Fungal mycelia were collected from the cellophane surface using a scraper, for weight and aflatoxin analyses. All fungal mycelia from each plate were transferred into a 50 ml tube containing 5 ml of methanol at room temperature, then incubated with continual agitation at 150 rpm, for 30 min. The supernatant was collected by centrifugation at 3,000 g and filtered through a syringe filter (RC 0.22 μm, Alltech). The presence of aflatoxin B1 was determined by HPLC with fluorescence detection, using a Waters 600 series HPLC equipped with a 600 pump, a 2,707 autosampler, and a 600 column thermostat set at 30°C. Detection was performed using a 2,475 Multi λ fluorescence detector set at 365 nm (λex) and 465 nm (λem), with a Waters Empower Windows xp operating system (Waters). The analytical column was a Luna 3u C18 (2) (150 × 4.6 mm, 3 μm) (Phenomenex) preceded by a SecurityGuard TM precolumn (C18, 4 × 3.0 mm, Phenomenex). The mobile phase consisted of methanol: water (55:45), eluted at a flow rate of 0.6 ml/min, with 20 μl of filtered extract injected into the HPLC per run. Aflatoxin B1 production was measured in μg/g of mycelia.
Securityguard precolumn
Manufactured by Phenomenex
Sourced in United States
The SecurityGuard precolumn is a protective device designed to extend the lifespan of analytical columns. It is installed upstream of the analytical column to remove particulates and other contaminants that could potentially damage the column.
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Lab products found in correlation
Agilent liquid chromatography system series 1100, by Agilent Technologies (5 mentions)
3200 triple quadrupole system, by Thermo Fisher Scientific (4 mentions)
2707 autosampler, by Waters Corporation (2 mentions)
Empower windows xp operating system, by Waters Corporation (2 mentions)
Luna 3u c18 2, by Phenomenex (2 mentions)
Integrated hplc system, by Jasco (2 mentions)
As 4050, by Jasco (2 mentions)
Pu 4180, by Jasco (2 mentions)
Fp 920, by Jasco (2 mentions)
F5 column, by Phenomenex (1 mentions)
C18 cartridge, by Phenomenex (1 mentions)
Ultimate 3000, by Thermo Fisher Scientific (1 mentions)
Luna c18 2 column, by Phenomenex (1 mentions)
Chromnav2, by Jasco (1 mentions)
Kinetex evo c18, by Phenomenex (1 mentions)
Kinetex column, by Phenomenex (1 mentions)
Micromass zq spectrometer, by Waters Corporation (1 mentions)
2795 hplc system, by Waters Corporation (1 mentions)
Pierce bca assay kit, by Thermo Fisher Scientific (1 mentions)
Turbo 5 ion source, by AB Sciex (1 mentions)
15 protocols using securityguard precolumn
1
Quantifying Aflatoxin B1 in Fungal Mycelia
2
Quantitative Comparison of Aflatoxin B1 Production
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The method for quantitative comparison of the production of aflatoxin B1 was conducted as previously described with some modification (Han et al. 2018 (link)). A conidial suspension (2×104 spores) was seeded centrally onto sterile cellophane sheets that were placed over a YES agar plate and incubated at 30° for 5 days. The fungal biomass was scraped from the plates, and extracted by incubation with 5 ml of methanol at room temperature with shaking at 150 rpm for 30 min. The supernatant was collected by centrifugation at 3,000 g and filtered through a syringe filter (RC 0.22 μm, Alltech, Deerfield, IL). The presence of aflatoxin B1 was determined by HPLC with fluorescence detection, using a Waters 600 series HPLC equipped with a 600 pump, a 2707 autosampler and a 600 column thermostat set at 30°. Detection was performed using a 2475 Multi λ fluorescence detector set at 365 nm (λex) and 465 nm (λem), with a Waters Empower Windows xp operating system (Waters, Milford, MA, USA). The analytical column was a Luna 3u C18 (2) (150×4.6 mm, 3 μm) (Phenomenex, Torrance, CA, USA) preceded by a SecurityGuard TM precolumn (C18, 4×3.0 mm, Phenomenex). The mobile phase consisted of methanol: water (55:45), eluted at a flow rate of 0.6 ml/min, with 20 μl of filtered extract injected into the HPLC per run. Aflatoxin B1 production was measured in μg/g of mycelia.
