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MB Sample ID: SA266594
| Local Sample ID: | S_102 |
| Subject ID: | SU002794 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
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Sample Preparation:
| Sampleprep ID: | SP002800 |
| Sampleprep Summary: | 279 newborn dried blood spot and blank samples and were randomized and extracted by 1000 µL methanol with 500 ng/mLtryptophan-d5 as internal standard via a multiple tube vortex mixer for 10 minutes at 5000 rpm. The blank and study samples were sonicated in an ice bath for 20 minutes. A volume of 700 µL of the supernatants was transferred into a pre-labeled 2.0 mL Low-bind Eppendorf tube labeled as the stock. Protein precipitate was pelleted using a centrifuge operating at 4° C for 5 minutes at 16,000 rcf. A volume of 70 µL of each blank sample stock was transferred to 15 mL conical tube to be combined to create a Blank Pool (BP). A volume of 70 µL of each study sample stock was transferred to 15 mL conical tube to be combined and create a Study Pool (SP). A total volume of 1400 µL of NIST Reference Plasma (NIST) was aliquoted into twenty-eight 50 µL aliquots. A volume of 600 µL supernatant (including experimental samples, BP, and SP) was transferred into a pre-labeled 2.0 mL Low-bind Eppendorf tube. All samples and pools were then dried by a SpeedVac overnight. For immediate analysis, 100 µL of water-methanol solution (95:5, v/v) was added to reconstitute the dried extracts, and the samples were thoroughly mixed on multiple tube vortex mixer for 10 min at 5000 rpm. Samples were centrifuged at 4°C for 4 min at 16,000 rcf. The supernatant was transferred to pre-labeled autosampler vials for data acquisition by LC-MS. Broad-spectrum metabolomics was conducted using a Thermo Scientific™ Vanquish™ UPHPLC - Q Exactive™ HF-X Orbitrap System. Metabolites were separated on an Acquity UPLC HSS T3 C18 (2.1 X 100 mm, 1.8 µm) operating at 50 C using a reversed phase gradient separation with Water with 0.1% Formic Acid (v/v) as mobile phase A and Methanol with 0.1% Formic Acid (v/v) as mobile phase B. A 5 µL was injected into the instrument, and MS was collected between 50-750 m/z in positive mode with the MS/MS data triggered by the Data-Dependent Acquisition. |
| Processing Storage Conditions: | 4℃ |
| Extraction Method: | Vortex with methanol containing 500ng/ml tryptophan-d5 as internal standard |
| Extract Storage: | -80℃ |
| Sample Resuspension: | Water-Methanol (95:5, v/v) |
| Sample Spiking: | Tryptophan-d5 stock solution at 500 ng/mL |
