The constituents of the studied extracts were separated on the on Gemini® column (3 μm i.d. C18 with TMS endcapping, 110 Å, 100 × 2 mm) guarded with pre-column (Phenomenex Inc, Torrance, CA, USA) following chromatographic conditions previously described [59 (link)]. The separation was performed on Agilent 1200 Infinity HPLC (Agilent Technologies, Santa Clara, CA, USA), whereas detection was obtained on Agilent 6530B QTOF (Agilent Technologies, Santa Clara, CA, USA). 2 mass spectra/s were registered in a scan range 100–1700 m/z for MS and MS/MS, applying collision energy of 10 and 40 eV. The drying gas temperature and flow were set at 300 °C and 12 L/min, whereas the sheath gas temperature and flow were set at 325 °C and 12 L/min, respectively. The ion source operated in negative mode with 40 psig, capillary V (+): 4000 V, skimmer 65 V. Freely available mass spectra databases (MassBank, PubChem, HMDB, Metlin, MoNA) were used for tentative identification of compounds, which was also supported by fragmentation patterns published in scientific literature.
Pre column
Manufactured by Phenomenex
Sourced in United States
A pre-column is a small column that is placed in front of the main analytical column in a high-performance liquid chromatography (HPLC) or gas chromatography (GC) system. Its primary function is to protect the main column from particulates, contaminants, and other substances that could potentially damage or reduce the lifespan of the main column.
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Lab products found in correlation
Luna c18 2 column, by Phenomenex (2 mentions)
Lcq fleet ion trap mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Lcq fleet system, by Thermo Fisher Scientific (2 mentions)
Surveyor ms pump autosampler pda detector, by Thermo Fisher Scientific (2 mentions)
Gemini c18 110a analytical column, by Phenomenex (2 mentions)
Agilent 1200 infinity hplc, by Agilent Technologies (1 mentions)
Agilent 6530b qtof, by Agilent Technologies (1 mentions)
Reversed phase x select hss t3 column, by Phenomenex (1 mentions)
0.45 m ptfe syringe filters, by Thermo Fisher Scientific (1 mentions)
Thermo excalibur 2, by Thermo Fisher Scientific (1 mentions)
Rezex roa organic acid h column, by Phenomenex (1 mentions)
Ultimate 3000 hplc system, by Thermo Fisher Scientific (1 mentions)
Hplc agilent 1 200 series, by Agilent Technologies (1 mentions)
Chlorogenic acid, by Merck Group (1 mentions)
P coumaric acid, by Merck Group (1 mentions)
Caffeic acid, by Merck Group (1 mentions)
Gemini c18 reverse phase column, by Phenomenex (1 mentions)
Agilent 1100, by Agilent Technologies (1 mentions)
Gemini c18 column, by Phenomenex (1 mentions)
Ptfe syringe filter, by Merck Group (1 mentions)
14 protocols using pre column
1
High-Resolution HPLC-QTOF Analysis of Plant Extracts
2
Targeted LC-MS/MS Analysis of Compounds
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HPLC separation was accomplished using a (Waters) reversed-phase X select HSS T3 column (2.1×150 mm, 2.5 µm), a (Phenomenex) precolumn, and in-Line filter disks (0.5 µm × 3.0 mm). Adopting conditions described by [17 (link)]. PeakViewTM software was used to compare retention time and m/z values obtained by MS and MS2 to identify compounds. PeakViewTM software’s XIC Manager was used to calculate peak area values. For each targeted analyte, extracted ion chromatograms (XICs) were automatically generated and compared to a user-defined threshold [51 (link)]. The LC-MS/MS analysis was carried out in Proteomics and Metabolomics Unit, Children’s Cancer Hospital (57357).
