Samples were prepared in accordance with previous studies54 , with some minor changes. Biofluids (rat plasma, human plasma or cardiomyocyte spent culture media) were thawed on ice and briefly vortexed (5 s). 50 µL of the biofluid was mixed with either (i) 150 µL 100% ice-cold acetonitrile (LC-MS grade, VWR International; polar metabolite extraction for HILIC analysis], or (ii) 150 µL 100% ice-cold isopropanol (LC-MS grade, VWR International; lipid extraction for RP C18). Samples were vortexed and centrifuged (20,000-g, 4 °C, 20 min) and 100 µL supernatant removed into a low recovery HPLC vial (Chromatography Direct, UK) for direct analysis. To create the QCs, 50 µL of each plasma sample (plasma QC) or 50 µL of each media sample (media QC) was pooled, vortexed (30 s) and split into several 50 µL aliquots. Each aliquot was prepared as for the samples (above).
Lc ms grade
Manufactured by Avantor
Sourced in United States, Germany, Italy, United Kingdom
LC-MS grade is a type of laboratory equipment used for liquid chromatography-mass spectrometry (LC-MS) analysis. It is designed to provide high-quality, ultra-pure solvents and reagents that are essential for accurate and reliable LC-MS measurements.
Automatically generated - may contain errors
Lab products found in correlation
Glu c, by Promega (2 mentions)
Tka x cad system, by Thermo Fisher Scientific (2 mentions)
Vacuum filtration unit, by Sartorius (1 mentions)
Ultrasonic bath, by Bandelin (1 mentions)
Lc ms grade, by Thermo Fisher Scientific (1 mentions)
Uhplc q tof ms, by Agilent Technologies (1 mentions)
Hplc grade, by Merck Group (1 mentions)
Butylated hydroxytoluene, by Merck Group (1 mentions)
Ammonium acetate, by Merck Group (1 mentions)
Butylated hydroxyl toluene, by Merck Group (1 mentions)
Fastprep 24, by MP Biomedicals (1 mentions)
Acquity uplc system, by Waters Corporation (1 mentions)
Pre stained protein ladder, by Thermo Fisher Scientific (1 mentions)
Porcine trypsin, by Promega (1 mentions)
Potassium dihydrogen phosphate, by Merck Group (1 mentions)
Sodium hydroxide (naoh), by Avantor (1 mentions)
Formic acid, by Avantor (1 mentions)
Acetic acid, by Avantor (1 mentions)
Ascorbic acid, by Avantor (1 mentions)
Sodium acetate trihydrate, by Merck Group (1 mentions)
23 protocols using lc ms grade
1
Metabolite and Lipid Extraction from Biofluids
2
Bacterial Metabolite Extraction and LC-MS Analysis
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Bacteria were cultivated as described above, if not indicated otherwise,
until the beginning of the stationary growth phase as described with
modifications.15 (link) Briefly, a 0.22 μM
cellulose-acetate membrane filter (Ø 47 mm, Sartorius) was washed
three times with 10 mL of ultrapure water, each using a vacuum filtration
unit (Sartorius), followed by the addition of 10 mL of cell culture
per OD600 unit and washing of the cells three times with
10 mL of ultrapure water. The filter with cells was transferred immediately
to a bottle containing 20 mL of extraction mixture chilled to −20
°C consisting of acetonitrile, methanol, water, and formic acid
(60:20:19.9:0.1 (v/v), LC–MS grade, VWR). Cells were disrupted
by three freeze–thaw cycles, that is, for 1 h at −70
°C, followed by 5 min of sonication in an ultrasonic bath (Bandelin).
