Blood was collected into EDTA tubes and processed to plasma from patients fasted at least 8 hours. Samples were stored at −80°C prior to analysis by Metabolon (Morrisville, NC, USA). Acylcarnitine species measurements were performed with Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy (UHPLC-MS; Waters Acquity UHPLC, Waters Corporation, Milford, MA USA) and Hybrid Quadrupole-Orbitrap mass spectometer (Q-Exactive Thermo Fischer Scientific, Waltham, MA, USA) platforms in electrospray ionisation-positive and -negative modes, as previously described.21 (link) Acylcarnitine species were identified by comparison to an in-house library of purified standards. Relative quantification of peaks was performed as area-under-the-curve detector ion counts by peak area integration. The mean relative standard deviation of internal standards for this study was 6%. The relative change in acylcarnitine species from baseline was assessed according to subgroups defined by treatment arm and the presence or absence of a PDFF response.
Waters acquity uhplc
Manufactured by Waters Corporation
Sourced in United States
The Waters Acquity UHPLC is a high-performance liquid chromatography (HPLC) system designed for the analysis of a wide range of samples. It utilizes ultra-high pressure technology to achieve superior separation and sensitivity performance compared to traditional HPLC systems.
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Lab products found in correlation
Waters oasis hlb, by Waters Corporation (1 mentions)
Linear trap quadrupole mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Thermo finnigan trace dsq, by Thermo Fisher Scientific (1 mentions)
Ltq mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Ltq orbitrap ftms hybrid mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Luna c18 column, by Phenomenex (1 mentions)
5 protocols using waters acquity uhplc
1
UHPLC-MS Quantification of Acylcarnitines
2
Bioavailability of Phenolic Metabolites
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A subgroup of seventeen subjects performed an additional bioavailability study, to
identify circulating phenolic metabolites following the administration of experimental
beverages. Halfway through the supplementation period (30 (sem 3) d), fasted
subjects were administered their respective treatment at INAF (SCP, n 8;
Control, n 9). Blood samples were collected using EDTA-containing
syringes before and 30, 60, 120, 240 and 360 min after the ingestion. During the
experiment, all subjects were kept fasted. Plasma samples were obtained by centrifugation
(3500 rpm, 10 min at 4°C). Plasma phenolic compounds were characterised by UHPLC–MS/MS as
previously described(24 ), with slight modifications. Acidified plasma samples (300 µl) were loaded
into preconditioned Waters OASIS HLB (Waters Ltd) µElution plates 2 mg–30 µm. The retained
phenolic compounds were eluted with 75 µl of acetone–ultrapure water–acetic acid solution
(70:29·5:0·5, v/v/v) in presence of rosmarinic acid as internal standard (1 µg/ml final
concentration). The eluted solutions were directly analysed by UHPLC–MS/MS, using a Waters
Xevo TQD MS (Waters Ltd) coupled to a Waters Acquity UHPLC (Waters Ltd). Phenolic
metabolites were separated and identified as previously reported(24 ).
identify circulating phenolic metabolites following the administration of experimental
beverages. Halfway through the supplementation period (30 (
subjects were administered their respective treatment at INAF (SCP, n 8;
Control, n 9). Blood samples were collected using EDTA-containing
syringes before and 30, 60, 120, 240 and 360 min after the ingestion. During the
experiment, all subjects were kept fasted. Plasma samples were obtained by centrifugation
(3500 rpm, 10 min at 4°C). Plasma phenolic compounds were characterised by UHPLC–MS/MS as
previously described(
into preconditioned Waters OASIS HLB (Waters Ltd) µElution plates 2 mg–30 µm. The retained
phenolic compounds were eluted with 75 µl of acetone–ultrapure water–acetic acid solution
(70:29·5:0·5, v/v/v) in presence of rosmarinic acid as internal standard (1 µg/ml final
concentration). The eluted solutions were directly analysed by UHPLC–MS/MS, using a Waters
Xevo TQD MS (Waters Ltd) coupled to a Waters Acquity UHPLC (Waters Ltd). Phenolic
metabolites were separated and identified as previously reported(
3
Comprehensive Metabolite Profiling by UHPLC-MS/GC-MS
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UHPLC/MS was performed using a Waters Acquity UHPLC (Waters Corporation, Milford, MA) coupled to a linear trap quadrupole mass spectrometer (Thermo Fisher Scientific, Inc, Waltham, MA) equipped with an electrospray ionization source. Two separate UHPLC/MS injections were performed on each sample: 1 optimized for positive ions and 1 for negative ions. Samples for GC/MS were analyzed on a Thermo-Finnigan Trace DSQ fast-scanning single-quadrupole MS (Thermo Fisher Scientific, Inc, Waltham, MA) operated at unit mass resolving power. Chromatographic separation followed by full-scan mass spectra were performed to record retention time, molecular weight (m/z), and MS/MS of all detectable ions presented in the samples.
4
UHPLC/MS Metabolite Profiling Protocol
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UHPLC/MS was carried out using a Waters Acquity UHPLC (Waters Corporation) coupled to an LTQ mass spectrometer (Thermo Fisher Scientific Inc.) equipped with an electrospray ionization (ESI) source and linear ion-trap (LIT) mass analyzer. Two separate UHPLC/MS injections were performed on each sample using separate dedicated columns: one optimized for positive ions and one for negative ions. The mobile phase for positive ion analysis was 0.1% formic acid in H2O (solvent A) and 0.1% formic acid in methanol (solvent B), while the mobile phase for negative ion analysis consisted of 6.5 mM ammonium bicarbonate, pH 8.0 (solvent A) and 6.5 mM ammonium bicarbonate in methanol (solvent B). The acidic extracts were monitored for positive ions and the basic extracts for negative ions in independent injections. The extracts were loaded via an autosampler (Waters Acquity), and gradient eluted (0% B to 98% B over 11 min) directly into the mass spectrometer at a flow rate of 350 μl/min. The MS analysis alternated between MS (99–1000 m/z) and data-dependent MS2 scans using dynamic exclusion.
5
Methanolic Extraction and LC-MS Analysis
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Methanolic extracts for LC‐MS analysis were obtained by extracting freeze‐dried biomass with aqueous methanol (75% v/v) acidified with formic acid (0.1% v/v) as described by De Vos et al., 2007 . Methanolic extracts and fractions obtained from proanthocyanidin purification were analysed by LC‐MS as described by van der Hooft et al. (2012 ). Briefly, separation of extracted compounds using a Waters Acquity (U)HPLC was over a Luna C18 column (150 mm × 2.0 mm; 3 μm particles; Phenomenex) with a water–acetonitrile gradient in 0.1% v/v formic acid. Detection was by both a Waters Photodiode Array (200–600nm) and a LTQ Orbitrap FT‐MS hybrid mass spectrometer (Thermo Fisher Scientific, Nieuwegein, the Netherlands) with negative electrospray ionisation, a mass resolution of 70 000 FWHM and a mass range of m/z 100–1300.
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