E‐7G‐4′S (395.7 ng) and E‐7S‐4′G (393.2 ng) were separated into the respective diastereomers by preparative HPLC. The HPLC apparatus used in this study was a Jasco 2000 plus system (Jasco, Tokyo, Japan) equipped with a model PU‐2089 gradient pump, CO‐2067 column oven, AS‐2059 auto sampler, and UV‐2075 UV‐visible detector. A Synergi Hydro‐RP column was employed (150 × 2.0 mm i.d., particle size 4 μm; Phenomenex, Torrance, CA) along with a guard cartridge (AQC 18, 4 × 2.1 mm i.d.). Each diastereomer mixture composed of E‐7G‐4′S or E‐7S‐4′G was eluted using a solvent system comprising 10 mmol/L ammonium acetate solution and acetonitrile (99:1). The flow rate was 0.4 mL/min at 45°C. The UV detection wavelength was set at 280 nm. Each diastereomer showing as 2 peaks for E‐7G‐4′S and E‐7S‐4′G on the HPLC chromatogram was divided into 2 fractions. The individual eluted solution from the preparative HPLC was evaporated. The 4 individual residues were divided into halves. One half of each fraction was used for enzymatic hydrolysis, while the other half was used for identification of plasma E‐7G‐4′S or E‐7S‐4′G.
Hydro rp column
Manufactured by Phenomenex
Sourced in United States
The Hydro-RP column is a versatile reversed-phase chromatography column designed for the separation and analysis of a wide range of polar and non-polar compounds. It features a silica-based stationary phase with a proprietary surface modification that provides excellent retention and selectivity for a variety of analytes.
Automatically generated - may contain errors
Lab products found in correlation
As 2059, by Jasco (1 mentions)
Co 2067, by Jasco (1 mentions)
Pu 2089, by Jasco (1 mentions)
Hplc cbm 20a system, by Shimadzu (1 mentions)
Prominence uflc system, by AB Sciex (1 mentions)
1260 infinity, by Agilent Technologies (1 mentions)
Uv 2600 spectrophotometer, by Shimadzu (1 mentions)
Agilent 1260 infinity, by Agilent Technologies (1 mentions)
6460 triple quadrupole tandem mass spectrometer, by Agilent Technologies (1 mentions)
6500plus qtrap mass spectrometer, by AB Sciex (1 mentions)
C18 ods guard column, by Phenomenex (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Agilent 1200 series hplc, by Agilent Technologies (1 mentions)
Prominence hplc system, by Shimadzu (1 mentions)
Diode array detector, by Agilent Technologies (1 mentions)
13 protocols using hydro rp column
1
Preparative HPLC Separation of Diastereomeric Plant Metabolites
2
Quantification of Acrylamide in Samples
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Samples were analysed on a Shimadzu CBM-20A HPLC system (Shimadzu Corporation, Kyoto, Japan) that consists of a degasser, quaternary pump, autosampler, and temperature controlled oven coupled to a Shimadzu LCMS-8050 triple quadrupole MS detector (Shimadzu Corporation, Kyoto, Japan). Separation of acrylamide was carried out on a Synergi Hydro-RP column (150 mm × 3 mm, 4 μm particle size) with a SecurityGuard Catridges C18 guard column (4 mm × 2 mm i.d.) (Phenomenex Inc., California, USA). The mobile phase was ultrapure water and methanol (99.5:0.5, v/v) at a flow rate of 0.2 mL/min. The oven temperature was 40°C and the injection volume was 10 μL. The Electrospray Ionization (ESI) source was operated in positive mode with an interface temperature of 300°C, desolvation line temperature of 250°C, and heat block temperature of 400°C. The nebulizing gas flow was 1 L/min, whereas the gas flow for heating and drying was 10 L/min. The transitions monitored for acrylamide was 72 > 55 and 72 > 27.1 while the transitions monitored for labelled acrylamide was 75 > 58.1 and 75 > 30.1. The dwell time for each transition was 247 ms. All samples and spiked samples were injected in duplicates. The calibration curve was constructed by plotting the response ratio (peak area for acrylamide: 72 > 55/peak area for labelled acrylamide: 75 > 58.1) against acrylamide concentration.
