Plasma metabolites were extracted from pre-aliquoted biospecimens (15 μL) with 45 μL of LCMS grade methanol (ThermoFisher) in a 96-well microplate (Eppendorf). Plates were heat sealed, vortexed for 5 min at 750 rpm, and centrifuged at 2,000 × g for 10 mins at room temperature. The supernatant (30 μL) was transferred to a 96-well plate, leaving behind the precipitated protein. The supernatant was further diluted with 60 μL of 100 mm ammonium formate, pH3 (Fisher Scientific). For Hydrophilic Interaction Liquid Chromatography (HILIC) positive ion analysis, 15 μL of the supernatant and ammonium formate mix were diluted with 195 μL of 1:3:8:144 water (GenPure ultrapure water system, Thermofisher): LCMS grade methanol (ThermoFisher): 100 mm ammonium formate, pH3 (Fisher Scientific): LCMS grade acetonitrile (ThermoFisher). For C18 analysis, 15 μL of the supernatant and ammonium formate mix were diluted with 90 μL water (GenPure ultrapure water system, ThermoFisher) for positive ion mode. Each sample solution was transferred to 384-well microplate (Eppendorf) for LCMS analysis.
Lc ms grade methanol
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, China, Canada, Germany, Belgium, France
LC-MS grade methanol is a high-purity solvent used in liquid chromatography-mass spectrometry (LC-MS) applications. It serves as a mobile phase component to facilitate the separation and analysis of compounds in complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Acetonitrile, by Thermo Fisher Scientific (83 mentions)
Formic acid, by Thermo Fisher Scientific (63 mentions)
Lc ms grade acetonitrile, by Thermo Fisher Scientific (14 mentions)
Formic acid, by Merck Group (11 mentions)
Ammonium acetate, by Thermo Fisher Scientific (10 mentions)
Lc ms grade water, by Thermo Fisher Scientific (10 mentions)
Ammonium acetate, by Merck Group (10 mentions)
Milli q system, by Merck Group (8 mentions)
Acetic acid, by Thermo Fisher Scientific (7 mentions)
Lc ms grade formic acid, by Merck Group (7 mentions)
Ammonium formate, by Thermo Fisher Scientific (7 mentions)
Isopropanol, by Thermo Fisher Scientific (6 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Milli q water purification system, by Merck Group (5 mentions)
Ammonium formate, by Merck Group (5 mentions)
Milli q water, by Merck Group (5 mentions)
Ethanol, by Thermo Fisher Scientific (5 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (5 mentions)
Hplc grade methanol, by Thermo Fisher Scientific (4 mentions)
Ethyl acetate, by Thermo Fisher Scientific (4 mentions)
172 protocols using lc ms grade methanol
1
Plasma Metabolite Extraction for LCMS Analysis
2
Metabolite Extraction and Preparation for LCMS
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Plasma and serum metabolites were extracted from pre-aliquoted biospecimens (15μL) with 45μL of LCMS grade methanol (ThermoFisher) in a 96-well microplate (Eppendorf). Plates were heat sealed, vortexed for 5 min at 750 rpm, and centrifuged at 2000 × g for 10 min at room temperature. The supernatant (30μL) was carefully transferred to a 96-well plate, leaving behind the precipitated protein. The supernatant was further diluted with 60μL of 100 mM ammonium formate, pH3 (Fisher Scientific). For Hydrophilic Interaction Liquid Chromatography (HILIC) positive ion analysis, 15μL of the supernatant and ammonium formate mix were diluted with 195μL of 1:3:8:144 water (GenPure ultrapure water system, Thermofisher): LCMS grade methanol (ThermoFisher): 100 mM ammonium formate, pH3 (Fisher Scientific): LCMS grade acetonitrile (ThermoFisher). For the HILIC negative ion analysis, 15μL of the supernatant and ammonium formate mix were diluted with 90μL of LCMS grade acetonitrile (ThermoFisher). For C18 analysis, 15μL of the supernatant and ammonium formate mix were diluted with 90μL water (GenPure ultrapure water system, ThermoFisher) for positive and negative ion modes, respectively. Each sample solution was transferred to 384-well microplate (Eppendorf) for LCMS analysis.
3
Steady-State Metabolomics of Cell Lines
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For steady-state metabolomics, 2 × 105 cells were plated the day before onto 6-well plates (5 or 6 replicates for each cell type) and extracted the day after. The experiment was repeated 3 times (N = 3). Before extraction, cells were counted using CASY cell counter (Omni Life Sciences) using a separate counting plate. After that, cells were washed at room temperature with PBS twice and then kept in a cold bath with dry-ice and methanol. Metabolite extraction buffer (MEB, 50% LC–MS grade Methanol (Fisher Scientific, 10284580), 30% LC–MS grade Acetonitrile (Fisher Scientific, 10001334) and 20% ultrapure water) was added to each well following the proportion 1 × 106 cells/1 ml of buffer. HEPES 100 ng/ml or Valine-d8 5 μM (CK isotopes, DLM-488) were used as internal standard for the MEB. After a couple of minutes, the plates were moved to the −80 °C freezer and kept overnight. The following day, the extracts were scraped and mixed at 4 °C for 10 min. After final centrifugation at max speed for 10 min at 4 °C, the supernatants were transferred into LC–MS vials.
