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These extracts were preserved at −20°C until used for sample derivatization.
These samples were preserved at −20°C until used for sample derivatization.
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The crude extract was filtered through 0.4 μm centrifuge tubes and the supernatant was used for sample cleanup and derivatization with EZ faast LCMS kit for free amino acids from Phenomenex, according to the kit protocol.
The use of the universal ELSD avoids the need for sample derivatization or use of specific detector such as pulsed amperometry.
The use of a universal detector avoids the need for sample derivatization or use of specific detector based on pulsed amperometry described to be difficult in routine assays.
The protocols for sample derivatization that utilize DNFB reagent include addition of acetonitrile [ 37, 38], which considerably improves the solubility of DNFB in aqueous samples.
The supernatants obtained from the deproteinized samples were diluted 1/750 in double-distilled water and then 20 µl was used for the derivatization procedure.
A mixture of isooctane/MSTFA 19/1 (v/ v) was used for manual derivatization and a mixture of isooctane/pyridine/MSTFA 14/5/1 (v/ v/ v) for automated derivatization.
An AccQ-Tag Ultra Derivatization Kit (Waters, Milford, MA, USA) was used for the derivatization of arginine and citrulline, and the derived compounds were further quantified by UPLC.
For derivatization of the sample 0.01 ml of the prepared reagent solution was used for each sample which contained 0.8 ml of a buffer (0.2 M, pH 8.8) and 0.01 ml of the supernatant.
The AccQTag Ultra Derivatization Kit (Nihon Waters K. K., Tokyo, Japan) was used for derivatization of free amino acids and ammonium.
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