Spherisorb ods2 column

DXA₃ was purified from lipid extracts of thrombin-activated platelets or COX-1 reactions using HPLC/UV on a Spherisorb ODS2 column (5 μm, 150 × 4.6 mm; Waters) with a gradient of 20–42.5% solvent B (acetonitrile, 0.1% formic acid) in solvent A (water, 0.1% formic acid) over 50 min, 42.5–90% solvent B from 50 to 60 min, 90% solvent B from 60 to 64.5 min, 90 to 20% from 64.5 to 65.5 min, and 20% solvent B from 65.5 to 75 min with a flow rate of 1 ml·min⁻¹, and fractions collected at 30-s intervals. DXA₃-containing fractions were identified using MS using m/z 351.2 → 165.1, and then H₂O was removed using Sep-Pak C18 cartridge purification (Waters). DXA₃ was stored in methanol at −80 °C, prior to derivatization and GC/MS analysis.

The nanoemulsions (500 μL) were freeze-dried and dissolved in 1.5 mL ethanol with sonication for 1 h. The solution was filtered through a 0.45 μm syringe filter, and the amount of lipiodol was determined at 260 nm using UV-visible absorption spectrophotometry (UV-1601, Shimadzu, Kyoto, Japan). As for the loading yield and efficiency of paclitaxel, the nanoemulsions (1 mL) were freeze-dried and dissolved in 1 mL acetonitrile with sonication for 1 h. The solution was filtered through a 0.45 μm syringe filter, paclitaxel in the filtrate was determined using reversed phase HPLC (Agilent 1100 series, Agilent Technologies, Palo Alto, USA) equipped with a Waters Spherisorb ODS2 column (C18, 5 μm, 4.6 mm × 250 mm). Acetonitrile was used as an isocratic mobile phase at a rate of 1 mL min⁻¹. The absorption of the eluted solution was determined at 227 nm using a standard curve of paclitaxel in acetonitrile.

The secreted amino acids (Asp, Glu, Gly and GABA) were analysed by HPLC performed following the procedure described by Márquez and coworkers [40] (link). Cells, after 10 culture days, were washed twice with 1 ml of Locke medium. The medium was removed and cells were stimulated for 15 min at 37°C with 0.5 ml of fresh Locke medium containing the different SNAP concentrations indicated in each case. After stimulation, we followed these steps: (i) the solution containing the released amino acids were collected (ii) the cells were lysed by adding 0.5 ml of distilled water and this suspension, containing the unreleased neurotransmitters, was centrifuged at 13000×g for 5 min. Supernatants were collected out.
The amino acid concentrations were determined by reverse-phase high-performance liquid chromatography using a precolumn derivation with dansyl chloride and UV detection at 254 nm. Peaks were integrated using a Spectraphysic integrator and then quantified and compared with standards for these neurotransmitters. The separation of dansyl derivatives was carried out using a Waters Spherisorb ODS 2 column (5 μM particle size; 15×0.46 cm).
Results were expressed as % of amino acid release as compared to the total amino acid content (amino acid in the medium plus amino acid inside the cells).

[¹⁴C]AA was oxidized using COX-1 as described for unlabeled AA above. The amount of [¹⁴C]DXA₃ was determined by comparison with a [¹⁴C]AA standard curve analyzed using LC separation with radiochemical detection (Berthold Technologies) using a Spherisorb ODS2 column (5 μm, 150 × 4.6 mm; Waters) with a gradient of 20–42.5% solvent B (acetonitrile, 0.1% formic acid) in solvent A (water, 0.1% formic acid) over 50 min, 42.5–90% solvent B from 50 to 60 min, 90% solvent B from 60 to 64.5 min, 90 to 20% from 64.5 to 65.5 min and 20% solvent B from 65.5 to 75 min with a flow rate of 1 ml·min⁻¹, and fractions were collected at 30-s intervals for LC/MS/MS confirmation of [¹⁴C]DXA₃. The same gradient was also used for LC/MS/MS detection of [¹⁴C]DXA₃ (m/z 353.2 → 165.1).

Microbial dry biomass was estimated gravimetrically in duplicate. Each sample of 50 mL was filtered through quantitative filter paper (VWR International, Leuven, The Netherlands). The fungal pellets were dried at 60 °C for 48 h. The samples were placed in a desiccator for 60 min and weighted afterwards. The concentration of biomass was calculated as grams of dry biomass per liter of medium. To determine glucose consumption in fungal cultures, the glucose concentration was measured using the commercial kit D-Glucose GOD-POD (NzyTech, Lisbon, Portugal).
APAP concentrations were quantified in the fungal culture supernatants by using a high-performance liquid chromatography system L-7100 (LaChrom HPLC System, Merck, Darmstadt, Germany) consisting of a quaternary gradient pump, and L-7400 UV detector. The whole system was controlled using the Merck HPLC System Manager software. The separation of the analytes was performed at ambient temperature in a Waters Spherisorb ODS2 column (5.0 µm, 4.6 × 250 mm) (Waters, Milford, MA, USA), using an isocratic condition with a mobile phase composed of water (pH 3.5, adjusted with orthophosphoric acid) and acetonitrile, at a 90:10 (v/v) and a flow rate of 1.0 mL/min. Detection was performed at 254 nm. Under these conditions, APAP, HQ, CAT, and APA could be separated within 12 min.

For residue analysis, plant samples (rice leaves, straw, grains, husk, and soil) were collected from the plants on which morphological data were recorded. Samples (in triplicate) that received maximum concentration treatment were considered for the study. After harvesting the plants, straws, leaves, and grains were separated. Furthermore, husks were removed from grains by hand hulling. Samples were grounded to a fine powder, and a total of 1.0 g of the powdered tissue was extracted in 10 ml of 2% alkaline water (200 mg NaOH in 10 ml water) for 2 h, followed by portioning of the extracts using dichloromethane. The dichloromethane extracts were then dried and later dissolved in methanol. Before HPLC injection, solutions were filtered through 0.45 μm membranes (Millipore, Billerica, MA). For extraction of calliterpenone, the HPLC analysis was performed as per the method described by our group (Verma et al., 2009 (link)) using an LC-10A HPLC system (Shimadzu, Japan) with acetonitrile:water (45:55) as mobile phase and detection was made at 220 nm using a Waters Spherisorb ODS-2 column (250 × 4.6 mm I.D., 10 μm).