Ergosterol was extracted from silage according to the method described by (Rousk and Bååth, 2007 ). Ground material (0.5 g DM) was mixed with 2 ml cyclohexane and 8 ml 10 % KOH which was vortexed for 10 s. Sonication was carried out in a heated water bath at 25°C for 25 min before incubating at 70°C for 90 min in a heat block and addition of 2 ml MQ. An additional 2 ml cyclohexane was added before a 5 min centrifugation (2284 × g). The upper phase of cyclohexane was extracted into a test tube, and then, extraction was repeated a further two times with 4 ml cyclohexane to the remaining sample, vortexed for 10 s and centrifuged at 2285 × g for 5 min; then, upper phase was extracted into the same test tube as the first. The extracted cyclohexane was evaporated until dry under N2 at 40°C and stored at 4°C in the dark. Samples were re‐suspended by adding 750 µl MeOH with glass syringe and vortexed to dissolve. Extracts were filtered through 0.45 µm sterile syringe filters (VWR international Ltd, Lutterworth, Leicestershire) syringe and run on HPLC Agilent Zorbax Rx‐C18 Column part number 866967‐902 at 1 min ml−1 flow rate (Mobile phase H2O:MeOH: 2:98 UV Absorption 282 nm).
Zorbax rx c18 column
Manufactured by Agilent Technologies
Sourced in Australia, United Kingdom, United States
The ZORBAX RX-C18 column is a reversed-phase high-performance liquid chromatography (HPLC) column used for the separation and analysis of a wide range of organic compounds. It features a silica-based stationary phase with octadecylsilane (C18) bonding, providing high-efficiency, reproducible separations.
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Lab products found in correlation
Sterile syringe filter, by Avantor (1 mentions)
1100 infinity, by Agilent Technologies (1 mentions)
Supelguard discovery c18 guard column, by Merck Group (1 mentions)
1260 infinity 2 lc system, by Agilent Technologies (1 mentions)
Ltq mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Xcalibur 3, by Thermo Fisher Scientific (1 mentions)
9 protocols using zorbax rx c18 column
1
Ergosterol Extraction from Silage
2
Quantitative Analysis of Hyaluronic Acid
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Quantitative analysis of HA was performed as previously described [13 (link)]. Briefly, 1 mL of the fermentation supernatant was collected and freeze-dried. The precipitate formed was resuspended with 1 mL methanol for HA extraction and the crude samples were subjected to HPLC analysis (Agilent Technologies 1100 Infinity) under the following conditions: C18 reversed-phase column [Agilent ZORBAX RX-C18 column (250 × 4.6 mm)] eluted with methanol/H2O (A/B) (1 mL/min, 30–90% A in 30 min, followed by 90–100% A in 10 min). The peak areas were used to quantify the production of HA according to the standard sample. To quantify HA more precisely, the extracted samples were analyzed by liquid chromatography-mass spectrometry (LC–MS).
3
HPLC Analysis of Organic Complexes
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An Agilent (Melbourne, VIC, Australia) Technologies 1260 Infinity instrument equipped with a Phenomenex OnyxTM Monolithic C18 reverse-phase column (100 4.6 mm, 5 µm pore size) (Sydney, NSW, Australia) was utilised for the complexes. The mobile phase consisted of solvents, A (0.06% TFA in d.i.H2O) and B (0.06% TFA in CH3CN/d.i.H2O (90:10)). An injection volume of 5 µL was utilised and eluted with a 0–100% linear gradient over 15 min with a flow rate of 1 mL.min−1, at the set wavelengths of 214 and 254 nm. An Agilent ZORBAX RX-C18 column (100 4.6 mm, 3.5 µm pore size) (Sydney, NSW, Australia) was utilised for the NHS ester of 5B3A using the same method described above.
4
Quantifying Thaxtomin A Production in S. scabies
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Mycelial suspensions of S. scabies strains (wild-type and different isolates of each mutant) were prepared from 48–72 h-old TSB-grown cultures by pelleting the mycelia, washing twice with sterile water, and resuspending in sterile water to an A600 of 1.0. Samples of 50 μl were plated out on small Petri dishes (5-cm diameter) containing 12.5 ml OBA or ISP-4 medium.
After incubation for 7 days at 28 °C, the medium was chopped into small cubes and soaked in 8 ml of methanol for 10 min. The supernatant was filtered through a 0.2-μm polytetrafluoroethylene (PTFE) filter and analyzed via HPLC on a Zorbax RX-C18 column (5 μm, 4.6 × 250 mm, Agilent Technologies) with a 1 ml/min flow rate of an isocratic mobile phase of 40:60 acetonitrile:water. Thaxtomin A was detected by measuring the absorbance at 380 nm. All experiments were repeated using different biological replicates of the Streptomyces strains, with three technical replicates per strain.