3
HPLC-FLD Quantification of Mycotoxin OTA
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OTA was quantified
with the previously reported HPLC-FLD method,48 (link) employing an integrated HPLC system (Jasco, Tokyo, Japan): autosampler
(AS-4050), binary pump (PU-4180), fluorescence detector (FP-920),
and ChromNAV2 software. Briefly, the isocratic elution was performed
with 1.0 mL/min flow rate at room temperature, using sodium borate
buffer (0.01 M, pH 10.0) and ACN (87:13 v/v%) as the mobile phase.
Samples (20 μL) were driven through a SecurityGuard precolumn
(C18, 4.0 × 3.0 mm; Phenomenex, Torrance, CA, US) linked to a
Kinetex EVO (C18, 150 × 4.6 mm, 5 μm; Phenomenex) analytical
column. The fluorescence detection of the mycotoxin was carried out
at 383 and 446 nm excitation and emission wavelengths, respectively.
with the previously reported HPLC-FLD method,48 (link) employing an integrated HPLC system (Jasco, Tokyo, Japan): autosampler
(AS-4050), binary pump (PU-4180), fluorescence detector (FP-920),
and ChromNAV2 software. Briefly, the isocratic elution was performed
with 1.0 mL/min flow rate at room temperature, using sodium borate
buffer (0.01 M, pH 10.0) and ACN (87:13 v/v%) as the mobile phase.
Samples (20 μL) were driven through a SecurityGuard precolumn
(C18, 4.0 × 3.0 mm; Phenomenex, Torrance, CA, US) linked to a
Kinetex EVO (C18, 150 × 4.6 mm, 5 μm; Phenomenex) analytical
column. The fluorescence detection of the mycotoxin was carried out
at 383 and 446 nm excitation and emission wavelengths, respectively.
4
Quantification of Tryptophan and Kynurenine
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Serum levels of tryptophan (trp) and kynurenine (kyn) were evaluated through modified liquid chromatography – tandem mass spectrometry method.
Samples were deproteinized using 50 μl of TCA 4% aqueous solution and following centrifuged at 14,000 rpm for 15 min. Supernatants were injected into chromatographic system to perform separation using an Agilent Liquid Chromatography System series 1100 (Agilent Technologies, USA), on a biphenyl column (100 × 2.1 mm, Kinetex 2.6 μm Biphenyl, 100 Å, Phenomenex, CA, USA) equipped with a security guard precolumn (Phenomenex, Torrance, CA, USA). Gradient elution was performed with a flow rate of 400 μl/min and mobile phases consisted of 0.1% aqueous formic acid and 100% acetonitrile.
The mass spectrometry method was performed on a 3200 triple quadrupole system (Applied Biosystems, Foster City, CA, USA), equipped with a Turbo Ion Spray source. The detector was set in the positive ion mode. The instrument was set in the Multiple Reaction Monitoring mode. Data were acquired and processed by the Analyst 1.5.1 Software.
Samples were deproteinized using 50 μl of TCA 4% aqueous solution and following centrifuged at 14,000 rpm for 15 min. Supernatants were injected into chromatographic system to perform separation using an Agilent Liquid Chromatography System series 1100 (Agilent Technologies, USA), on a biphenyl column (100 × 2.1 mm, Kinetex 2.6 μm Biphenyl, 100 Å, Phenomenex, CA, USA) equipped with a security guard precolumn (Phenomenex, Torrance, CA, USA). Gradient elution was performed with a flow rate of 400 μl/min and mobile phases consisted of 0.1% aqueous formic acid and 100% acetonitrile.
The mass spectrometry method was performed on a 3200 triple quadrupole system (Applied Biosystems, Foster City, CA, USA), equipped with a Turbo Ion Spray source. The detector was set in the positive ion mode. The instrument was set in the Multiple Reaction Monitoring mode. Data were acquired and processed by the Analyst 1.5.1 Software.
5
Serum Metabolite Profiling by LC-MS/MS
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Blood samples were collected from the participants before the second HD session of the week and centrifuged obtaining serum samples and stored at −80 °C until analysis. Sixty microliters of serum were treated by 60 µL of deproteinizing solution. The mixture was mixed for 30 s and centrifuged at 14.000× g rpm for 15 min. 10 µL of clean supernatant was injected into the chromatographic system. Compounds were detected using a LC–MS/MS analytical method [16 (link),17 (link)]. Chromatographic separation of analytes was made utilizing an Agilent Liquid Chromatography System series 1100 (Agilent Technologies, Santa Clara, CA, USA), on a F5 column (Phenomenex, Torrance, CA, USA) equipped with a security guard pre-column (Phenomenex, Torrance, CA, USA). The mobile phase included a solution of 0.1% aqueous formic acid and 100% acetonitrile; elution was realized at a flow rate of 300 μL/min, using an elution gradient, as previously described [17 (link)]. The mass spectrometry was realized on a 3200 triple quadrupole system (Applied Biosystems, Foster City, CA, USA) supplied with a Turbo Ion Spray source, and the detector was set in the positive ion mode, as previously described [17 (link)]. The instrument was set in the Multiple Reaction Monitoring (MRM) mode. Data were collected and analyzed by the Analyst 1.5.1 Software.
