For detecting metabolites, a triple-quadrupole mass spectrometer (TSQ Vantage,
Thermo Scientific) coupled to an HPLC was used, which allows one to detect each
metabolite simultaneously using multiple reaction monitoring (MRM). Metabolites
were separated chromatographically using a Synergi Fusion column (150 × 2.0 mm,
4 μm, Phenomenex), using a Shimadzu Prominence HPLC (high-performance liquid
chromatography) autosampler coupled to the mass spectrometer. Extracted
metabolites were measured using targeted LC-MS/MS methods, detailed method
described previously 23 (link)42 (link). A library of common metabolites was
constructed using standards, and metabolites were detected using a TSQ Vantage
(Thermo Scientific) triple quadrupole-linear ion trap mass spectrometer for
quantitative optimized detection of daughter ions upon collision-induced
fragmentation of the parent ion. For each metabolite, parameters for
quantitation of the two most abundant daughter ions (that is, two MRMs per
metabolite) were included. To quantify metabolites, the area under each peak was
quantitated by using the Xcaliber software, inspected for accuracy, and
normalized against total ion count, after which relative amounts were
quantified.
Thermo Scientific) coupled to an HPLC was used, which allows one to detect each
metabolite simultaneously using multiple reaction monitoring (MRM). Metabolites
were separated chromatographically using a Synergi Fusion column (150 × 2.0 mm,
4 μm, Phenomenex), using a Shimadzu Prominence HPLC (high-performance liquid
chromatography) autosampler coupled to the mass spectrometer. Extracted
metabolites were measured using targeted LC-MS/MS methods, detailed method
described previously 23 (link)42 (link). A library of common metabolites was
constructed using standards, and metabolites were detected using a TSQ Vantage
(Thermo Scientific) triple quadrupole-linear ion trap mass spectrometer for
quantitative optimized detection of daughter ions upon collision-induced
fragmentation of the parent ion. For each metabolite, parameters for
quantitation of the two most abundant daughter ions (that is, two MRMs per
metabolite) were included. To quantify metabolites, the area under each peak was
quantitated by using the Xcaliber software, inspected for accuracy, and
normalized against total ion count, after which relative amounts were
quantified.
Free full text: Click here