4
Headspace Analysis of Volatile Fatty Acids
5
Vitamin D Metabolite Quantification
6
Perfluorinated Compounds Analysis Protocol
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Native standards of PFBA, PFPrS,
and PFBS and mass-labeled standards of PFBA and PFBS were purchased
from Wellington Laboratories (Guelph, ON, Canada). TFA was purchased
from Sigma-Aldrich (Munich, Germany). PFPrA was from Sigma-Aldrich
(Oakville, ON, Canada). TFMS was from Sigma-Aldrich (Stockholm, Sweden),
and potassium salt of PFEtS was obtained from Kanto Chemical Co.,
Inc. (Portland, OR, USA). Mass-labeled standard of TFA (13C2-TFA) was purchased from Toronto Research Chemicals
Inc. (Toronto, ON, Canada). Mass-labeled standards for PFPrA, TFMS,
PFEtS, and PFPrS were not available commercially. The purity of all
standards was above 97%. Ammonium acetate (≥99.0%) was purchased
from Sigma-Aldrich (Stockholm, Sweden). Glacial acetic acid (EMPROVE
EXPERT, Ph. Eur., JP, USP) was purchased from Merck (Darmstadt, Germany).
Analytical reagent-grade ammonia solution, HPLC grade methanol (≥99.8%),
and LC–MS-grade methanol (≥99.9%) were from Fisher Scientific
(Ottawa, ON, Canada).
and PFBS and mass-labeled standards of PFBA and PFBS were purchased
from Wellington Laboratories (Guelph, ON, Canada). TFA was purchased
from Sigma-Aldrich (Munich, Germany). PFPrA was from Sigma-Aldrich
(Oakville, ON, Canada). TFMS was from Sigma-Aldrich (Stockholm, Sweden),
and potassium salt of PFEtS was obtained from Kanto Chemical Co.,
Inc. (Portland, OR, USA). Mass-labeled standard of TFA (13C2-TFA) was purchased from Toronto Research Chemicals
Inc. (Toronto, ON, Canada). Mass-labeled standards for PFPrA, TFMS,
PFEtS, and PFPrS were not available commercially. The purity of all
standards was above 97%. Ammonium acetate (≥99.0%) was purchased
from Sigma-Aldrich (Stockholm, Sweden). Glacial acetic acid (EMPROVE
EXPERT, Ph. Eur., JP, USP) was purchased from Merck (Darmstadt, Germany).
Analytical reagent-grade ammonia solution, HPLC grade methanol (≥99.8%),
and LC–MS-grade methanol (≥99.9%) were from Fisher Scientific
(Ottawa, ON, Canada).
7
Analytical Chemistry Reagents Protocol
8
Analysis of Dicationic Reagent Structure
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The dicationic reagent 1,5-pentanediyl-bis(1-butylpyrrolidinium) difluoride ([C5(bpyr)2]F2) at the concentration of 2.5 mM in 50% methanol/H2O (v/v) was purchased from AZYP, LLC (Arlington, TX, USA). The dicationic structure was observed at 162.1747 m/z (z = 2) and 343.3483 m/z (z = 1) with high ion abundance (Figure 1 A). The dicationic chemical structure was shown in Figure 1 B. [C5(bpyr)2]F2 was diluted to 10 µM with 50% methanol/H2O in this experiment. LC/MS grade methanol, formic acid, and water were purchased from Fisher Chemical (Fair Lawn, NJ, USA). Nitrogen gas for purging the enclosure was purchased from Arc3 Gases (Raleigh, NC, USA).
9
Plasma Metabolite Profiling Workflow
10
Quantifying Oxycodone and Diazepam in Rat Plasma
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Reference standards of oxycodone hydrochloride (100% purity) and diazepam (99.8% purity) were purchased from the United States Pharmacopeia (Bethesda, MD, USA). Internal standards (ISs) oxycodone-d6 and diazepam-d5 were obtained from Cerilliant (Round Rock, TX, USA). LC-MS grade methanol, acetonitrile and water were purchased from Fisher Scientific (Waltham, MA, USA). GR grade ammonium formate was procured from Sigma-Aldrich (St. Louis, MO, USA). MultiScreen® solvinert filter plates (0.45 μm low-binding Hydrophilic PTFE) were used to precipitate the plasma proteins and were obtained from Merck Millipore Ltd (Tullagreen, IRL). Control Sprague Dawley male rat plasma (K3-EDTA) was obtained from BioreclamationIVT (Hicksville, NY, USA).
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