The extracts were transferred to glass flasks and lyophilized at 1
mbar and −90 °C, followed by reconstitution in a total
of 1 mL of water (LC–MS grade, VWR). The extracts were prepared
for LC–MS measurement by centrifugation for 20 min at 17,000g and room temperature to remove cell debris, followed by
transfer of the supernatant to a fresh tube and centrifugation for
20 min at 17,000g and room temperature. Extracts
were stored at −20 °C until measurement.
until the beginning of the stationary growth phase as described with
modifications.15 (link) Briefly, a 0.22 μM
cellulose-acetate membrane filter (Ø 47 mm, Sartorius) was washed
three times with 10 mL of ultrapure water, each using a vacuum filtration
unit (Sartorius), followed by the addition of 10 mL of cell culture
per OD600 unit and washing of the cells three times with
10 mL of ultrapure water. The filter with cells was transferred immediately
to a bottle containing 20 mL of extraction mixture chilled to −20
°C consisting of acetonitrile, methanol, water, and formic acid
(60:20:19.9:0.1 (v/v), LC–MS grade, VWR). Cells were disrupted
by three freeze–thaw cycles, that is, for 1 h at −70
°C, followed by 5 min of sonication in an ultrasonic bath (Bandelin).
The extracts were transferred to glass flasks and lyophilized at 1
mbar and −90 °C, followed by reconstitution in a total
of 1 mL of water (LC–MS grade, VWR). The extracts were prepared
for LC–MS measurement by centrifugation for 20 min at 17,000g and room temperature to remove cell debris, followed by
transfer of the supernatant to a fresh tube and centrifugation for
20 min at 17,000g and room temperature. Extracts
were stored at −20 °C until measurement.
3
Metabolomic Profiling of Coffee Beverages
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For the metabolomics analysis, a total of 44 samples were analyzed, when considering Espresso (20 replicates), Neapolitan (eight replicates), Moka (eight replicates), and Filter (eight replicates) preparations. In this regard, one mL of each coffee beverage (as resulting from Moka, Neapolitan pot, Espresso, and Filter preparations) were extracted in five mL of 70% aqueous methanol (LC-MS grade, VWR, Milan, Italy) acidified with 0.1% formic acid. Regarding the starting ground coffee samples of C. arabica and C. canephora var. robusta, four replicates (1 g) of each sample were extracted using an Ultra-Turrax homogenizer (IKA T25, Staufen., Germany) using the same extraction solution. The extracts were then centrifuged (Eppendorf 5810R, Hamburg, Germany) at 10,000× g for 10 min at 4 °C and filtered using 0.22 μm cellulose syringe filters into amber vials.
4
Metabolite Extraction and LC-MS Analysis
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Bacteria were cultivated as
described above. 2 ml of culture was centrifuged for 3 min at 6500g and room temperature. The supernatant was processed using
a 1 mL/100 mg C18 solid-phase extraction column (Macherey-Nagel, CHROMABOND).
Bound analytes were eluted with 500 μL of water/methanol 50:50
(v/v), followed by elution with 500 μL of methanol. The fractions
were combined and dried under reduced pressure at 60 °C. The
dried extract was reconstituted in 200 μL water and subjected
to LC–MS/MS analysis as described below. All solvents used
were LC–MS grade (VWR). Data were normalized to the cell density
using the OD600 of the cultures.
described above. 2 ml of culture was centrifuged for 3 min at 6500g and room temperature. The supernatant was processed using
a 1 mL/100 mg C18 solid-phase extraction column (Macherey-Nagel, CHROMABOND).
Bound analytes were eluted with 500 μL of water/methanol 50:50
(v/v), followed by elution with 500 μL of methanol. The fractions
were combined and dried under reduced pressure at 60 °C. The
dried extract was reconstituted in 200 μL water and subjected
to LC–MS/MS analysis as described below. All solvents used
were LC–MS grade (VWR). Data were normalized to the cell density
using the OD600 of the cultures.
5
Phytohormone Extraction and Analysis
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The sources of the phytohormone standards were jasmonic acid-d6 (HPC Standards GmbH, Cunnersdorf, Germany), SA-d4 (Sigma–Aldrich), ABA-d6 (Santa Cruz Biotechnology, Dallas, TX, USA), and jasmonic acid-13C6-isoleucine conjugate [synthesized as described by Kramell et al. (1988) (link) using 13C6-Ile (Sigma–Aldrich)].
The sources of the solvents used for the phytohormone extraction were methanol (LiChrosolv®, LC-MS grade, Merck KGaA, Germany), acetonitrile (LC-MS grade, VWR Chemicals, USA), and formic acid (LC-MS grade, Fisher Scientific, Belgium).