6
Carotenoid Analysis by RP-HPLC
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UV/Vis spectra of the whole extracts were recorded between 300 and 600 nm by means of a spectrophotometer Genesys 180.
The analysis of the carotenoids was carried out by reverse-phase high-performance liquid chromatography (RP-HPLC) using a Dionex Ultimate 3000® HPLC system equipped with a quaternary pump and a diode array detector (Dionex, Sunnyvale, CA, USA) as follows: aliquots of extracts (50 μL) were injected into a Luna C18 (2) column (250 × 4.6 mm, 5.0 μm) equipped with a SecurityGuard™ pre-column containing a C18 cartridge (Phenomenex Inc. Castel Maggiore, Bologna, Italy). The separation was performed at a flow rate of 1 mL/min by applying the following linear gradient: from 0 to 100% solvent B in 45 min, 100% solvent B for a further 10 min, and from 100% solvent B to 100% solvent A in 5 min. The mobile phase was degassed ultrapure water/methanol (1:9 v/v) (solvent A) and ethyl acetate/methanol (1:9 v/v) (solvent B). Peak elution was monitored at 490 nm, and the online spectrum of each peak was registered between 300 and 600 nm. The percentage of each carotenoid detected in the extracts was determined by dividing the area of the carotenoid by the sum of the areas of all identified carotenoids.
The analysis of the carotenoids was carried out by reverse-phase high-performance liquid chromatography (RP-HPLC) using a Dionex Ultimate 3000® HPLC system equipped with a quaternary pump and a diode array detector (Dionex, Sunnyvale, CA, USA) as follows: aliquots of extracts (50 μL) were injected into a Luna C18 (2) column (250 × 4.6 mm, 5.0 μm) equipped with a SecurityGuard™ pre-column containing a C18 cartridge (Phenomenex Inc. Castel Maggiore, Bologna, Italy). The separation was performed at a flow rate of 1 mL/min by applying the following linear gradient: from 0 to 100% solvent B in 45 min, 100% solvent B for a further 10 min, and from 100% solvent B to 100% solvent A in 5 min. The mobile phase was degassed ultrapure water/methanol (1:9 v/v) (solvent A) and ethyl acetate/methanol (1:9 v/v) (solvent B). Peak elution was monitored at 490 nm, and the online spectrum of each peak was registered between 300 and 600 nm. The percentage of each carotenoid detected in the extracts was determined by dividing the area of the carotenoid by the sum of the areas of all identified carotenoids.
7
Quantification of Ochratoxin A by HPLC
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An integrated HPLC system (Jasco, Tokyo, Japan) was applied, which is built up from an autosampler (AS-4050), a binary pump (PU-4180), and a fluorescence detector (FP-920). Chromatographic data were evaluated employing ChromNAV2 software (Jasco, Tokyo, Japan). OTA was quantified as it has been recently reported [55 (link)]. Briefly, samples (20 μL) were driven through a SecurityGuard precolumn (C18, 4.0 × 3.0 mm; Phenomenex, Torrance, CA, USA) linked to a Kinetex EVO-C18 (150 × 4.6 mm, 5 μm; Phenomenex) analytical column with 1.0 mL/min flow rate, at room temperature. Isocratic elution was performed, where sodium borate buffer (10 mM, pH 10.0) and ACN (87:13 v/v%) were applied in the mobile phase. OTA was detected at 446 nm (λex = 383 nm). Samples were measured in triplicates.
The linearity of the method was determined between 10 nmol/L and 1.0 μmol/L (4.0–403.8 μg/L) concentrations (R2 = 0.9996). Limit of detection (2 nmol/L or 0.8 μg/L) and limit of quantification (6 nmol/L or 2.4 μg/L) values were defined as the lowest concentrations when the signal-to-noise ratios reached 3 and 10, respectively. The intraday repeatability was evaluated based on the intraday coefficient of variation (0.55%; n = 7).