After incubation for 7 days at 28 °C, the medium was chopped into small cubes and soaked in 8 ml of methanol for 10 min. The supernatant was filtered through a 0.2-μm polytetrafluoroethylene (PTFE) filter and analyzed via HPLC on a Zorbax RX-C18 column (5 μm, 4.6 × 250 mm, Agilent Technologies) with a 1 ml/min flow rate of an isocratic mobile phase of 40:60 acetonitrile:water. Thaxtomin A was detected by measuring the absorbance at 380 nm. All experiments were repeated using different biological replicates of the Streptomyces strains, with three technical replicates per strain.
5
HPLC Analysis of Tocols in Grains
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Tocols in the grain, digesta, and micellar fraction were identified and quantified using a SpectraSystem HPLC instrument (Thermo Separation Products, Inc., Waltham, MA, USA) equipped with a quaternary gradient pump (P4000), an autosampler (AS3000), and an FL detector (FL3000). Tocol separation was achieved with the isocratic elution of the mobile phase on two sequentially connected C18 columns: a Vydac 201TP54 column (5 µm, 4.6 × 150 mm; Hichrom, Reading, UK) and a Zorbax RX-C18 column (5 µm, 4.6 × 150 mm; Agilent Technologies, Santa Clara, CA, USA). The aforementioned columns were protected by a Supelguard Discovery C18 guard column (5 µm, 4 × 20 mm; Supelco, Bellefonte, PA, USA). The mobile phase used was an acetonitrile:dichloromethane:methanol (75:20:5, v/v/v) solution containing 0.1% BHT and 0.05% triethylamine. The flow rate was 1.8 mL/min and 30 µL of the sample was injected. The tocols were monitored at an extinction of 290 nm and an emission of 330 nm.
Tocols were identified by comparing their retention times and were quantified via external standardization with calibration curves, using commercially available standards (Sigma-Aldrich, St. Louis, MO, USA; purity ≥ 96%; r2 ≥ 0.98 for all tocols). The total tocol content was calculated by summing the contents of the individual tocols.
Tocols were identified by comparing their retention times and were quantified via external standardization with calibration curves, using commercially available standards (Sigma-Aldrich, St. Louis, MO, USA; purity ≥ 96%; r2 ≥ 0.98 for all tocols). The total tocol content was calculated by summing the contents of the individual tocols.
6
Quantitative Analysis of JA and ABA in Leaves
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The JA and ABA levels in the leaves were estimated using Liquid Chromatography-Mass Spectrometry/Mass-Spectrometry (LC-MS/MS) analysis. About 50 mg leaf tissue was homogenized in 500 µl solution of propanol, water and conc. HCl (2: 1: 0.002 vol/vol). The samples were vigorously shaken on a shaker at 4°C for 30 min. One ml dichloromethane was added to each sample followed by shaking for 30 min at 4°C. The mix was then centrifuged at 10000 rpm at 4°C. The lower phase was filtered and used for quantitative analysis of JA and ABA (Pan et al., 2010 (link)). The analysis was performed in Agilent Q-TOF LC/MS 6520 series system (Agilent Technologies, U.S.A.) with ZORBAX RX-C18 column (4.6×150 mm, 5 µm, Agilent) at 24°C. The mobile phase used was water with 0.1% formic acid (solution A) and methanol with 0.1% formic acid (solution B) on a gradient elution mode with 2 µl injection volume and 0.4 ml/min flow rate. The gradient elution program used was: 1% B at 0 min to 11 min, 40% B up to next 2 min, 70% B for next 2 min, 99% B for next 1 min and then again 1% B for another 4 min. The detected mass range was 100-2000 m/z.
7
Quantification of β-apo-10'-carotenal via ScBCO2 Conversion
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The ScBCO2-catalyzed conversion of β-carotene to β-apo-10′-carotenal was followed by HPLC using a Zorbax RX-C18 column (4.6 × 250 mm, 5 μm particle size, Agilent Technologies). The column was eluted with methanol/water (7:3 v/v) containing 0.1 % ammonium acetate (solvent B) and methanol (solvent A). The gradients were as follows: 100 % B to 0 % B over 12 min; 0 % solvent B for 8 min; and then 100 % A for 8 min at a flow rate of 0.5 ml/min. The reaction products were characterized by LC–MS. For quantification, a curve correlating peak area to moles of the reaction product was attained with β-apo-10′-carotenal as standard sample. A concentrated stock was prepared by dissolving β-apo-10′-carotenal in chloroform at 100 mg/l. This was diluted in chloroform to generate standards at 1, 2, 3, and 4 mg/l. The stocks were stored at −20 °C. Sample and standards were analyzed using an LTQ XL ultra-HPLC-ion trap MS equipped with an atmospheric pressure chemical ionization ion source (Thermo Electron Corp, USA). Mass fragmentation spectra were monitored for masses in the range of 50–800 atomic mass units on the LC–MS system. The chromatography conditions used for the LC–MS analysis were identical to those described above for the HPLC analysis.