The sources of the solvents used for the phytohormone extraction were methanol (LiChrosolv®, LC-MS grade, Merck KGaA, Germany), acetonitrile (LC-MS grade, VWR Chemicals, USA), and formic acid (LC-MS grade, Fisher Scientific, Belgium).
6
Untargeted Metabolomics Analysis of Grape Juice
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The untargeted metabolomics
analysis was directly conducted on grape juice following centrifugation
(8000g for 15 min) and filtration (cellulose membrane,
0.22 μm) into amber glass vials. Analysis was done using ultra-high-pressure
liquid chromatography coupled to a quadrupole-time-of-flight mass
spectrometer (UHPLC/QTOF-MS) from Agilent Technologies (Santa Clara,
CA, USA), as previously reported.29 (link) Briefly,
the chromatographic separation was achieved using a Pursuit 3 pentafluorophenyl
(PFP) column (2.0 × 100 mm, 3 μm) from Agilent Technologies
(Santa Clara, CA, USA) and a binary gradient from 6 to 94% acetonitrile
(LC–MS grade, VWR, Milan, Italy) in 33 min with a flow rate
of 200 μL min–1. The samples were injected
randomly, with an injection volume of 6 μL, and SCAN acquisition
was adopted (100–1000 m/z, 30,000 fwhm, 1 Hz). The raw data were processed using Agilent Profinder
B.06 software, as previously described,24 (link) to putatively annotate compounds based on isotopic spacing and ratio
according to the “find-by-formula” algorithm (5 ppm
mass accuracy for monoisotopic mass and 0.05 min for retention time
alignment). The PlantCyc 12.6 database25 (link) was used as a reference, and level 2 confidence was achieved concerning
the COSMOS Metabolomics Standards Initiative.26 (link) The Metabolomics data were deposited into the EMBL-EBI MetaboLights
database with the identifier MTBLS7841.
analysis was directly conducted on grape juice following centrifugation
(8000g for 15 min) and filtration (cellulose membrane,
0.22 μm) into amber glass vials. Analysis was done using ultra-high-pressure
liquid chromatography coupled to a quadrupole-time-of-flight mass
spectrometer (UHPLC/QTOF-MS) from Agilent Technologies (Santa Clara,
CA, USA), as previously reported.29 (link) Briefly,
the chromatographic separation was achieved using a Pursuit 3 pentafluorophenyl
(PFP) column (2.0 × 100 mm, 3 μm) from Agilent Technologies
(Santa Clara, CA, USA) and a binary gradient from 6 to 94% acetonitrile
(LC–MS grade, VWR, Milan, Italy) in 33 min with a flow rate
of 200 μL min–1. The samples were injected
randomly, with an injection volume of 6 μL, and SCAN acquisition
was adopted (100–1000 m/z, 30,000 fwhm, 1 Hz). The raw data were processed using Agilent Profinder
B.06 software, as previously described,24 (link) to putatively annotate compounds based on isotopic spacing and ratio
according to the “find-by-formula” algorithm (5 ppm
mass accuracy for monoisotopic mass and 0.05 min for retention time
alignment). The PlantCyc 12.6 database25 (link) was used as a reference, and level 2 confidence was achieved concerning
the COSMOS Metabolomics Standards Initiative.26 (link) The Metabolomics data were deposited into the EMBL-EBI MetaboLights
database with the identifier MTBLS7841.
7
Lipid Extraction from Human Brain Tissue
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Lipids were extracted from the human brain tissue using a modified methyl tert-butyl ether (MTBE) method described previously [27 (link),28 (link)]. Brain tissue (~10 mg) was homogenized in 300 µL of methanol (MeOH; LC-MS Grade, VWR International, Tingalpa, QLD, Australia) containing 0.01% butylated hydroxytoluene (Sigma Aldrich, North Ryde, NSW, Australia) and internal standards (1 nmol LPC 14:0, 1 nmol LPE 14:0, 40 nmol PC 19:0/19:0, 40 nmol PE 17:0/17:0, 5 nmol PS 17:0/17:0; Avanti Polar Lipids Inc., Alabaster, AL, USA) using 1.4 mm ceramic beads in a FastPrep24 homogenizer at 6 m/s for 40 s. The homogenate was transferred to a 2 mL glass vial, and the beads washed with 100 µL MeOH. MTBE (920 µL; HPLC Grade, Bio-Strategy, Murarrie, NSW, Australia) was added to each sample, then rotated for 1 h at room temperature (25 °C). Ammonium acetate (230 µL of 150 mM; HPLC Grade, Sigma Aldrich, North Ryde, NSW, Australia) was added to each sample before being vortexed and centrifuged at 2000× g for 5 min. The top organic phase was removed from each sample and transferred to a new 2 mL glass vial for storage at −20 °C. Extracts were diluted 500-fold in a 2:1 MeOH: chloroform (v/v) solution with 5 mM ammonium acetate for mass spectrometric analysis.
