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Atlantis hilic silica column

Manufactured by Waters Corporation
Sourced in United States, Canada

The Atlantis HILIC silica column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of polar and hydrophilic compounds. The column utilizes a hydrophilic interaction liquid chromatography (HILIC) stationary phase to provide effective retention and separation of these types of analytes.

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26 protocols using atlantis hilic silica column

1

Quantifying Serum TMAO Levels

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In this study, serum TMAO was measured by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The sample was prepared by adding 10 uL of TMAO d9 (Toronto Research Chemicals Inc., Toronto, Canada) to 100 uL serum; 300 uL of acetonitrile precipitated the protein, vortexed for 1 min, centrifuged at 1,000 rpm, 4°C for 5 min; and 200 uL of the remaining supernatant was injected into a Waters Atlantis HILIC Silica column for analysis.
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2

HPLC/ESI-QTOF-MS Analysis of Compounds

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The separation samples were analyzed by an HPLC/ESI-QTOF-MS system in positive ion mode using a 6530B Accurate-Mass-QTOF-MS mass spectrometer with an ESI-Jet Stream® ion source and Atlantis HILIC silica column (150 × 2.1mm, dp = 3μm) (Waters Milford, MA, USA). The liquid chromatography system was equipped with DAD (diode array detector), autosampler, binary gradient pump, and column oven. Gradient of solvents: acetonitrile (95%) with 10 mM ammonium formate (0.2%) (solvent A) and acetonitrile (50%) with 10 mM ammonium formate (0.2%) (solvent B) was used as the mobile phase. The following gradient procedure was adopted: 0–10 min, 100% using solvent A; 10–40 min, 92% A and 8% B; 40–45 min, 64% A and 36% of solvent B. ESI-QTOF-MS analysis was performed according to the following parameters: total time of analysis was 45 min, with a stable flow rate at 0.25 mL/min. Injection volume for analyzed extracts was 10 μL. Dual spray jet stream ESI, (+)-positive ion mode, gas (N2) flow rate: 12 L/min., nebulizer pressure: 35 psig, vaporizer temp.: 300 °C; m/z range 100–1000 mass units, with acquisition Mode AutoMS2, collision induced dissociation (CID): 0, 10, 40 and 60 eV with MS scan rate 1 spectrum per s, 2 spectra per cycle, skimmer: 65 V, fragmentor: 150 V and octopole RF Peak: 750 V.
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3

HILIC-Based LC-MS/MS Analysis of Sialic Acids

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LC–MS/MS analyses were performed on an LC-20 AC HPLC system (Shimadzu, Kyoto, Japan) coupled with a Sciex API 4000 Q Trap mass spectrometer (AB Sciex, Foster City, CA, USA). HILIC chromatography was established on an XBridge Amide column (3.5 μm, 100 × 2.1 mm; Waters Co., Milford, MA, USA) for ManNAc (method 1), and on an Atlantis HILIC Silica column (5 μm, 50 × 3.0 mm; Waters Co., Milford, MA, USA) for Neu5Ac (method 2) and CMP-Neu5Ac (method 3). The HPLC columns were kept at ambient temperature.
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4

Quantification of TMAO in Plasma and Urine

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The concentrations of TMAO in the fasting plasma and urine were detected on a liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) system consisting of an Agilent 1260 high‐performance liquid chromatography (HPLC) and 6420 triple quadrupole mass spectrometer with an electrospray ionization source (ESI) (Agilent). One hundred microliters of the specimen was mixed with internal standard (IS) working solution (d9‐TMAO), and 300 μL of acetonitrile was added to the sample and then vortexed, and the mixture was centrifuged at  15,294 × g for 5 min at 4°C. Finally, the supernatant was transferred into an autosampler vial, and 2 μL was injected into the HPLC‐MS/MS for analysis. Chromatographic separation was performed on the Waters Atlantis HILIC Silica column (3.0 × 100 mm, 3.0 μm), which was equilibrated with 30% solution A (10 mmol/L ammonium formate in water, pH 3.0) and 70% solution B (acetonitrile) under isocratic elution with the flow rate of 0.3 ml/min. The ESI was operated in positive mode, and the mass spectrometer was run in multiple‐reaction monitoring mode. The settings for the mass transition of TMAO and IS were m/z 76.1 → 58.3 and m/z 85.1 → 66.3.
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5

