Sciex 4000 qtrap ms instrument

Standard stock solutions were prepared based on the method description provided by Han et al. (2013) [26 (link)]. The standard stock solutions of all targeted carboxylic acids were prepared in 80% methanol at concentrations of 200–1000 μM. A total of 50 μL of each standard solution or the supernatant of each sample was mixed and reacted with 50 μL of a 200 mM 3-nitrophenylhydrazines (NPH) solution at 30 °C. Following the reaction, 450 μL of water was added to each solution, and 10 μL of the resulting solution was injected into a C18 liquid chromatography (LC) column (2.1 × 100 mm, 1.8 μm) for quantification of the carboxylic acid by ultrahigh LC-multiple reaction monitoring/mass spectroscopy (UPLC-MRM/MS), and ion detection was performed on an Agilent 1290 UHPLC coupled to a Sciex 4000 QTRAP MS instrument (AB Sciex, Concord, ON, Canada) [26 (link)].

A standard solution of all targeted carboxylic acids was prepared using 80% methanol, with concentrations ranging from 200 to 1000 μM. For each sample, 50 µL of both the standard solution and the sample supernatant were mixed with an equal volume of 200 mM 3-nitrophenyl hydrazines (NPH) solution and 150 mM carbodiimide hydrochloride (EDC)-6% pyridine solution. The mixture was then incubated at 30 °C for 40 min. After the reaction, 450 µL of water was added to each solution, and 10 µL of the resulting solution was injected into a C18 liquid chromatography (LC) column (2.1 × 100 mm, 1.8 μm) for quantification of carboxylic acids using ultrahigh LC-multiple reaction monitoring/mass spectroscopy (UPLC-MRM/MS) with (−) ion detection. The UPLC-MRM/MS was performed on an Agilent 1290 UHPLC system coupled to a Sciex 4000 QTRAP MS instrument (AB Sciex, Concord, ON, Canada). The metabolite-dependent parameters used in the UPLC-MRM/MS were based on the procedure described by Han et al. [24 (link)].