Synergi fusion rp c18 column

Separations used either a Synergi Fusion-RP C₁₈ column (ID: 2 mm, length: 150 mm, particle size: 4 µm, pore size: 80 Å, Phenomenex) or Aqua C₁₈ column (ID: 2 mm, length: 150 mm, particle size: 3 µm, pore size: 125 Å, Phenomenex) using a System Gold HPLC equipped with a 507e autosampler (100 µL injection volume), a 125NM binary gradient pump, and a 166 UV/VIS detector. Eluents were water (eluent B1) and aqueous acetonitrile (60% v/v, eluent B2 or 40% v/v, eluent B3) containing sodium or potassium phosphate buffer or ammonium acetate (10 mmol/L). Eluents were prepared from aqueous stock solutions (0.1 mol/L) adjusted to pH 7.2, in case of ammonium acetate with ammonia. Solvents were filtered (pore size, 0.2 µm; Pall Corp., Ann Arbor, MI) and sonicated for 15 min prior to use. Peptides were eluted using a linear acetonitrile gradient from either 5 to 95% eluent B2 in 30 min, 5 to 70% eluent B2 in 65 min, or 7.5 to 95% eluent B3 in 58 min. Unless otherwise indicated, separations were performed at 60 °C with a flow rate of 0.2 mL/min and the absorbance was recorded at 214 nm. Fractions were analyzed by matrix-assisted laser desorption/ionization time-of-flight MS (MALDI-TOF/TOF–MS) on a 5800 proteomic analyzer (ABSciex GmbH, Darmstadt, Germany) operating in reflector mode and using a solution of α-cyano-4-hydroxycinnamic acid (4 g/L) in eluent A2 as matrix.

LC-MS/MS analysis method was developed and optimized by using synthetic standards to achieve maximal sensitivity for target detection, including the LC gradient and the retention time, m/z of the transmitted parent ion, m/z of the monitored product ion, fragmentor voltage, and collision energy. The ribonucleosides were resolved a Synergi Fusion-RP C18 column (100 × 2.0 mm, 2.5 µm, Phenomenex, Torrance, CA, USA) with a gradient starting with 100% phase A (5 mM ammonium acetate, pH 5.3), followed by 0–10% phase B (acetonitrile) 0–10 min; 10–40% phase B, 10–14 min; 40–80% phase B, 14–15 min; 80–90% phase B, 15–15.1 min; 90% phase B, 15.1–18 min at 35 °C and a flow rate of 0.35 mL/min. The HPLC column was coupled to an Agilent 6490 Triple Quad mass spectrometer with an electrospray ionization source in positive mode with the following parameters: gas temperature, 200 °C; gas flow, 11 L/min; nebulizer, 20 psi; sheath gas temperature, 300 °C; sheath gas flow, 12 L/min; capillary voltage, 1800 V. MRM mode was used for detection of product ions derived from the precursor ions for all the modified ribonuleosides. Quantitative analysis was performed by normalizing MS signals by UV signals of canonical ribonuleosides.

Protein precipitation by methanol was applied for preparation of LC-MS/MS samples. Briefly, plasma sample (50 μL) was mixed with 200 μL of methanol containing 10 ng of IS. The mixture was centrifuged at 10,000 g for 10 min and 10 μL of supernatant then injected for analysis by the LC-MS/MS system. Validation of the bioanalytical method of bioactive triterpenoid determination was based on previous study by our groups [26 (link)]. In brief, LC-MS/MS was performed on a Nexera ultra high-performance liquid chromatograph and 8060 triple quadrupole mass spectrometers (Shimadzu Co., Ltd., Japan). The stationary phase was Synergi Fusion-RP C18 column (Phenomenex Inc., USA) with 40 °C oven temperature. The mobile phase was 100% methanol and 0.2% formic acid in water with gradient elution. The flow rate was 0.5 mL/min and the volume of injection 10 μL. The analysis was conducted in a negative mode with mass-to-charge ratios of madecassoside, asiaticoside, and glycyrrhetinic acid of 973.40/503.30, 957.40/469.20, and 469.35/409.40, respectively.