Quantitative analysis of TTX and analogue

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The TTX used in this study was commercially obtained (FUJIFILM Wako Pure Chemicals, Osaka, Japan), while 5,6,11-trideoxyTTX was chemically synthesized [52 (link)]. TTX extracts were prepared from the homogenates for the left half the body with skin of the pufferfish and toxic goby, and they were filtered through a membrane of pore-size 0.45 μm (SupraPure Syringe Filter, Recenttec, Taipei, Taiwan). LC–MS experiments were performed on a Shimadzu LC-20AD solvent delivery system and a SCIEX X500R Q-TOF Mass Spectrometer with an ESI ion source. The filtered solution was subjected to LC–MS/MS analysis with multiple reaction monitoring (MRM) using an Atlantis HILIC Silica column (Waters, Milford, MA, USA) with gradient elution, following the method used in a previous study [28 (link)]. The optimized transitions for TTX were m/z 320 > 302 for qualification and m/z 320 > 162 for quantification, and those for 5,6,11-trideoxyTTX were m/z 272 > 254 and m/z 272 > 162. The calibration curve generated with 1–50 ng/mL of TTX standards (TTX and 5,6,11-trideoxyTTX) showed good linearity and precision. The other TTX analogues were not analyzed because they were in very small amounts.
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6

Quantitative Determination of Carnosine and Anserine

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The carnosine and anserine contents were determined using the method of Mora et al. (2007) (link) with slight modifications. Breast meat (0.1 g) was homogenized with 900 µL of 0.01 N HCl for 2 min and centrifuged at 12,000 × g at 4°C for 10 min. Supernatants were filtered through a 0.45-μm syringe filter. The filtrate (250 µL) was mixed with 750 µL of acetonitrile and stored at 4°C for 20 min. Then, the sample was centrifuged at 10,000 rpm at 4°C for 10 min, filtered through a 0.2-μm syringe filter, and stored at −20°C until use. carnosine and anserine were separated on an Atlantis HILIC silica column (4.6 × 150 mm, 3 µm, Waters Corporation, Milford, MA) equipped with high-performance liquid chromatography (HPLC 1260, Agilent Technology, Santa Clara, CA). Mobile phase A containing 0.65 mM ammonium acetate in 75% acetonitrile at pH 5.5 and mobile phase B containing 4.55 mM ammonium acetate in 30% acetonitrile at pH 5.5 were used. The separation conditions were determined by the linear gradient of phase B from 0 to 100% for 13 min at 1 mL/min. Twenty microliters of the sample obtained after the addition of acetonitrile were injected. Dipeptides were detected at a wavelength of 210 nm. Quantification was performed using the external standard carnosine and anserine (Sigma-Aldrich, St. Louis, MO) at 25°C.
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7

Quantitative Analysis of Glycosphingolipids

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Quantitative analysis of SLs was performed by liquid chromatography and high-resolution or tandem mass spectrometry (LC/HR-MS or LC/MS/MS). Tissue homogenization, GlcCer and PC extractions, and analyses were performed as previously described with modifications for GM3 analysis as follows (67 ). Briefly, 10 μL of tissue homogenate (liver, brain or kidney) or 10 μL of serum were extracted with a solvent consisting of equal volume of acetonitrile and methanol. GM3 was separated with isocratic elution using a mobile phase consisting of acetonitrile:methanol:water (61.75:33.23:5 vol/vol) in 10 mM of ammonium acetate, pH 6.8 for 2 min using a Waters Acquity UPLC and Waters Atlantis HILIC Silica column (3 μm, 2.1 × 100 mm) at room temperature. The flow rate was 0.4 mL/min. The eluent was analyzed by a Q Exactive mass spectrometer (ThermoFisher Scientific) equipped with a Heated ElectroSpray Ionization source. Data were collected in full MS negative mode using the following parameters: resolution of 70 000, scan range of 1100–1700 m/z, AGC target of 3e6, and max IT of 256 ms. Quantitation of different isoforms with various acyl chain lengths was achieved by MS1 extraction of ion chromatograms. GM3 standard was purchased from Matreya, LLC.
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8

