The concentration of FNB in the samples’ post-release studies was analyzed using reverse-phase high-performance liquid chromatography (RP-HPLC) analysis (Thermo Fisher Vanquish HPLC system, Thermo Fisher, Waltham, MA, USA). A stainless-steel C-18 column (250 × 2.1 mm, 2 µm particle size) (Avantor ACE® EXCEL 3 C-18-AR column, VWR International, Radnor, PA, USA) was used for the analysis. The mobile phase was prepared using deionized water with 0.1% ortho-phosphoric acid (pH 2.5) and acetonitrile (ACN) at a 70:30 ratio. The flow rate was set to 1 mL/min and the injection volume was set at 10 µL. The run time for each sample was 10 min at 30 °C. The retention time for the drug was observed at 7.450 min. The detector used in this analysis was anultraviolet–visible (UV–Vis) spectrophotometer (Vanquish HPLC system, Thermo Fisher, Waltham, MA, USA) at 290 nm. The estimation of the collected data was performed using a calibration curve ranging from 1 to 128 µg/mL (R2 = 0.999).
Vanquish hplc system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Vanquish HPLC system is a high-performance liquid chromatography (HPLC) instrument designed for analytical and preparative applications. The system features advanced technology to provide reliable and precise separation and detection of a wide range of analytes.
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Lab products found in correlation
Chromeleon software, by Thermo Fisher Scientific (3 mentions)
Xcalibur software, by Thermo Fisher Scientific (3 mentions)
Q exactive hf orbitrap mass spectrometer, by Thermo Fisher Scientific (3 mentions)
Xbridge beh amide column, by Waters Corporation (2 mentions)
Q exactive hybrid mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Orbitrap q exactive high field mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Compound discoverer 3, by Thermo Fisher Scientific (1 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Altis triple quadrupole mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Accucore c30 column, by Thermo Fisher Scientific (1 mentions)
Orbitrap velos pro mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Tsq altis mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Xcalibur, by Thermo Fisher Scientific (1 mentions)
Milli q advantage, by Merck Group (1 mentions)
Xs205du, by Mettler Toledo (1 mentions)
Hypersil gold c18 column, by Thermo Fisher Scientific (1 mentions)
Hypersil gold c18, by Thermo Fisher Scientific (1 mentions)
Sequencing grade modified porcine trypsin, by Promega (1 mentions)
Q exactive plus, by Thermo Fisher Scientific (1 mentions)
Vwd 3100, by Phenomenex (1 mentions)
19 protocols using vanquish hplc system
1
RP-HPLC Analysis of FNB Concentration
2
Untargeted Metabolomic Profiling of Tissue Samples
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Tissue homogenates (100 μl) were added to 350 μl methanol (LC–MS grade), cooled to –80 °C, and maintained on dry ice during the addition. The mixture was vortexed vigorously and centrifuged at 13,300 rpm for 15 min at 4 °C to sediment proteins. Aliquots were subsequently dried in a vacuum centrifuge and stored at –80 °C until LC–MS/MS analysis. Prior to analysis, samples were resuspended in 52 μl water (LC–MS grade), centrifuged at 13,300 rpm for 15 min at 4 °C to remove any particulates, and transferred to glass sample vials. Untargeted HPLC–MS/MS data acquisition was performed as published (26 , 27 , 28 ). Full-scan MS and data-dependent MS/MS data were acquired using a Thermo Fisher Scientific Vanquish HPLC system coupled to a Thermo Fisher Scientific Q-Exactive mass spectrometer operating in positive ionization mode as described (29 (link)). Raw instrument data (.raw files) were exported to Compound Discoverer 3.1 (Thermo Fisher Scientific) for deconvolution, alignment, and annotation, as described (29 (link)). The peak areas of [12C6]lysine and [13C6]lysine were retrieved and used to calculate the RIA.