8
Quantitative Analysis of Cannabinoids
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The LC-ESI-MS system used in this study was a modular Agilent 1260 Infinity II LC system comprised of the following components: a vacuum degasser, a quaternary pump (G7111B), an autosampler (G7129A), an integrated column compartment (G7130-60030), and a single quadrupole liquid chromatography/mass selective detector (LC/MSD 6125B) with electrospray ionization (ESI) (C1960-64217). The chromatographic separation of cannabinoids was performed on an Agilent Zorbax RX-C18 column (4.6 mm × 150 mm, 3.5 μm). The mobile phase was composed of 0.2% aqueous formic acid (A) and methanol (B). Gradient elution was as follows: 75–90% B in 0–13 minutes and 90% B in 13–26 minutes. The post-run time was 4 minutes. The flow rate was 0.6 mL/min. The column temperature was set at 30 °C. The injection volume was 5 µL. The ESI-MS system was operated in positive ionization mode. Mass to charge ratios (M/z) of fragment ions for each compound were listed in Supplementary Table 18 . The instrument settings were set as follows: the capillary voltage was 3 kV, the nebulizer (N2) pressure was 50 psi, the drying gas temperature was 350 °C, the drying gas flow was 12 L/min, and the fragmentor voltage was 70 V.
9
Structural Characterization of HN Compound
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1) 1H NMR analysis of HN
The structure of HN was determined using 1H nuclear magnetic resonance (1H NMR, Mettler Toledo, Switzerland). Briefly, 20 μL of HN (50 mg/mL) was dissolved in 0.5 mL of deuterium DMSO and placed into a nuclear magnetic tube with a diameter of 5 mm. The hydrogen spectrum data for HN were collected using 1H NMR with the following parameters: each sample was scanned 64 times, the acquisition time was 2.9089 s, the working frequency was 400.05 MHz, and the spectral width was 8221.82 Hz.
2) LC-MS/MS analysis of HN
LC-MS/MS was carried out on an Agilent 1100 LC system (Santa Clara, CA, USA) coupled to an LTQ mass spectrometer (Thermo, MA, USA). Samples were separated in an Agilent ZORBAX RX-C18 column (4.6 mm × 250 mm, 5 μm, 100 Å) at a flow rate of 0.4 mL/min with an 80% mobile phase B in 20 min. Mobile phase A was 0.1% acetic acid solution in water. The injection sample volume was 5 μL, and the column was kept at 25°C. The LC eluent was directed to the MS for a full scan (50–1000 m/z) in positive ionization mode. The MS parameters were set as follows: capillary temperature of 325°C, source current of 4 μA, source voltage of 3.5 kV, aux gas flow rate of 6 arbitrary units (arb), sheath gas flow rate of 25 arb, and isolation width of 1.5 m/z. The MS data were analyzed using Xcalibur 3.0 software (Thermo, USA).
The structure of HN was determined using 1H nuclear magnetic resonance (1H NMR, Mettler Toledo, Switzerland). Briefly, 20 μL of HN (50 mg/mL) was dissolved in 0.5 mL of deuterium DMSO and placed into a nuclear magnetic tube with a diameter of 5 mm. The hydrogen spectrum data for HN were collected using 1H NMR with the following parameters: each sample was scanned 64 times, the acquisition time was 2.9089 s, the working frequency was 400.05 MHz, and the spectral width was 8221.82 Hz.
2) LC-MS/MS analysis of HN
LC-MS/MS was carried out on an Agilent 1100 LC system (Santa Clara, CA, USA) coupled to an LTQ mass spectrometer (Thermo, MA, USA). Samples were separated in an Agilent ZORBAX RX-C18 column (4.6 mm × 250 mm, 5 μm, 100 Å) at a flow rate of 0.4 mL/min with an 80% mobile phase B in 20 min. Mobile phase A was 0.1% acetic acid solution in water. The injection sample volume was 5 μL, and the column was kept at 25°C. The LC eluent was directed to the MS for a full scan (50–1000 m/z) in positive ionization mode. The MS parameters were set as follows: capillary temperature of 325°C, source current of 4 μA, source voltage of 3.5 kV, aux gas flow rate of 6 arbitrary units (arb), sheath gas flow rate of 25 arb, and isolation width of 1.5 m/z. The MS data were analyzed using Xcalibur 3.0 software (Thermo, USA).
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