Qualitative LC/MS Analysis of PAs–PANOs

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The PAs–PANOs extract was examined by qualitative LC/MS analysis. The scientific equipment used was an Agilent | 6500 Series Accurate-Mass Quadrupole Time-of-Flight (Agilent Technologies Inc., Santa Clara, CA, USA) device equipped with ESI-Jet Stream ion source and Atlantis HILIC silica column (150 × 2.1 mm, dp = 3 μm) (Waters Milform, MA, USA). The chromatograph used a diode array detector autosampler, dual grading pump, and column heater. An RP-18 stable phase was used in conjunction with a gradient elution mobile phase of 0.1% formic acid in MeOH with a stable flow of 0.25 mL/min. For this particular experiment, a positive charge ESI ion source was used [40 (link)].
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9

Quantitative LC-MS/MS Analysis of NAD+ and NMN

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LC–MS/MS analysis was conducted using the NexeraX2 system (Shimadzu Corporation, Kyoto, Japan) coupled with the 4000 Qtrap system (AB Sciex, Framingham, MA, USA). The samples (3–5 μL) were injected into an Atlantis HILIC Silica column (5 μm, 4.6 mm × 50 mm) (Waters, Milford, MA, USA) and maintained at 50 °C. Elution was conducted at a flow rate of 1 mL/min under a gradient condition of 0.05% (v/v) formic acid containing 10 mmol/L ammonium formate aqueous solution (mobile phase A) and acetonitrile (mobile phase B). The following two types of gradient conditions were used: (1) optimization of the extraction buffer, 0–5 min: 97–40% B, 5–7 min: 40% B, 7–7.1 min: 97% B, and 7.1–10 min: 97% B; (2) other studies, 0–1 min: 80% B, 1–3 min: 80–40% B, 3–4.5 min: 40% B, 4.5–4.6 min: 40–80% B, and 4.6–7 min: 80% B. Multiple reaction monitoring mode (positive ion mode) was used to monitor ions as follows: precursor ion/product ion; NAD+ (664.1/136.1), d4-NAD+ (668.2/136.1), NMN (335.1/123.1), d4-NMN (339.1/126.9), and d5-NMN (340.1/127.0). As NAD+ and NMN are endogenous substances, calibration standards were prepared using water instead of blood at 10 concentrations in the range of 0.25–250 μM (optimization of the extraction buffer) or 0.25–200 μM (other studies).
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10

Quantitative Sphingolipid Analysis by LC-MS/MS

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Quantitative analysis of sphingolipids was performed by using liquid chromatography and tandem mass spectrometry. Briefly, tissues were homogenized in water to give a final concentration of approximately 100 mg/mL (w/v). The homogenate was extracted with 1 mL of a solution of acetonitrile:methanol:water (97:2:1, v/v/v) at room temperature. Extracts were injected onto an Atlantis HILIC silica column (Waters Corp, Milford, MA) for separation of GlcCer and GalCer, and these molecules were detected by using multiple reaction monitoring (MRM) mode tandem mass spectrometry with an AB Sciex API-5000 mass spectrometer (AB Sciex, Framingham, MA). For other lipid analysis, extracts were injected onto an Acquity BEH C8 column (Waters Corp., Milford, MA), and MRM mode detection was performed using an AB Sciex API-5000 mass spectrometer. For GlcSph analysis, homogenate was extracted with 1 mL of acetonitrile:methanol:water (48.5:50.5:1, v/v/v), and extracts were injected onto an Acquity BEH HILIC column to resolve GlcSph from psychosine, (Waters Corp., Milford, MA) and detected using MRM mode with an Agilent 6490 mass spectrometer. Except for phosphatidylcholine, all analytes were quantitated against standards obtained from Matreya, LLC (Pleasant Gap, PA).
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