The AbsoluteIDQ Bile Acid Kit (Biocrates Life Sciences AG) was used for bile acid analyses. Liquid chromatography-mass spectrometry (LC-MS/MS) measurements were carried out by MRM acquisition on a Waters Acquity UPLC System and a QTRAP 5500 (AB Sciex). Data were processed with Analyst Software (1.6.2) and MetIDQ Software (Biocrates Life Sciences AG). For measurements of amino acids and amines, the AbsoluteIDQ p180 Kit (Biocrates Life Sciences AG) was used on a QTRAP mass spectrometer (MS) applying electrospray ionization (ESI) (ABI Sciex API5500Q-TRAP). After separation through a precolumn (Security Guard, Phenomenex, C18, 4 × 3 mm; Phenomenex) and hyphenated reverse phase column (Agilent, Zorbax Eclipse XDB C18, 3.0 × 100 mm, 3.5 µm), analytes were quantified by multi reaction monitoring (MRM) which was standardized by applying spiked-in isotopically labelled standards in positive and negative mode. For data processing, MetIQ software (Biocrates Life Sciences AG) was used. The isotope-labeled chemical derivatization method described by Han et al. [89 (link)] was modified for quantification of SCFA. SCFA were chromatographically separated on an Acquity UPLC BEH C18 column (1.7 μm) (Waters) using H2O (0.01% FA) and acetonitrile (0.01% FA). Analytes were quantified and identified by the scheduled MRM method.
Metiq software
Manufactured by Biocrates
MetIQ software is a data analysis platform developed by Biocrates for the processing and analysis of metabolomics data. The software provides tools for data management, statistical analysis, and visualization of metabolomics data.
Automatically generated - may contain errors
Lab products found in correlation
Absolute idq p180 kits, by Biocrates (3 mentions)
Qtrap 5500, by AB Sciex (2 mentions)
Absoluteidq p180 kit, by Biocrates (2 mentions)
Acquity uplc beh c18 1.7 μm column, by Waters Corporation (1 mentions)
Acquity uplc system, by AB Sciex (1 mentions)
Absoluteidq bile acid kit, by Biocrates (1 mentions)
Zorbax eclipse xdb c18, by Agilent Technologies (1 mentions)
Metidq software, by Biocrates (1 mentions)
Prominence uflc xr, by Shimadzu (1 mentions)
Sciex qtrap 6500, by AB Sciex (1 mentions)
Absoluteidq 180 kit, by Biocrates (1 mentions)
Qtrap 6500, by AB Sciex (1 mentions)
6 protocols using metiq software
1
Bile Acid, Amino Acid, and SCFA Quantification
2
Targeted Metabolomic Analysis of Jejunal Citrulline
3
Targeted Metabolomic Analysis of Plasma Samples
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The targeted metabolomic analysis has been described previously [25] (link). We used the Biocrates Absolute-IDQ P180 kits (Biocrates, Life Science AG, Innsbruck, Austria), a validated targeted assay that allows for simultaneous detection and quantification of 188 metabolites (40 acylcarnitines, 21 amino acids, 21 biogenic amines, 15 sphingolipids, 90 glycerophospholipids, and 1 hexose) in 10 μL of plasma samples in a high-throughput manner. Please refer to Supplementary Method for the details of the analysis. We used seven p180 kits to analyze all the plasma samples, once for each sample. As part of the quality control, three concentrations of quality controls (1 QC1, 4 QC2, and 1 QC3) were included in each kit to monitor imprecisions (% coefficient variances [CVs]) of measuring these 188 metabolites. We calculated means, standard deviations (SDs), and CVs of 3 levels of QCs for all the 188 metabolites, and imprecisions of >83% of the metabolites were less than 25% for all three concentrations of quality controls (Supplementary Table 1 ). Initial data analysis was performed using the MetIQ software (Biocrates). The targeted metabolomic profile was measured in blood samples obtained at the ARIC Brain MRI visit in 2004–2006 and repeated in blood samples obtained at the ARIC visit 5/ARIC-NCS in 2011–2013.
4
Targeted Metabolomics Analysis of Plasma
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Targeted metabolomics analysis of plasma samples was performed using the Biocrates Absolute-IDQ P180 kits (Biocrates; Life Science AG, Innsbruck, Austria). The plasma samples were processed as per the manufacturer instructions and analyzed on a triple-quadrupole mass spectrometer (AB Sciex QTRAP 6500). As part of the quality control, three concentrations of quality controls were included in each kit to monitor imprecisions (% coefficient variances [CVs]) of measuring these 188 metabolites. Means, standard deviations (SDs), and CVs of all the 188 metabolites in seven kits (the number of kits needed for analysis of the 441 samples) were calculated, and imprecisions >80% of the metabolites were <20% for all three concentrations of QCs (Supplementary Table 1 ). Data analysis was performed using the MetIQ software (Biocrates).
5
Targeted Quantitative Metabolomics Approach
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We performed a targeted quantitative approach using a combined direct flow injection and liquid chromatography (LC) tandem mass spectrometry (MS/MS) assay (AbsoluteIDQ 180 kit, Biocrates, Innsbruck, Austria), as detailed elsewhere [9 (link)]. This method combines derivatisation and extraction of analytes with the selective mass-spectrometric detection using multiple reaction monitoring (MRM) pairs. Isotope-labelled internal standards are integrated into the platform for absolute quantification of metabolites. MRM detection was used for quantification applying spectra parsing algorithm integrated into the Metiq software (Biocrates Life Science AG, Innsbruck, Austria). Concentrations were calculated and evaluated by comparing measured analytes in a defined extracted ion count section to those of specific labelled internal standards or non-labelled ones, provided by the kit. This strategy allows simultaneous quantification of up to 186 metabolites. Metabolites were excluded from further analysis if: (1) fewer than 20% of missing values (non-detectable peak) for each quantified metabolite, (2) 50% of all sample concentrations for the metabolite had to be above the limit of detection (LOD). In total, 130 of the 186 metabolites were used for statistical analysis.
6
Targeted Plasma Metabolomics Analysis
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Plasma metabolites were measured as part of a targeted metabolomics panel using the Biocrates Absolute-IDQ P180 kits (Biocrates, Life Science AG, Innsbruck, Austria), as described elsewhere [14 (link)]. Fasting, frozen never thawed plasma samples were processed per the manufacturer instructions and analyzed on a triple-quadrupole mass spectrometer (QTRAP 6500, AB Sciex, Framingham, MA, USA). Three levels of quality controls were included in each kit to monitor imprecisions (% coefficient of variation [CVs]). Means, standard deviations (SDs), and CVs in 7 kits (the number of kits needed for analysis of the 441 samples) were calculated, and CVs of majority of the metabolites (i.e., > 80%) were less than 20%. We excluded those metabolites that had very high CVs [e.g., > 30%], which left 131 plasma metabolites in the secondary analysis. Metabolite concentrations were quantified by the MetIQ software (Biocrates).
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