Betaine and proline in intra/extracellular metabolites was analyzed by LC–MS/MS under multiple reaction monitoring (MRM) methods. Ten microliters of each prepared sample was analyzed by an Agilent 6420 Triple Quadrupole LC/MS (CA, USA) coupled to an Agilent 1260 Infinity Binary LC (CA, USA). Separations were performed using an Agilent Zorbax HILIC Plus column (4.6 × 100 mm, 3.5 μm) and a flow rate of 500 μL min−1. Eluent A was water containing 0.1% v/v formic acid, and B was 100% acetonitrile. The gradient applied was as follows: 0–3 min, 80% eluent B; 3–10 min, linear decrease to 20% eluent B; 10–13 min, 20% eluent B; 13–14 min, linear increase to 80% eluent B; 14–23 min, 80% eluent B. The flow rate was at 0.5 mL min−1. The capillary temperature was 300 °C, and an electrospray ionization spray voltage was used at 4 kV.
Zorbax hilic plus column
Manufactured by Agilent Technologies
Sourced in Canada
The Zorbax HILIC Plus column is a hydrophilic interaction liquid chromatography (HILIC) column designed for the separation and analysis of polar and hydrophilic compounds. The column utilizes a proprietary stationary phase to provide selectivity for a wide range of analytes.
Automatically generated - may contain errors
Lab products found in correlation
1260 infinity binary lc, by Agilent Technologies (1 mentions)
6420 triple quadrupole lc ms, by Agilent Technologies (1 mentions)
Vanquish hplc system, by Thermo Fisher Scientific (1 mentions)
Orbitrap q exactive hf x mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Positive and negative ion calibration solutions, by Thermo Fisher Scientific (1 mentions)
Betaine hydrochloride, by Merck Group (1 mentions)
Hplc grade solvents, by Thermo Fisher Scientific (1 mentions)
Vanquish horizon uhplc system, by Thermo Fisher Scientific (1 mentions)
Prominence uflc system, by Shimadzu (1 mentions)
Api4000 mass spectrometer, by Shimadzu (1 mentions)
5 protocols using zorbax hilic plus column
1
Betaine and Proline Quantification by LC-MS/MS
2
HILIC-MS-based Metabolomics Analysis
3
Urinary Metabolite Profiling by LC-MS/MS
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A panel of 17 urinary metabolites were measured with urine samples collected at baseline and post-treatment from 4 of the 5 study participants. Mass spectrometry (MS) protocols were slightly modified from83 (link),84 (link). Briefly, for LC/MS/MS we utilized a Thermo Q Exactive HF-X Orbitrap mass spectrometer with a Thermo Vanquish HPLC system, auto-injecting a 5 μL urine sample. For chromatography, we used an Agilent ZORBAX HILIC PLUS column with a mobile phase of components A (10 mM ammonium bicarbonate, 0.05% formic acid in Millipore water, pH=4.2) and B (0.1% formic acid in acetonitrile), with flow rate flow rate of 0.3 mL/min. The gradient ran for 12 minutes. MS settings included a 4300 V spray voltage, nitrogen gas, ion transfer tubes, and auxiliary heater at 320°C and 30°C, respectively. PRM mode was positive polarity. Data were processed using Xcalibur Quant Browser, comparing peak areas to internal standards (A/IS ratio) and a standard curve (0.01–100 μM) for concentration determination.
4
High-Resolution LC-MS Analysis of Betaine
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High-resolution LC−MS analysis was performed on a ThermoFisher Scientific Vanquish Horizon UHPLC System coupled with a Thermo Q Exactive HF hybrid quadrupole-orbitrap high-resolution mass spectrometer equipped with a HESI ion source. 1 μL extract was injected and separated using a water-acetonitrile gradient on an Agilent Zorbax Hilic Plus column (150 mm × 2.1 mm, particle size 1.8 μm) maintained at 40°C with a flow rate 0.3 mL/min. HPLC grade solvents were purchased from Fisher Scientific. Solvent A: 0.1% formic acid in water; Solvent B: 0.1% formic acid in acetonitrile. Analytes were separated using a gradient profile as follows: 2 min (95% B) → 20 min (50% B) → 22 min (10% B) → 25 min (10% B) → 27 min (95% B) →30 min (95% B). Mass spectrometer parameters: spray voltage 3.0 kV, capillary temperature 380°C, prober heater temperature 300°C; sheath, auxiliary, and spare gas 60, 20, and 2, respectively; S-lens RF level 50, resolution 240,000 at m/z 200, AGC target 3 × 106. The instrument was calibrated with positive and negative ion calibration solutions (ThermoFisher). Each sample was analyzed in positive and negative modes with m/z range 100–700. As a reference for betaine, betaine hydrochloride was purchased from Sigma-Aldrich. HRMS (ESI) m/z: calculated for C5H12NO2+, [M + H]+: 118.0863, found: 118.0860.
5
Quantification of Citrulline by LC-MS/MS
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