Subjects fasted for more than eight hours before sampling. The plasma was separated from the whole blood according to the standard protocol. The JF was obtained by inserting syringes into the articular cavity of the knee and aspirating before TKR surgery. The collected samples were transferred to polyethylene tubes and placed in a liquid nitrogen tank for subsequent analysis. The samples were processed and analyzed according to the manual included in the AbsoluteIDQ p180 assay kit of Biocrates (Biocrates life sciences ag, Austria).
Absoluteidq p180 assay kit
Manufactured by Biocrates
Sourced in Austria
The AbsoluteIDQ p180 assay kit is a targeted metabolomics platform developed by Biocrates. The kit enables the quantification of up to 188 metabolites from various chemical classes, including acylcarnitines, amino acids, biogenic amines, glycerophospholipids, and sphingolipids, in a single analytical run.
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Lab products found in correlation
6 protocols using absoluteidq p180 assay kit
1
Metabolomic Analysis of Knee Fluid
2
Plasma and Tissue Metabolite Profiling
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The metabolic profiling of the plasma and JF samples was conducted using Water’s triple quadruple liquid mass spectrometer combined with the Biocrates AbsoluteIDQ p180 assay kit. This powerful method can measure 186 metabolites (including 90 glycerophospholipids, 40 acylcarnitines, 21 amino acids, 19 biogenic amines, 15 sphingolipids, and one hexose). More information on these metabolites is presented in S1 Table
The metabolite concentrations obtained from the AbsoluteIDQ kit were used for the analyses. For further analysis, we selected the metabolites that could be measured in more than 80% of the total samples[11 (link)] , and half of the minimum value was used to replace the blank values of the metabolites [12 (link)] .
The metabolite concentrations obtained from the AbsoluteIDQ kit were used for the analyses. For further analysis, we selected the metabolites that could be measured in more than 80% of the total samples
3
Quantitative Metabolomics Using BIOCRATES
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A mass spectrometric-based metabolomic approach was carried out at BIOCRATES Life Sciences AG (Eduard-Bodem-Gasse 8, A-6020 Innsbruck, Austria) using the BIOCRATES ABSOLUTEIDQ p180 Assay Kit; only 40 samples per group were randomly selected (due to budget limitations) and analysed. The samples cannot be remeasured, and the data doesn’t exist for the excluded 10 samples per group. The assay allows the quantification of 188 metabolites. The kit plates were used for the quantification of amino acids, biogenic amines, acylcarnitines, (lyso-) phosphatidylcholines, sphingomyelins, and hexoses. The fully automated assay was based on phenylisothiocyanate (PITC) derivatization in the presence of internal standards followed by Flow Injection Analysis Tandom Mass Spectrometry (FIA-MS/MS) (acylcarnitines, (lyso-) phosphatidylcholines, sphingomyelins, hexoses) and LC-MS/MS (amino acids, biogenic amines) using a SCIEX 4000 QTRAP (SCIEX, Darmstadt, Germany) or a Waters TQ-S micro (Waters, Vienna) instrument with electrospray ionization (ESI). Metabolites naming and abbreviation was following the description of Römisch-Margl et al.42 (link). The experimental metabolomics measurement technique is described in detail by patents EP 1 897 014 B1 and EP 1 875 401 B143 ,44 .
4
Targeted Metabolite Profiling in Serum
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Before analysis, all serum samples were processed as described, with samples thawed on ice, then centrifuged; the supernatant was subjected to further analyses6 (link),9 (link). The AbsoluteIDQ p180 kit assay (Biocrates Life Sciences AG, Innsbruck, Austria) permitted targeted, fully automated quantification of 188 analytes (42 amino acids or biogenic amines and their derivatives) based on phenylisothiocyanate derivatization in the presence of internal standards followed by liquid chromatography mass spectrometry using a TSQ-Vantage (Thermo Fisher Scientific, Waltham, MA) instrument with electrospray ionization.
5
Comprehensive Metabolomic Profiling Protocol
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GC-TOF—study design information was entered into the miniX database (a simplified version of the SetupX database).27 All plasma samples were aliquoted and stored at −80 °C until use, at which point 30 μl of each sample was thawed, extracted and derivatized.28 (link) All metabolites were measured as peak height. A total of 288 metabolites were measured (128 known and 160 unknown metabolites). GC-TOF MS data acquisition and processing were conducted as previously described.29 (link) Additional information regarding the GC-TOF methods can be found in the Supplementary Material .
Biocrates P180—the Biocrates AbsoluteIDQ p180 kit assay (Innsbruck, Austria) was used for the quantification of amino acids, acylcarnitines, sphingomyelins, phosphatidylcholines, hexoses and biogenic amines. Additional information regarding the Biocrates p180 methods can be found in theSupplementary Material .
Biocrates P180—the Biocrates AbsoluteIDQ p180 kit assay (Innsbruck, Austria) was used for the quantification of amino acids, acylcarnitines, sphingomyelins, phosphatidylcholines, hexoses and biogenic amines. Additional information regarding the Biocrates p180 methods can be found in the
6
Targeted Metabolite Quantification in Serum
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Before analysis, all serum samples were processed as described, with samples thawed on ice and centrifuged; the supernatant was subjected to further analyses.22 (link) The AbsoluteIDQ® p180 kit assay (Biocrates Life Sciences AG, Innsbruck, Austria) permitted targeted, fully automated quantification of 188 metabolites (142 lipids comprising PC, SM, and acylcarnitines) based on phenylisothiocyanate derivatisation in the presence of internal standards followed by flow injection analysis tandem mass spectrometry using a SCIEX 4000 QTRAP® instrument (SCIEX, Darmstadt, Germany) with electrospray ionisation.
The SCIEX 4000 QTRAP instrument was calibrated periodically after each cleaning cycle, at least once a year, using a chemical standards kit with low/high concentrations of polypropylene glycol polymers (SCIEX, MS Chemical Kit 1, Low-High Conc. PPGs, part number 4406127). The solutions were measured according to the kit instructions and mass calibration and resolution were manually adjusted to meet the specifications of the manufacturer. Measurements were validated by meeting criteria specified by the manufacturer.
The SCIEX 4000 QTRAP instrument was calibrated periodically after each cleaning cycle, at least once a year, using a chemical standards kit with low/high concentrations of polypropylene glycol polymers (SCIEX, MS Chemical Kit 1, Low-High Conc. PPGs, part number 4406127). The solutions were measured according to the kit instructions and mass calibration and resolution were manually adjusted to meet the specifications of the manufacturer. Measurements were validated by meeting criteria specified by the manufacturer.
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