3
LCMS-based Quantitation of Chloroquine
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Drug concentrations were determined using an LCMS system consisting of a Thermo Scientific (Waltham, MA) Vanquish HPLC System interfaced to a Thermo Scientific Altis Triple Quadrupole Mass Spectrometer with an electrospray source. All drugs were separated using gradient elution methodology on a Biphenyl phase (Restek, 2.1 mmid, 100 mm length, 1.8 µm particle diameter) with binary mobile phase. Samples (50 µL) were diluted with methanol (200 µl) and centrifuged to precipitate proteins. The supernatant was diluted with a solution similar to the initial mobile phase that contained the internal standard, cisapride. The MS was set to positive ion mode and Selective Reactive Monitoring (SRM) with argon used for collision induced dissociation. The initial mobile phase was 20% methanol. Immediately following injection, the organic phase concentration was increased linearly to reach 95% methanol at 8 min. Then the mobile phase was returned to the initial condition (20% methanol) and held for 2 min to condition the column for the next injection. Both aqueous and organic phases contained 0.1% formic acid. SRM (parent/daughter) transitions used were m/z 320.19 → 247.125 for chloroquine and 466.12 → 184.042 for cisapride. Quantitation was based on a 7-point calibration curve.
4
Targeted Lipidomics of Prostate Cancer
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A Vanquish HPLC system
(Thermo Fisher Scientific) coupled with an Orbitrap Velos Pro mass
spectrometer (Thermo Fisher Scientific) was deployed for the LC-MS
analysis of healthy and cancerous mouse prostate samples. A solvent
system of ACN/water (60:40 v/v) (solvent A) and IPA/ACN (90:10 v/v)
(solvent B), both containing 0.1% formic acid and 10 mM ammonium formate,
was used. An aliquot of 5 μL of the sample was injected into
the Accucore C30 column (Thermo Fisher Scientific, C30, 2.1 mm ×
150 mm, 2.6 μm). A flow rate of 0.200 mL/min and a column temperature
of 40 °C were used. The elution gradient was modified from a
previously reported method40 (link) and was as
follows: 30%–43% B at 0–5 min, 43–50% B at 5–5.1
min, 50–70% B at 5.1–14 min, 70–99% B at 14.1–21
min, 99% B at 21–24 min, 99–30% B at 24–24.1
min, and 30% B at 24.1–33 min. Data were acquired with a top
3 data dependent acquisition (DDA) method using a m/z range of 100–1000, a full MS resolution
of 30 000, a MS/MS resolution of 15 000, and HCD with
an NCE of 35. Additionally, CID with an NCE of 40 was used to visualize
triacylglycerol (TG) diagnostic ions that were not produced under
HCD.
(Thermo Fisher Scientific) coupled with an Orbitrap Velos Pro mass
spectrometer (Thermo Fisher Scientific) was deployed for the LC-MS
analysis of healthy and cancerous mouse prostate samples. A solvent
system of ACN/water (60:40 v/v) (solvent A) and IPA/ACN (90:10 v/v)
(solvent B), both containing 0.1% formic acid and 10 mM ammonium formate,
was used. An aliquot of 5 μL of the sample was injected into
the Accucore C30 column (Thermo Fisher Scientific, C30, 2.1 mm ×
150 mm, 2.6 μm). A flow rate of 0.200 mL/min and a column temperature
of 40 °C were used. The elution gradient was modified from a
previously reported method40 (link) and was as
follows: 30%–43% B at 0–5 min, 43–50% B at 5–5.1
min, 50–70% B at 5.1–14 min, 70–99% B at 14.1–21
min, 99% B at 21–24 min, 99–30% B at 24–24.1
min, and 30% B at 24.1–33 min. Data were acquired with a top
3 data dependent acquisition (DDA) method using a m/z range of 100–1000, a full MS resolution
of 30 000, a MS/MS resolution of 15 000, and HCD with
an NCE of 35. Additionally, CID with an NCE of 40 was used to visualize
triacylglycerol (TG) diagnostic ions that were not produced under
HCD.
