HPLC analysis was carried out on a model Accela ultra HPLC system (Thermo Fisher, San Jose, CA) consisting of a Kinetex C18 column (150 × 3 mm, 2.6 μm) coupled to a UV/VIS photo diode array detector (Thermo Scientific, San Jose, CA). Gradient elution was performed at a flow rate of 250 μL/minute using a linear gradient with MeOH (A) against 5mM ammonium bicarbonate in water (B) starting at 20% A, holding for 1 minute then linear gradient to 60% A over 13 minutes linear gradient, back to 20% A over 2 minutes, then holding for 8 minutes for equilibration. Detection was performed from 300-600 nm and data were predominantly extracted from the 488 nm trace.
Kinetex c18 column
Manufactured by Thermo Fisher Scientific
Sourced in Germany
The Kinetex C18 column is a high-performance liquid chromatography (HPLC) column designed for the separation and analysis of a wide range of analytes. It features a silica-based stationary phase with a C18 modification, providing effective reversed-phase separation capabilities. The column is characterized by its reliable performance and consistent results.
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2 protocols using kinetex c18 column
1
HPLC Analysis of Compound Separation
2
UHPLC-MS/MS Quantification of Analytes
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Analytes were separated on a Phenomenex Kinetex C18 column (100 x 2.1 mm, 2.6 um) using a Thermo Fisher Ultimate 3000 high performance liquid chromatography (UHPLC) system and detected by an Applied Biosystems 5500 Q Trap linear ion trap triple quadrupole mass spectrometer (Sciex, Darmstadt/ Germany) with Analyst software (Version 1.6). The following mobile phase gradient of eluent A and B was adjusted: 0-1 min: 2% B; 1-2 min: increase to 40% B; 2-9.3 min: increase to 60% B; 9.3-9.5 min: increase to 98% B; 9.5-10.5 min: holding at 98% B; 10.5-10.8 min: decrease to 2% B; 10.8-12 min: holding at 2% B. The flow rate was 0.4 mL/min. The column temperature was maintained at 35°C and the injection volume was 10 µL. The MS instrument was operated in positive electrospray ionization and multiple reaction monitoring mode. Quantification was achieved using the most abundant transition of each precursor to the respective product ion and two MRM transitions served for identification. Further MS operation parameters were as previously reported by Steuer et al. [36] (link).
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