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MB Sample ID: SA184841
Local Sample ID: | 1022386202 |
Subject ID: | SU002044 |
Subject Type: | Human |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Gender: | Male and female |
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Sample Preparation:
Sampleprep ID: | SP002050 |
Sampleprep Summary: | Serum samples were randomized and sample preparation was carried out as described previously (Castilloet al. 2011). In summary, 400 µL of MeOH containing ISTDs (heptadecanoic acid, deuterium-labeled DL-valine, deuterium-labeled succinic acid, and deuterium-labeled glutamic acid, c = 1 µg/mL) was added to 30 µl of the serum samples which were vortex mixed and incubated on ice for 30 min after which they were centrifuged (9400 × g, 3 min) and 350 µL of the supernatant was collected after centrifugation. The solvent was evaporated to dryness and 25 µL of MOX reagent was added and the sample was incubated for 60 min at 45 °C. 25 µL of MSTFA was added and after 60 min incubation at 45 °C 25 µL of the retention index standard mixture (n-alkanes, c=10 µg/mL) was added. The analyses were carried out on an Agilent 7890B GC coupled to 7200 QTOF MS. Injection volume was 1 µL with 100:1 cold solvent split on PTV at 70 °C, heating to 300 °C at 120 °C/minute. Column: Zebron ZB-SemiVolatiles. Length: 20m, I.D. 0.18mm, film thickness: 0.18 µm. With initial Helium flow 1.2 mL/min, increasing to 2.4 mL/min after 16 mins. Oven temperature program: 50 °C (5 min), then to 270°C at 20 °C/min and then to 300 °C at 40 °C/min (5 min). EI source: 250 °C, 70 eV electron energy, 35µA emission, solvent delay 3 min. Mass range 55 to 650 amu, acquisition rate 5 spectra/s, acquisition time 200 ms/spectrum. Quad at 150 °C, 1.5 mL/min N2 collision flow, aux-2 temperature: 280 °C. Calibration curves were constructed using alanine, citric acid, fumaric acid, glutamic acid, glycine, lactic acid, malic acid, 2-hydroxybutyric acid, 3-hydroxybutyric acid, linoleic acid, oleic acid, palmitic acid, stearic acid, cholesterol, fructose, glutamine, indole-3-propionic acid, isoleucine, leucine, proline, succinic acid, valine, asparagine, aspartic acid, arachidonic acid, glycerol-3-phosphate, lysine, methionine, ornithine, phenylalanine, serine and threonine purchased from Sigma-Aldrich (St. Louis, MO, USA) at concentration range of 0.1 to 80 µg/mL. An aliquot of each sample was collected and pooled and used as quality control samples, together with a NIST SRM 1950 serum sample and an in-house pooled serum sample. Relative standard deviations (% RSDs) of the metabolite concentrations in control serum samples showed % RSDs within accepted analytical limits at averages of 27.2% and 29.2% for in-house QC abd pooled QC samples. |
Processing Storage Conditions: | -20? |
Extract Storage: | -80? |