3
HPLC-MS/MS Quantification of Analytes
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A Shimadzu (Columbia, MD, USA) Prominence UFLC system coupled to a 4000 Q TRAP tandem mass spectrometer system (AB SCIEX, Concord, ON, Canada) was used as previously described [27 (link)]. Analytes were separated via HPLC using a HydroRP column (4 μm, 250 × 2 mm) purchased from Phenomenex (Torrance, CA, USA) with mobile phase delivered at a binary flow rate of 0.3 mL/min using the following gradient program: 100% mobile phase A (0.01% formic acid in ultrapure water) for 0.5 min, followed by a linear gradient of 0–60% B from 0.5 to 6.0 min, then holding at 60% B from 6.0 to 7.0 min, and, finally, back to 100% A from 7.0 to 7.5 min, followed by a 4.5 min re-equilibration with 100% A before the next injection. The column oven temperature was set at 40 °C, and the injection volume was 20 μL.
Eluted analytes were detected and quantified using positive electrospray ionization in MRM mode. All mass spectrometer conditions were optimized for quantification of the analytes, and the following parameters were set accordingly. The ion source temperature was set to 600 °C, and the ion spray voltage was 5500 V. The curtain gas pressure was 15 psi, and the ion source gases (GS1 and GS2) were set to 20 and 45 psi, respectively.
Eluted analytes were detected and quantified using positive electrospray ionization in MRM mode. All mass spectrometer conditions were optimized for quantification of the analytes, and the following parameters were set accordingly. The ion source temperature was set to 600 °C, and the ion spray voltage was 5500 V. The curtain gas pressure was 15 psi, and the ion source gases (GS1 and GS2) were set to 20 and 45 psi, respectively.
4
Chromatographic Separation of Analytes
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Chromatographic separation was performed using 1260 Infinity high-performance liquid chromatograph (Agilent Technologies, Santa Clara, CA, USA). Analytes were eluted from a Synergi Hydro-RP column (4 µm, 150 mm × 2.0 mm, 80Å, Phenomenex, Torrance, CA, USA) at a flow rate of 300 µL/min at 50 °C. The gradient elution of solvent A (0.2% formic acid in water) and solvent B (0.2% formic acid in methanol) was programmed as follows: 0–2 min with 97% solvent A, 2 min–4.5 min linear from 97% to 3% solvent A, 4.5 min–5 min with 3% solvent A, 5 min–5.5 min from 3% to 97% solvent A, 5.5 min–10 min with 97% solvent A. Injection volume was constant and equal to 10 µL.
5
Synthesis of Compounds 4 and 5
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Next, 2,2-dimethylpropanoyl chloride (0.5 mL) was added to an aqueous solution of 6 (12.0 mg) at room temperature, and the solution was stirred for 12 h. After the evaporation of the solvent, the product was dissolved in MeOH and purified by reversed-phase HPLC (Phenomenex Hydro-RP column) eluted with CH3OH-H2O (50:50) to yield compound 4 (3.7 mg) and compound 5 (6.4 mg).
6
Quantification of Organic Acids in Samples
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Quantification of organics acids was achieved on a Phenomenex Hydro-RP column (4.6 × 150 mm, 4 μm). Samples were carried through the column following an isocratic elution mode of solvent A (H2O; 0.1% formic acid and at a flow rate of 0.400 mL min−1, for a total run time of 25 min. Injection was performed in a full loop mode with 5 μL of the samples. Samples were analyzed in negative ion mode with capillary voltage set at 3.5 kV, nebulizer at 50 psi and dry gas at 11 L. min−1, with a dry temperature of 325°C. Fragmentation was performed in Auto acquire MS mode. Quantification was based on calibration curves obtained using acidified deionized water standard solutions of organic acids. The organic acid content was calculated using the following formula:
7
Analytical Methods for Chemical Quantification
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Free chlorine
and TBO concentrations
were measured with a Shimadzu UV-2600 spectrophotometer using the
DPD method at 515 nm23 and the TBO method
at 633 nm, respectively.34 (link) Test compounds
were quantified by high-performance liquid chromatography–tandem
mass spectrometry (HPLC–MS/MS) in the multiple reaction monitoring
(MRM) mode using an Agilent 1200 series HPLC system with a Hydro-RP
column (150 × 3 mm, 4uM; Phenomenex, Aschaffenburg, Germany)
coupled to a 6460 triple quadrupole tandem mass spectrometer, as described
previously.35 (link)pCBA was
quantified on a high-performance liquid chromatography (HPLC) system
equipped with UV detection at 254 nm (Agilent 1260 Infinity). Analytical
details and compound specific parameters are provided inTable S2 .