An Agilent^® 1050 HPLC system (Waldbronn, Germany) was used to determine the efficiency in removing CBZ, KET and DCF in selective pressure experiments (Section 2.2.2) and in degradation experiments by microbial isolates (Section 2.4.2). This system was provided with a diode array detector (DAD; 190–700 nm) and a Synergi Fusion RP C18 column (4 μm, 4.6 Å~150 mm; Phenomenex^®, Madrid, Spain). A volume of 10 μL of each sample was injected at a gradient flow rate of 0.9 mL/min, using a buffer of 85% of acetonitrile and 15% of H₂O-H₃PO₄ (0.01%) for elution. Peak areas from determined absorbance at 278 nm were interpolated into a generated standard curve using 10–100 μM from each compound.

Anthracene was extracted from the plant tissues using a modified protocol of [60 (link)]. Briefly, dry plants were homogenised with a mixture of n-hexane–acetone (2:1 v/v) followed by three sonication cycles (30 min at 60 °C). The samples were mixed with 6 mL of HPLC water and vortexed during 1 min. The upper n-hexane layer was pipetted out and evaporated using a rotary evaporator at 40 °C (Heidolph Rotary Evaporator, Laborota 4000). The final residue was re-suspended in acetonitrile and analysed using a High Performance Liquid Chromatograph (Agilent ^®, Hewett-Packard 1050, Palo Alto, CA, USA) equipped with a diode array detector (DAD; 190–700 nm). Separation of the compounds were carried out using a Synergi Fusion RP C18 column (4 μm particle size, 4.6 mm internal diameter × 150 mm length; Phenomenex^®, Madrid, Spain) in isocratic mode (85% B) using B: acetonitrile and A: water (0,01% phosphoric acid, pH 2) as mobile phase. The temperature of the column was set at 23 °C and the flow rate was maintained at 0.9 mL min⁻¹. Anthracene was quantified at 251 nm. Results were expressed as mmol PAH mg⁻¹ dry weight.

Both compounds (DFC/BPA) were measured by HPLC employing an Agilent^® 1050 HPLC system (Waldbronn, Karlsruhe, Germany) equipped with a diode array detector (DAD; 190–700 nm) and a Synergi Fusion RP C18 column (4 μm, 4.6 Å~ 150 mm; Phenomenex^®, Madrid, Spain). Ten microliters of each sample (DFC/BPA) was injected. For DFC analyses, an isocratic flow rate of 0.9 mL min^-1 and a buffer acetonitrile/water ratio of 85:15 (containing H3PO4 0.01%) were used for elution. For BPA, an isocratic flow rate of 1 mL min^-1 and a buffer acetonitrile/water ratio of 65:35 (containing H₃PO₄ 0.01%) were used. Peak areas of UV detection at 278 nm were interpolated into a generated standard curve using 10–100 μM. The removal rate was calculated according to Olicón-Hernandez et al. (2019) [31 (link)].

CPu tissue block in experiment 2 was weighed and homogenized in 80% acetonitrile containing 0.1% formic acid (5 μL). The homogenates were centrifuged at 14,000g for 10 min at 4°C. The supernatants were collected and stored at −80°C. DA, 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) were determined by the use of LC-MS/MS. LC separation was performed on the Agilent 1200 LC system (Agilent, Santa Clara, USA) using a Synergi Fusion-RP C18 column (50 mm × 3.0 mm, 4 μm) provided by Phenomenex. MS/MS detection was carried out using the 3200 QTRAP LC-MS/MS System (Applied Biosystems, Foster City, CA, USA). The multiple-reaction monitoring mode was used for the quantification. The principal validation parameters of the LC-MS/MS were set up as showed in Table 1.