The monosaccharide composition of UPPs 1–3 was measured based on the method reported previously. In brief, 5 mg of each sample (UPPs 1–3) in 1 mL of 2.5 M trifluoroacetic acid (TFA) was hydrolyzed at 121 °C for 2 h in a sealed ampule. The hydrolysate was analyzed by ICS-5000 ion chromatography (ThermoFisher Scientific, MA, U.S.A.) coupled with Dionex™ CarboPac™ PA20 column (150 mm × 3 mm, 6.5 μm) and an electrochemical detector. The monosaccharide compositions were authenticated by comparison with the standards’ retention times. The quantitative analysis of each monosaccharide was conducted based on the linear regression equation.
Ics 5000 ion
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ICS-5000+ ion chromatography system is a high-performance liquid chromatography instrument designed for the separation and analysis of ionic compounds. It features advanced hardware components and software control to enable precise and reproducible ion analysis.
Automatically generated - may contain errors
Lab products found in correlation
As ap autosampler, by Thermo Fisher Scientific (2 mentions)
Atc 3 trap column, by Thermo Fisher Scientific (2 mentions)
Ionpac 4x250 mm as11 hc ion exchange analytical column, by Thermo Fisher Scientific (2 mentions)
Ag11 hc 4mm guard column, by Thermo Fisher Scientific (2 mentions)
Asrs 300 4mm in, by Thermo Fisher Scientific (2 mentions)
Citric acid, by Merck Group (2 mentions)
Dionex carbopac pa20 column, by Thermo Fisher Scientific (1 mentions)
Orbitrap fusion tribrid mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Carbopac pa 20, by Thermo Fisher Scientific (1 mentions)
6 protocols using ics 5000 ion
1
Monosaccharide Composition Analysis of UPPs
2
Comprehensive 13C Metabolite Analysis
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Polar fractions were reconstituted in 30 μl nanopure water, and analyzed on a Dionex ICS-5000+ion chromatograph interfaced to an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) operating at a resolution setting of 500,000 (FWHM at m/z 200) on MS1 acquisition to capture any and all 13C isotopologues40 (link). The Orbitrap Fusion was tuned and calibrated according to the manufacturer’s default standard recommendations, to routinely achieve a mass accuracy of 0.5 ppm or better. The chromatograph was outfitted with a Dionex IonPac AG11-HC-4 µm RFIC&HPIC guard (2 × 50 mm) guard column upstream of a Dionex IonPac AS11-HC-4 µm RFIC&HPIC (2 × 250 mm) column. An m/z range of 80–700 except for the organ extracts where the m/z range was 50-750. Peak areas were integrated and exported to Excel via the TraceFinder 3.3 (Themo) software package. Peak areas were corrected for natural abundance distribution of each of the isotopologues41 (link), after which fractional enrichment and µmoles metabolites/g protein were calculated to quantify 13C incorporation into various pathways.
3
Monosaccharide Analysis via Ion Chromatography
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Processed DhPs (5 mg) were hydrolyzed with 1 mL of 2.0 M TFA at 120 °C for 2 h in a sealed tube and the solution was then dried with nitrogen. After rinsing with methanol 3 times, residue of hydrolyzed DhPs was dissolved in ddH2O for analysis. The samples and standards were analyzed using an ICS5000 ion chromatography system (Thermo Fisher, Waltham, MA, USA) equipped with a pulsed amperometric detector. The column was Dionex CarboPac PA20 (150 × 3.0 mm, 10 μm) and the column temperature was 30 °C. An A solvent containing 0.1 M NaOH in water and B solvent containing 0.1 M NaOH and 0.2 M NaAc in water were used to produce a gradient profile from 5-20-40-40-5-5 % B from 0.0-30.0-30.1-45.0-45.1-60.0 min, at a flow rate of 0.5 mL/min. After analysis, chromatographic peaks of respective monosaccharides were manually integrated and the concentrations were calculated using an external calibration curve fitted with linear regression.
4
Quantification of Human Milk Oligosaccharides
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HMOs in milk samples were quantified using HPAEC-PAD with an ICS-5000+ ion chromatography detection system (Thermo Scientific). This system was comprised of a CarboPac PA-1 (4 × 250 mm, 6.5 μm) column, a CarboPac PA-1 (4 × 50 mm) precolumn, and a pulsed amperometric detector with a gold electrode. The analysis was performed according to a previously published method with minor modifications (29 ). HMO standards were included as controls to quantitatively calculate concentrations of 24 HMOs in the milk samples. All HMO standards were purchased from Carbosynth. Table 1 summarizes details of the 24 HMOs tested.
5
Citrate Quantification by Ion Chromatography
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Citrate concentration was analysed by ion chromatography (IC) with suppressed conductivity detection (ICS-5000+ ion chromatographic system; AS-AP autosampler; ATC-3 trap column installed between pump and injection valve; IonPac 4x250 mm AS11-HC ion-exchange analytical column coupled with a AG11-HC 4mm guard column; anion self-regenerating suppressor ASRS 300 (4mm) in auto suppression recycle mode; Dionex, Thermo Fischer Scientific, USA). Ten µL sample was injected. Separation was performed with NaOH in laboratory grade I water as eluent, with a flow rate of 1.5 mL min H 2 O. An external standard of citrate (Sigma Aldrich, citric acid, ACS reagent, ≥ 99.5%) was used for identification (retention time) and quantification after the construction of a calibration curve (R 2 =0.99).
6
Citrate Quantification by Ion Chromatography
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Citrate concentration was analysed by ion chromatography (IC) with suppressed conductivity detection (ICS-5000+ ion chromatographic system; AS-AP autosampler; ATC-3 trap column installed between pump and injection valve; IonPac 4x250 mm AS11-HC ion-exchange analytical column coupled with a AG11-HC 4mm guard column; anion self-regenerating suppressor ASRS 300 (4mm) in auto suppression recycle mode; Dionex, Thermo Fischer Scientific, USA). Ten µL sample was injected. Separation was performed with NaOH in laboratory grade I water as eluent, with a flow rate of 1.5 mL min -1 and constant temperature of 30°C, with the following gradient: E1 (NaOH 1mM) and E2 (NaOH 60mM); 0-15 min 20% E1 80% H2O, 15-25 min 15% E2 85% H2O, 25-35 min 30% E2 70% H2O, 35-45 min 60% E2 40% H2O. An external standard of citrate (Sigma Aldrich, citric acid, ACS reagent, ≥ 99.5%) was used for identification (retention time) and quantification after the construction of a calibration curve (R 2 =0.99). Seven standards with the following concentrations were used: 2.60. 5.20, 26, 52, 130, 260, 520 µmol L -1 . Limit of quantification was 0.014 µmol L -1 .
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