The extracted metabolites (three biological and two technical replicates per treatment) were applied with C18, 2.7 μm (particle size) HPLC column, 15 cm × 100 μm ID (Supelco analytical, Sigma-Aldrich) and 96 min gradient ranging with 400 nL min-1 flow rate. The eluent was analyzed by LTQ-Orbitrap XL Hybrid Ion Trap-Orbitrap Mass Spectrometer (Thermo Fisher Scientific, Germany) with full scan range 130–1,800 m/z, enabled dynamic exclusion, exclusion duration 60 s, exclusion list size 500, repeat duration 30 s, repeat count 1, CID activation type and minimum required signal 50,000.
Supelco analytical
Manufactured by Merck Group
Supelco analytical is a line of laboratory equipment and consumables offered by Merck Group. It provides a range of products for analytical applications, including chromatography columns, sample preparation devices, and other accessories. The core function of Supelco analytical is to facilitate the analysis and separation of chemical compounds in various research and industrial settings.
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2 protocols using supelco analytical
1
Metabolite Profiling by HPLC-MS
2
UHPLC-MS Quantification of Sugars
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Ultra-high performance liquid chromatography Orbitrap Exactive – Thermo Scientific (UHPLC-MS) was performed using a NH4 column apHera 15 cm × 4.6 mm (Supelco Analytical– Sigma Aldrich). UHPLC parameters were: 40 °C temperature column, 10 μl injection volume, 0.3 ml/min flow injection, isocratic mode (ACN: H2O/70:30) and 19 min total retention time. Mass spectrometer parameters were: 2.5 KV spray voltage, 10 eV collision energy, negative mode ESI, 100 °C capillary temperature, 250 °C aux gas heater temperature, and a resolution of 70000.
The limit of quantification for the instrument was established with glucose, sucrose and fructose standards (Sigma Aldrich – Merck) and calibration curves for each of the main sugars. The range of calibration curve was from 25 to 200 μg/L for sucrose and from 50 to 400 μg/L for glucose and fructose, respectively. The lower limit of quantification was 9.82 μg/L glucose, 8.05 μg/L fructose and 10.69 μg/L sucrose, and the lower limit of detection was 2.95 μg/L glucose, 2.42 μg/L fructose and 3.2 μg/L sucrose.
The limit of quantification for the instrument was established with glucose, sucrose and fructose standards (Sigma Aldrich – Merck) and calibration curves for each of the main sugars. The range of calibration curve was from 25 to 200 μg/L for sucrose and from 50 to 400 μg/L for glucose and fructose, respectively. The lower limit of quantification was 9.82 μg/L glucose, 8.05 μg/L fructose and 10.69 μg/L sucrose, and the lower limit of detection was 2.95 μg/L glucose, 2.42 μg/L fructose and 3.2 μg/L sucrose.
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