flow rate of 0.2 mL min⁻¹. The gradient elution was set as follows: 0–10 min, 100% A–20% A; 10–11 min, 20% A; 11–12 min, 20%–100% A. The sample injection volume was 5 μL, and the column temperature was 35 °C. All the compounds were identified by comparing the dynamic multiple reaction monitoring (MRM) information with reference standards.

Enzymatic reactions and hairy root extracts were filtered and analyzed on UPLC (Agilent 1290 Infinity II, Santa Clara, CA, United States) with a Eclipse XDB-C18 column (4.6 × 150 mm, 5-μm) maintained at 30 °C. Separation was performed with 0.1% (v/v) formic acid/water (A) and 100% methanol (B) at 1 mL/min flow rate. The linear gradient was 5%–80% B over 16 min, 80%–100% B from 16 to 18 min, 100% B for 2 min, and equilibrated at 5% B for 1 min. The flavonoid compounds were detected at 280 nm with a diode array detector. Flavonoids contents were measured by comparing area of individual peak with standard curves obtained with standard compounds (daidzein and genistein).
The enzymatic products were also detected on LC-MS/MS on an ion-trap TOF mass spectrometer attached to UPLC (Agilent, Santa Clara, CA, United States). Separation was performed on a Eclipse Plus C18 column (2.1 mm × 100 mm, 1.8-μm) using the same solvent system as described above for UPLC. The linear gradient was 5%–60% B over 5 min, 60%–100% B from 5 to 10 min, 100% B for 2 min, and equilibrated at 5% B for 0.1 min. The flow rate was maintained at 0.4 mL/min, and the column temperature was maintained at 30 °C. Flavonoid compounds were detected under negative mode, and the m/z spectra were collected from 500 to 1000 as described previously [44 (link)].

The procedure was based on the method published by Kobus et al. [12 (link)]. Phenolic acids and flavonols were analyzed using Agilent UPLC equipped with a Bin Pump Infinity DAD 1290 detector (for phenolics acids λ = 260 nm and 310 nm and for flavonols λ = 370 nm). Separated compounds of phenolic acids and individual flavonols were determined as chlorogenic acid, ferulic acid, vanillic acid, gallic acid, o-coumaric acid, p-coumaric acid, cinnamic acid, syringic acid, p-hydroxybenzoic acid, caffeic acid, catechin, epicatechin, quercetin, rutin, and kaempferitrin. Compounds were identified using the standards dissolved in methanol.
The single-reference method was applied to determine a relationship between peak area and the concentration of the analyzed standard.

The amino acid contents were determined using the O-phthalaldehyde (OPA) precolumn derivatization method [49 (link)]. Mycelial samples (0.03 g) were ground and added to 500 µL of NaOH-sodium borate buffer (pH 9.5) for ultrasonic extraction for 30 min. Then, 200 µL of supernatant was mixed with 100 µL of OPA reagent solution (30 mM OPA, 70 mM 2-mercaptoethanol, 50 mM sodium borate, pH 9.5) for 1 min. Next, 200 µL of 100 mM KH₂PO₄ buffer was added to terminate the reaction. The supernatant was analysed using a UPLC (Agilent Technologies, Santa Clara, CA, USA) equipped with a C₁₈ column (1.8 μm, 50 mm × 2.1 mm I.D.). A gradient elution was performed with 20 mM aqueous acetic acid (mobile phase A, 85–20%) and 20 mM aqueous acetic acid/methanol/acetonitrile (1:2:2, v/v; mobile phase B, 15–80%). A fluorescence detector was used to monitor fluorescence at excitation and emission wavelengths of 340 and 455 nm, respectively.

A QTRAP 4500 series MS/MS system (SCIEX, USA) equipped with a UPLC (Agilent) system and Intrada IMTAKT amino acid column (3.0 × 150 mm, 3.0 µm) was used to analyze the amino acid content. The injection amount was 2 μL. The flow rate was set at 0.3 mL/min. The column temperature was maintained at 40 °C. A gradient elution of solvent A (water) and solvent B (ACN) was used as follows: 0–4 min, 20% A and 80% B; 4–14 min, 0% A and 100% B; 14–16 min, 0% A and 100% B; 16.1–20 min, 80% A and 20% B.
The MS analysis conditions used the positive ion mode. The curtain gas and collision gas were set to 30 and 3, respectively. The ion spray voltage and ion source temperature were set to 5500 and 550, respectively.

An X500R series QTOF-MS system (SCIEX, Pte, Ltd.) equipped with a UPLC (Agilent) system and CORETECS T3 column (2.1 × 150 mm, 1.6 µm, Waters, Milford, MA, USA) was used to analyze ginsenoside metabolites. The injection amount was 1 μL. The flow rate was set at 0.35 mL/min. The column temperature was maintained at 30 °C. A gradient elution of solvent A (0.1% formic acid + water) and solvent B (0.1% formic acid + ACN) was used as follows: 0–10 min, 85% A and 15% B; 10–40 min, 50% A and 50% B; 40–45 min, 50% A and 50% B; 45–50 min, 30% A and 70% B; 50–60 min, 5% A and 95% B; 60–65 min, 85% A and 15% B; 65–80 min, 85% A and 15% B.
QTOF-MS analysis was conducted in the TOF-MS mode with a mass range of 100–1500 m/z. The ion source gas and curtain gas were set to 50 and 30, respectively. The ion source temperature was maintained at 500 °C. The declustering potential (DP) and collision energy (CE) values were set to 50 and 15 V, respectively. The spray voltage and CE spread were adjusted to 4500 and 10 V, respectively. The analysis was performed in the positive mode.

The whole body adult whiteflies were homogenized for amino acid analysis by UPLC using the protocol described previously [25 (link), 56 (link)]. Briefly, samples were injected into an Agilent UPLC with a PDA detector and AccQ-Tag Ultra 2.1 x 100 mm column. Amino acids are determined by comparing their retention time with standards, protein-amino acids μl^-1 (Waters amino acid hydrolysate standard #088122, supplemented with asparagine, tryptophan, and glutamine) and quantified with standard curves. Proteins were quantified using a Lowry Protein Assay Kit (Sangon, Biotech) following manufacturer’s instructions using bovine serum albumin as a standard. Amounts of individual amino acids were normalized to the total protein content.