A targeted, quantitative metabolite profiling approach was employed that combined RPLC–MS with DFI–MS (RPLC–DFI–MS/MS) to determine the concentrations of a wide range of metabolites. These analyses were performed using an in-house quantitative metabolomics assay (TMIC Prime) [73 (link),74 (link)]. This assay was used with an Agilent 1260 series ultra high-performance liquid chromatography (UHPLC) system (Agilent Technologies, Palo Alto, CA, USA) coupled with an AB SCIEX QTRAP® 4000 mass spectrometer (Sciex Canada, Concord, ON, Canada) to identify and quantify up to 143 compounds (including amino acids, biogenic amines, glucose, organic acids, acylcarnitines, PCs, LysoPCs, SMs, and SM(OH)s). The absolute quantification of water-soluble compounds including amino acids, organic acids, and biogenic amines was ensured by using two separate UHPLC injections with C18 column separations. On the other hand, glucose and the various lipid classes (acylcarnitines, PCs, LysoPCs, SMs, etc.) are measured by the column-free DFI method (both +ve and –ve mode). While initially designed and calibrated for human metabolomics studies, the measurable ranges of metabolite concentrations available through the TMIC Prime assay match very closely with the known or expected metabolite concentrations in bovine biofluids and tissues (as determined via orthogonal NMR experiments and high levels of agreement with published literature data).
The detection of each metabolite in the TMIC Prime assay relies on multiple reaction monitoring (MRM). The assay incorporates both isotope-labelled internal standards and other quality control (QC) standards into its 96-well filter plate to ensure accurate compound quantification. The first 14 wells in the 96-well plate are used for building calibration curves and QCs, while the other 82 wells are used for sample analysis. For all biofluids analyzed with this assay, both the original sample (without dilution) and diluted samples (10×) were analyzed to ensure correct calibration and quantification. In brief, 10 µL of each sample (the filtered biofluid or the water-soluble or lipid-soluble extract of the tissue) was carefully pipetted into an appropriate sample well of the upper 96-well filter plate and dried using a stream of nitrogen gas. Amino acid and biogenic amine derivatization were done by adding 50 µL of a 5% solution of phenyl-isothiocyanate (PITC) to each well and incubating for 20 min. After incubation and PITC derivatization, the samples were dried down using a nitrogen gas evaporator. The metabolites were then extracted by adding an ammonium acetate/methanol mixture (5 mM ammonium acetate in methanol) to the upper 96-well filter plate, shaking at 330 rpm for 30 min, and then centrifuging the plates so that the extract bled into the lower 96-deep well plate.
The resulting extract was split for RPLC–MS (150 µL) and DFI–MS (150 µL) analyses followed by a dilution step with 150 µL of water for RPLC–MS analysis and with 400 µL of the MS running solvent for DFI–MS analysis. All LC–MS analyses were conducted on an AB SCIEX QTRAP® 4000 mass spectrometer equipped with an Agilent 1260 series UHPLC system. The Analyst software 1.6.2 (Concord, ON, Canada) was used to control the entire assay’s workflow.
The detection of each metabolite in the TMIC Prime assay relies on multiple reaction monitoring (MRM). The assay incorporates both isotope-labelled internal standards and other quality control (QC) standards into its 96-well filter plate to ensure accurate compound quantification. The first 14 wells in the 96-well plate are used for building calibration curves and QCs, while the other 82 wells are used for sample analysis. For all biofluids analyzed with this assay, both the original sample (without dilution) and diluted samples (10×) were analyzed to ensure correct calibration and quantification. In brief, 10 µL of each sample (the filtered biofluid or the water-soluble or lipid-soluble extract of the tissue) was carefully pipetted into an appropriate sample well of the upper 96-well filter plate and dried using a stream of nitrogen gas. Amino acid and biogenic amine derivatization were done by adding 50 µL of a 5% solution of phenyl-isothiocyanate (PITC) to each well and incubating for 20 min. After incubation and PITC derivatization, the samples were dried down using a nitrogen gas evaporator. The metabolites were then extracted by adding an ammonium acetate/methanol mixture (5 mM ammonium acetate in methanol) to the upper 96-well filter plate, shaking at 330 rpm for 30 min, and then centrifuging the plates so that the extract bled into the lower 96-deep well plate.
The resulting extract was split for RPLC–MS (150 µL) and DFI–MS (150 µL) analyses followed by a dilution step with 150 µL of water for RPLC–MS analysis and with 400 µL of the MS running solvent for DFI–MS analysis. All LC–MS analyses were conducted on an AB SCIEX QTRAP® 4000 mass spectrometer equipped with an Agilent 1260 series UHPLC system. The Analyst software 1.6.2 (Concord, ON, Canada) was used to control the entire assay’s workflow.
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