and TBO concentrations
were measured with a Shimadzu UV-2600 spectrophotometer using the
DPD method at 515 nm23 and the TBO method
at 633 nm, respectively.34 (link) Test compounds
were quantified by high-performance liquid chromatography–tandem
mass spectrometry (HPLC–MS/MS) in the multiple reaction monitoring
(MRM) mode using an Agilent 1200 series HPLC system with a Hydro-RP
column (150 × 3 mm, 4uM; Phenomenex, Aschaffenburg, Germany)
coupled to a 6460 triple quadrupole tandem mass spectrometer, as described
previously.35 (link)pCBA was
quantified on a high-performance liquid chromatography (HPLC) system
equipped with UV detection at 254 nm (Agilent 1260 Infinity). Analytical
details and compound specific parameters are provided in
8
HPLC Method for Urine Profiling
9
NAD+ Quantification in Small Intestinal Crypts
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Small intestinal crypts were isolated and sent to Tsinghua University (Beijing, China) for determining NAD+ content using a liquid chromatography-tandem mass spectrometry (LC–MS/MS). The ACQUITY UPLC H-Class system was coupled a 6500plus QTrap mass spectrometer (AB SCIEX, USA), equipped with a heated electrospray ionization (HESI) probe. Extracts were separated by a synergi Hydro-RP column (2.0 × 100 mm, 2.5 μm, phenomenex). The mobile phase consisted of a binary solvent system: mobile phase A (2 mM triisobutylamine adjusted with 5 mM acetic acid in water) and mobile phase B (methanol). A 15-min gradient with flow rate of 250 μL/min was used as follows: 0–1.5 min, 5%B; 1.5–9 min, 5–35% B; 9.5–12 min, 98% B;12.1–15 min, 5%B. Column chamber and sample tray were held at 35 °C and 10 °C, respectively, and data were acquired in multiple reaction monitor (MRM) mode. The nebulizer gas (Gas1), heater gas (Gas2), and curtain gas were set at 50, 50, and 35 psi, respectively. The ion spray voltage was −4500 V in negative ion mode. The optimal probe temperature was determined to be 450 °C. The SCIEX OS 1.6 software was applied for metabolite identification and peak integration.
10
HPLC Quantification of Phenolic Compounds in Green Banana Flour
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The targeted phenolic compounds in green banana flour were quantified using Agilent 1200 series HPLC (Agilent Technologies, Santa Clara, CA, USA) coupled with a photodiode array (PDA) reader by the previous published protocol of Suleria et al. with a few adjustments. A 0.45 µm syringe filter (PVDF, Millipore, MA, USA) was utilized to filter sample extracts. A Synergi Hydro-RP column (250 4.6 mm i.d.) with a 4 µm particle size (Phenomenex, Lane Cove, NSW, Australia) was guarded by a Phenomenex C18 ODS guard column (4.0 × 2.0 mm i.d.). The volume of injection of samples or standards was 20 µL. The mobile phases A and B were composed of water/acetic acid (98:2, v/v) and acetonitrile/water/acetic acid (55:43:2, v/v/v). The range of gradient profile was 10–25% B (0–20 min), 25–35% B (20–30 min), 35–40% B (30–40 min), 40–55% B (40–70 min), 55–80% B (70–75 min), 80–90% B (75–77 min), 90–100% B (77–79 min), 100–10% B (79–82 min), isocratic 10% B (82–85 min). The flow rate was 0.8 mL/min, and the column temperature was ambient. At the PDA detector, the wavelengths 280, 320, and 370 nm were chosen at the same time. The (2010) version of Empower 3 Software was utilized for device management, gathering data, and chromatography analysis.
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