Separation was performed using a Shimadzu LC-20AD chromatograph (Shimadzu Corporation, Kyoto, Japan). Samples were kept in the autosampler in vials at 4°C, and 5 μL samples were injected on the column. The Thermo Accucore C18 column (2.6 μm, 100 × 4.6 mm, Thermo Fisher Scientific Inc. Waltham, MA, USA) was kept at 35°C. Aqueous phase A was deionized water containing 0.1% FA as a modifier. Organic phase B was 100% MeOH. Starting gradient conditions were 39% A/61% B from 0 to 1 min, reaching 14% A/86% B at 2 min and maintaining for 5.5 min, then returned to 39% A/61% B at 8 min, and retained 3.5 min for equilibration. The flow rate was set at 0.3 mL/min. For MS/MS analysis, a QTRAP 4000 mass spectrometer was operated in electrospray ionization- (ESI-) positive ion multiple reaction monitoring (MRM) mode with the curtain gas set to 25 psi, ion spear voltage set to 5000 V, source temperature set to 600°C, ion source gas set to 170 psi, ion source gas set to 270 psi, declustering potential set to 80 V, entrance potential set to 10 V, and collision cell exit potential set to 10 V. Other compound specific settings were listed in Table 1 .
Thermo accucore c18 column
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Thermo Accucore C18 column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of compounds. It features a solid-core particle technology that provides efficient and rapid separations. The column is packed with 2.6 μm diameter particles and is suitable for use in both normal-phase and reversed-phase HPLC applications.
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2 protocols using thermo accucore c18 column
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LC-MS/MS Quantification of Analytes
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HPLC-MS/MS Analysis of 14 Analytes
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The extracting solutions were analyzed using a Thermo Q-Exactive, equipped with an electrospray ionization interface (ESI), which was coupled to a UltiMate 3000 UPLC. A Thermo Accucore C18 column, 2.6 μm, 4.6 × 150 mm (Thermo Scientific), was installed and operated at 25°C, and the injection volume was 10 μL.
The mobile phase consisted of (A) 0.05% formic acid in water and (B) methanol, and the gradient was applied: 0-4 min, 5%B-20%B; 4-5.5 min, 20%B-40%B; 5.5-10.5 min, 40%B-100%B; 10.5-12.9 min, 100%B; 12.9-15 min, 100%B-5%B. The flow rate was maintained at 0.4 mL/min, and at this condition of HPLC, 14 analytes were well separated (Figure 1). Q-Exactive work in simultaneous acquisition of positive and negative modes: the spray voltage of the ESI interface was set to ±2500 V. The capillary temperature was set to 200°C and the vaporizer temperature to 450°C. While the sheet gas pressure (nitrogen) was 50 units, the auxiliary gas flow was 12 units. The instrument work in parallel reaction monitor modes used a mass resolving power of 17 500 full width at half maximum, covering the mass range of 50-750 Da, while the isolation width of the precursor was 4 Da and collision energy was 35 eV. The instrumental parameters used for the targeted acquisition are given in Table 1. The C-trap capacity was set to 5 000 000 charges, and a maximum ion collection time of 15 ms was defined.
The mobile phase consisted of (A) 0.05% formic acid in water and (B) methanol, and the gradient was applied: 0-4 min, 5%B-20%B; 4-5.5 min, 20%B-40%B; 5.5-10.5 min, 40%B-100%B; 10.5-12.9 min, 100%B; 12.9-15 min, 100%B-5%B. The flow rate was maintained at 0.4 mL/min, and at this condition of HPLC, 14 analytes were well separated (Figure 1). Q-Exactive work in simultaneous acquisition of positive and negative modes: the spray voltage of the ESI interface was set to ±2500 V. The capillary temperature was set to 200°C and the vaporizer temperature to 450°C. While the sheet gas pressure (nitrogen) was 50 units, the auxiliary gas flow was 12 units. The instrument work in parallel reaction monitor modes used a mass resolving power of 17 500 full width at half maximum, covering the mass range of 50-750 Da, while the isolation width of the precursor was 4 Da and collision energy was 35 eV. The instrumental parameters used for the targeted acquisition are given in Table 1. The C-trap capacity was set to 5 000 000 charges, and a maximum ion collection time of 15 ms was defined.
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