3
PLA Quantification in Microbial Cultures
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For PLA quantification in microbial cultures in liquid Ym synthetic medium, 1 mL of culture media was withdrawn at each sampling time and filtrated through 0.45 µm PTFE syringe filters (Thermo Fisher Scientific) to eliminate microorganisms from the supernatant before injection to HPLC apparatus. Regarding micro-malting assays, PLA was quantified in the same extracts as for the T-2 toxin quantification. Analyses of PLA were performed using a Luna C18(2) column (5 µm, 250 × 4.6 mm) and a pre-column with the same characteristics (Phenomenex). The PLA was detected and quantified using HPLC-DAD according to the methodology previously described by Kawtharani et al. [20 (link)]. PLA quantification was calculated according to a standard calibration curve with concentrations ranging between 0.01 and 1 g/L. In micro-malting experiments, PLA concentrations are expressed in g/g.
4
Comprehensive LC-MS/MS Analysis of Metabolites
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The analyses were performed using a Thermo Scientific LCQ FLEET system consisting of an LCQ FLEET ion trap mass spectrometer, a Surveyor MS Pump/Autosampler/PDA Detector (Thermo Fisher Scientific, Waltham, MA, USA) through an ESI source. The separation was obtained by using a Gemini® C18 110 A analytical column (150 × 2.00 mm i.d., 5 μm) and the pre-column (Phenomenex, Torrance, CA, USA). The mobile phase consisted of aqueous formic acid at 0.1% and acetonitrile (solvent B) at 0.3 mL/min (the injection volume was 10 µL). A linear solvent gradient was used as follows: from 10% B to 95% B in 25 min with a final plateau of 3 min at 100% B. The ion trap operated in data-dependent, full scan (60–2000 m/z), and MSn mode to obtain fragment ions m/z with a collision energy of 35% and an isolation width of 3 m/z. The negative and positive parameters of the ion mode ESI source have been optimized to an ionization voltage of 5.0 kV, a capillary temperature of 320 °C, a capillary voltage of 32 V, a sheath gas flow rate of 25 arbitrary units (AU), and an auxiliary gas flow rate of 10 AU. Data was acquired using Thermo Excalibur 2.2 software (Thermo Fisher Scientific, MA, USA).
5
HPLC Analysis of Metabolite Profiles
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Supernatants from cultures were syringe filtered (0.2 µm) before analysis using HPLC. Samples from the successive cultivations in microplate were analyzed using an HPLC Agilent 1,200 Series (Agilent) equipped with an Aminex HPX-87H column coupled with a pre-column (Bio-Rad). The other samples were analyzed using either Vanquish or Ultimate 3000 HPLC system (Thermo Scientific) equipped with a REZEX ROA-Organic Acid H+ column coupled with a pre-column (Phenomenex). For all experiments, the eluent solution was 5 mM H2SO4, running at 0.5 mL/min for 40 min at 50°C. Compounds were detected by an RI detector and quantified from standard curves using the Chromeleon software.
6
Quantification of Phenolic Acids in Blue Honeysuckle
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The HPLC with DAD detector and Luna 5 µm C18(2), 250x4.6 mm column with precolumn (Phenomenex, Torrance, California, USA) were used to analyze the content of phenolic acids in juices from blue honeysuckle berry fruits. The mobile phase of flow rate of 1 mL/min consisted of 5 % HCOOH (Eluent A) and 100 % ACN (Eluent B), both of the HPLC purity. The following linear gradient was set: 0–30 min 10–25 % B, 30–33 min 25–50 % B, 33–38 min 50 % B, 38–40 min 50–10 % B, 40–55 min 10 % B. The temperature in column oven was equal to 25 °C. The peaks were analyzed at 320 nm based on the standards from chlorogenic acid, caffeic acid and p-coumaric acid (Sigma-Aldrich). Determination was carried out in three repetitions and the results were expressed in mg/100 mL. The sum of individual phenolic acids was presented as total phenolic acids (TPA) [5] (link).