The linearity of the method was determined between 10 nmol/L and 1.0 μmol/L (4.0–403.8 μg/L) concentrations (R2 = 0.9996). Limit of detection (2 nmol/L or 0.8 μg/L) and limit of quantification (6 nmol/L or 2.4 μg/L) values were defined as the lowest concentrations when the signal-to-noise ratios reached 3 and 10, respectively. The intraday repeatability was evaluated based on the intraday coefficient of variation (0.55%; n = 7).
8
Quantification of SPHINX31 in Mouse Plasma
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Three Dba/2J mice were given i.p. injections of 0.8 mg/kg SPHINX31 and sacrificed after 24 h when blood was taken by cardiac puncture into EDTA tubes. Plasma was isolated by centrifugation, and an equal volume (100 µl) acetonitrile added. An internal standard of 100 µg/ml of a related compound (compound 3 from Batson et al) was added to samples to account for any loss of material during preparation. The solutions were centrifuged for 15 min at 4 °C and the supernatant taken for analysis. Solutions were evaporated at 37 °C for eight hours and resuspended in 30 µl acetonitrile ready for analysis by LC MS, using a Waters 2795 HPLC system. Detection was achieved by positive ion electrospray (ESI + ) mass spectrometry using a Waters Micromass ZQ spectrometer in single ion monitoring (SIM) mode, at 352 m/z units ([M+H]+). Chromatography (flow rate 1 mL·min−1) was achieved using a Phenomenex Kinetex column (2.6 μ, C18, 100 Å, 4.6 × 50 mm) equipped with a Phenomenex Security Guard precolumn (Luna C5 300 Å). Peaks occurring at these times in the SIM chromatograms per compound were integrated using Water MassLynx software. The chromatograms produced clear peaks at the expected molecular weights. The integrated area under the peaks and read from a standard curve led to quantification of the circulating concentration of SPHINX31.
9
Quantifying Endocannabinoids in Lung Tissues
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LC-MRM measurements were performed as described before8 (link). In detail, frozen lung lobes were homogenized in cold extraction tubes with ceramic beads containing spiking solution (deuterated endocannabinoids in acetonitrile), 0.1 M formic acid (buffer) and ethylacetate/hexane (9:1). After homogenization with Precellys 24 samples were centrifuged and kept at −20 °C for 10 min to freeze the aqueous phase. Then, the upper organic phase was recovered, evaporated under a gentle stream of nitrogen at 37 °C and reconstituted in a solution of water/acetonitrile (1:1). The aqueous phase was used for determination of protein concentration. For LC-MRM, samples were injected and separated on a Phenomenex Luna 2.5-µm C18(2) HST column combined with a SecurityGuard precolumn (Phenomenex Inc., USA). Separated endocannabinoids were flow-through analyzed by MRM with the 5500 QTrap triple-quadrupole linear ion trap mass spectrometer armed with a Turbo V Ion Source (AB Sciex Germany GmbH, Germany). For quantification of the endocannabinoids in lung tissues, triplicate calibration curves were used. Protein concentrations of lung samples were determined using the Pierce BCA Assay Kit (Thermo Fisher Scientific, USA) and the endocannabinoid concentrations were normalized to total protein.
10
Serum Metabolite Analysis by LC-MS/MS
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Blood samples were collected within 24 h after admission to the Emergency Room, centrifuged to obtain serum samples, and stored at −80 °C until analysis. Sixty microliters of serum sample were treated using 60 μl of deproteinizing solution. The mixture was mixed for 30 s and centrifuged at 14.000 rpm for 15 min. Ten microliters of clean supernatant were injected into the chromatographic system. Compounds were detected using a liquid chromatography–tandem mass spectrometry (LC–MS/MS) analytical method [19 (link)]. Chromatographic separation of analytes was performed using an Agilent Liquid Chromatography System series 1100 (Agilent Technologies, USA), on a F5 column (100 × 2.1 mm, Kinetex 2.6 μm F5, 100 Å, Phenomenex, CA, USA) equipped with a security guard pre-column (Phenomenex, Torrance, CA, USA). The mobile phase consisted of a solution of 0.1% aqueous formic acid (A) and 100% acetonitrile (B); elution was performed at flow rate of 300 μl/min, using an elution gradient. The mass spectrometry method was performed on a 3200 triple quadrupole system (Applied Biosystems, Foster City, CA, USA) equipped with a Turbo Ion Spray source. The detector was set in the positive ion mode. The instrument was set in the Multiple Reaction Monitoring (MRM) mode. Data were acquired and processed by the Analyst 1.5.1 Software.
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