8
Extraction and Analysis of EVOO Samples
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In this work, five commercial EVOO samples were acquired from local supermarkets (Piacenza, Italy). These EVOO samples were chosen because they are representative of typical olive cultivars spread in the Mediterranean basin, namely Leccino and Frantoio (normally grown in Italy), Picual and Picholine marocaine (characteristic of Spain and Northern Africa) and Kalamon (typical of Greece). The selected EVOO samples were immediately placed in dark and cold storage (10 ± 2 °C) until further analyses. Regarding the extraction procedure, the method reported by Mohamed and co-authors [12 (link)] was applied. Briefly, three replicates from each sample (3 g each) were extracted in 3 mL of 80% methanol solution (v/v) (LC-MS grade, VWR, Milan, Italy) using a vortex mixer. The suspension was kept at room temperature for 30 min and then centrifuged at 6000× g for 10 min at 4 °C. The extracts were collected in 2 mL amber glass vials and then stored at −18 °C until analysis.
9
Brain Lipid Extraction and Analysis
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Lipids were extracted using a modified methyl tert-butyl ether (MTBE, HPLC grade; Fischer Scientific, Massachusetts, USA) method as described previously50 (link),51 (link). Brain tissue (10 mg) was homogenized using a bead homogenizer (FastPrep-24, MP Bio, Sydney, Australia) at 6 m/s for 40 s, using 600 mg of 1.4 mm ceramic beads in 300 µL of methanol (LC–MS grade; VWR International, Pennsylvania, USA) containing 0.01% butylated hydroxyl-toluene (BHT; Sigma Aldrich, Missouri, USA) and internal standards (2 nmol CE 22:1, Avanti Polar Lipids, Alabama, USA). The homogenate was transferred into 2 mL glass vials. MTBE (920 µL) was added to each sample and rotated for 1 h at room temperature. Ammonium acetate (Sigma Aldrich, Missouri, USA) was added (230 µL of 150 mM), samples were vortexed for 20 s before being centrifuged at 2000 × g for 5 min. The top organic phase was removed from each sample without disturbing the bottom aqueous phase, and transferred into a new 2 mL glass vial for storage at -20 °C. This extract was diluted 500-fold in methanol: chloroform (2:1 v/v, with 5 mM Ammonium acetate) for targeted mass spectrometric analysis of CE.
10
Negative Ion Mass Spectrometry Protocol
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Negative
ion electrospray mass spectrometry data were collected with a Waters
LCT Premier XE time-of-flight instrument controlled by MassLynx version
4.1. Samples from the NMR tubes were transferred to GC vials and injected
into the multimode ionization source with a Waters Acquity UPLC system.
The flow injection solvent was a 50/50 MeOH/MeCN mixture (LCMS grade,
VWR Scientific), and water blanks were run between all samples. The
lock mass standard for accurate mass determination was leucine enkephalin
(Sigma catalog no. L9133).
ion electrospray mass spectrometry data were collected with a Waters
LCT Premier XE time-of-flight instrument controlled by MassLynx version
4.1. Samples from the NMR tubes were transferred to GC vials and injected
into the multimode ionization source with a Waters Acquity UPLC system.
The flow injection solvent was a 50/50 MeOH/MeCN mixture (LCMS grade,
VWR Scientific), and water blanks were run between all samples. The
lock mass standard for accurate mass determination was leucine enkephalin
(Sigma catalog no. L9133).
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