7
Metabolite Separation by HILIC-LC-MS/MS
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Resuspended metabolites were separated by HILIC with a Gemini reverse-phase C18 column (50 mm × 4.6 mm with 5 μm diameter particles) from Phenomenex together with precolumn (C18, 3.5 mm, 2 mm × 20 mm). Mobile phase A was composed of 100% H2O (10 mmol/L tributylamine aqueous solution, adjusted to pH 4.95 with 15 mmol/L acetic acid), and mobile phase B was composed of 100% methanol. We used a multistep gradient with buffer A and B as follows: from 0 to 5 minutes, 95% buffer A; from 5 to 15 minutes, linear change from 95% to 90% buffer A; from 15 to 22 minutes, linear change from 90% to 85% buffer A; from 22 to 26 minutes, 10% buffer A; and from 30 to 33 minutes, 95% buffer A, and maintained for 7 minutes. The flow rate was 0.2 mL/minute for 0 to 15 minutes and 30 to 40 minutes, and 0.3 mL/minute for 15 to 30 minutes. Targeted MS/MS analysis was performed on an Agilent triple quadrupole LC-MS/MS instrument (Agilent Technologies 6460 QQQ). The capillary voltage was set to 4.0 kV. The drying gas temperature was 350°C, the drying gas flow rate was 10 L/minute, and the nebulizer pressure was 45 psi. Relative metabolite abundance was quantified by integrated peak area for the given MRM transition. Data presented are representative of 3 independent biological experiments each containing 3 technical replicates for a given condition.
8
Quantitative HPLC Analysis of BU Epimers
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The analysis was carried out by HPLC-DAD (Agilent 1100, Agilent, Santa Clara, CA, USA) using an RP-C18 column (LC 150 × 4.6 mm, 5 µm) with pre-column (Phenomenex, Torrance, CA, USA). The mobile phase was composed of phosphate buffer pH 3.2:acetonitrile:ethanol [29 (link)]. The elution was carried out in gradient (Table 2 ). The flow rate was 1.7 mL/min, the wavelength was set at 240 nm and the injection volume at 10 µl. The retention times of the 22R and 22S epimers were 8.8 min and 9.1 min, respectively. A calibration curve was prepared twice solubilizing in ethanol 10 mg of BU in a 100 mL volumetric flask. Dilution to obtain 0.1, 0.2, 0.5, 1 and 2 μg/mL concentration were performed (22R: R2 = 0.99930; 22S: R2 = 0.99999).
9
Analysis of Polar Lipid Fractions
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The polar fractions obtained above were analysed using high-performance size exclusion chromatography (HPSEC) following the method of Dobarganes et al. [16 (link)]. Separation was performed on a liquid chromatographic system, Varian (Paolo Alto, CA, USA), consisting of a ternary pump (230 Pro-Star) and an autosampler (430 Pro-Star). Components were separated on Phenogel columns SEC/GPC (300 × 7.8 mm; 100 and 500 Å) that were connected in series, and were equipped with a pre-column (50 × 7.8 mm) (Phenomenex, USA). An RI detector was used (Knauer, Germany). Using this system, the following compounds were separated and quantified: triglyceride polymers and dimers (TGP and TGD), oxidized triacylglycerols (oxTAG), diacylglycerols (DAG) and free fatty acids (FFA).
10
Quantification of T-2 Toxin by HPLC-DAD
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After the incubation period, cultures were filtrated with Nalgene™ Rapid-Flow™ Filters of 0.45 μm pore size (Thermofischer Scientific, Waltham, MA, USA) to remove microorganisms. Filtrates were then extracted with 70 mL of ethyl acetate and shaken on a Universal Shaker SM 30 B Control Edmund Bühler® (Thermofischer Scientific, Waltham, MA, USA) set at 150 rpm overnight. The organic phase was recovered and evaporated until dry under a rotavapor set at 60 °C. Samples were resuspended with 2 mL of acetonitrile/water (30/70, v/v) mixture and filtered through 0.45 µm PTFE syringe filters (Sigma Aldrich, St. Quentin Fallavier, France). Samples were conserved at 4 °C until further analysis. T-2 toxin was analyzed by Gemini C18 columns, 150 mm × 4.6 mm, 3 μm and a pre-column with the same characteristics (Phenomenex). As for PLA, T-2 toxin was detected and quantified using HPLC-DAD (Dionex, Sunnyvale, CA, USA) according to the methodology described by Medina et al. [59 (link)]. T-2 toxin quantification was calculated according to a standard calibration curve with concentrations ranging between 0.2 and 50 μg/mL.
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