5
HILIC-MS Metabolite Profiling of Polar Extracts
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Extracted polar metabolite samples were analyzed by LC-MC. Separation was achieved by hydrophilic interaction liquid chromotograhpy (HILIC) using a Vanquish HPLC system (ThermoFisher Scientific). The column was an Xbridge BEH amide column (2.1 mm x 150 mm, 2.5 µM particular size, 130 Å pore size, Waters Co.) run with a gradient of solvent A (20 mM ammonium hydroxide, 20 mM ammonium acetate in 95:5 acetonitrile:Water, pH 9.5) and solvent B (100% acetonitrile) at a constant flow rate of 150 uL/min. The gradient function was: 0 min, 90% B; 2 min, 90% B; 3 min, 75% B; 7 min, 75% B; 8 min, 70% B; 9 min, 70% B; 10 min, 50% B; 12 min, 50% B; 13 min, 25% B; 14 min, 25% B; 16 min, 0% B; 20.5 min, 0% B; 21 min; 90% B; 25 min, 90% B. Autosampler temperature was 4°C, column temperature 30°C and injection volume 2 µL. Samples were injected by electrospray ionization into a QExactive HF orbitrap mass spectrometer (ThermoFisher Scientific) operating in negative ion mode with a resolving power of 120,000 at m/z of 200 and a full scan range of 75–1000. Data were analyzed using the MAVEN software package and specific peaks assigned based on exact mass and comparison with known standards (Melamud et al., 2010 (link)). Extracted peak intensities were corrected for natural isotopic abundance (Su et al., 2017 (link)).
6
Amino Acid and Dipeptide Quantification by LC-MS/MS
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Detection and quantification of amino acids and dipeptides was done by LC–MS/MS, using a Vanquish HPLC system coupled to a TSQ Altis mass spectrometer (both Thermo Fisher Scientific), employing the Selected Reaction Monitoring (SRM) mode and positive polarity. In brief, dried samples were resolved in 0.1% formic acid in water and 1 µL was injected onto a Kinetex (Phenomenex) C18 column (100 Å, 150 × 2.1 mm), employing a 9-min-long linear gradient from 100% A (1% acetonitrile, 0.1% formic acid in water) to 90% B (0.1% formic acid in acetonitrile) at a flow rate of 100 µL/min. Retention times, SRM transitions, and optimal collisional energies were determined by authentic standards and degenerate dipeptide libraries. All data interpretation was performed using Xcalibur (Thermo Fisher Scientific).
7
SCFA Analysis from Fecal Samples
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SCFAs will be analyzed from fecal samples collected on day 13 using LC-MS as described (59 (link)). In brief, 10 C2–C6 straight-chain SCFAs (e.g., butyrate, acetate, propionate) and branched-chain SCFAs (e.g., isobutyric acid, isovaleric acid) will be measured from fecal samples homogenized in propanol. Following centrifugation (4000 × g, 4°C, 10 min), the supernatant is mixed with 3-nitrophenylhydrazine (3NPH) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide, and incubated (30 min, 40°C) to generate 3NPH-SCFA derivatives. The sample is then analyzed on a Vanquish HPLC system equipped with a Q-Exactive hybrid mass spectrometer operated with electrospray ionization in negative mode (ThermoFisher Scientific). SCFAs will be quantified against area ratios of derivatized authentic standards prepared in parallel relative to 3NPH-[13C]-butyrate (internal standard).
8
Analytical HPLC Protocol for Sample Analysis
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A Thermo Fisher Vanquish HPLC system with a diode array detector was used in this investigation. A chromatographic column (ZHONG PU RED RD‐C18; 4.6 mm × 250 mm, 5 μm; Zhong Pu Technology Co., Ltd.; China), an electronic analytical balance (XS205DU; Mettler‐Toledo Co., Ltd.; Switzerland), a Milli‐Q system (Milli‐Q Advantage; Millipore Co., Ltd.; America), and an ultrasonic extractor (SB‐50; Ningbo Biotechnology Co., Ltd.; China) were also used in the HPLC analysis.
9
Comprehensive Metabolite Profiling by HILIC-MS
10
Quantifying ISG15-TRAF2 Binding Interactions
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Binding assays were performed with ISG15 (1–157 aa) and TRAF2 variants (1–185 aa) on a Vanquish HPLC system (Thermo Fisher Scientific) using an AdvanceBio size‐exclusion chromatography column (Agilent Technologies). As a positive control for ISG15 binding, the influenza B virus NS1B protein (1–103 aa) was used. Prior to analytical sizing, the column was pre‐equilibrated with SEC buffer (50 mM Tris pH 7.5, 150 mM NaCl, 2 mM DTT). ISG15 (30 µM) was mixed with TRAF2 variants or NS1B (25 µM) prior to injection on the column. Fractions were mixed with SDS sample buffer and resolved on a 4–20% gradient SDS/PAGE. Gels were visualized by Coomassie staining.
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