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MB Sample ID: SA250657
| Local Sample ID: | ReCHARGE_HHEAR_U1_10 |
| Subject ID: | SU002598 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002598 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| ReCHARGE_HHEAR_U1_10 | SA250657 | FL031974 | HHEAR Urine Pool 1 (HHEAR_U1_20) | Sample Type |
| ReCHARGE_HHEAR_U1_10 | SA250657 | FL031974 | 3 | Batch |
Collection:
| Collection ID: | CO002591 |
| Collection Summary: | NA |
| Sample Type: | Urine |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR002610 |
| Treatment Summary: | NA |
Sample Preparation:
| Sampleprep ID: | SP002604 |
| Sampleprep Summary: | Study samples (2 mL aliquots) were shipped from the University of California, Davis to the NC HHEAR Hub on dry ice. The NC HHEAR Hub thawed and transferred 50 µL of the study samples to a new set of tubes and used them for the analysis. An additional 10 µL was taken from the original study sample and transferred to another tube to make the total study pool for this project, and then distributed with 50 µL per aliquot, used as quality control study pools (QC study pools) throughout the whole analysis. All sample aliquots (50 µL each) and QC study pools (50 µL each) were stored at -80° C until the day of sample preparation. HHEAR Urine Pool 1 (50 µL each), HHEAR Urine Pool 2 (50 µL each), CHEAR reference urine (50 µL each) and NIST urine (SRM 3672) reference material (50 µL each) were provided by the NC HHEAR Hub. LC-MS grade water (50 µL) was used as blanks. All samples were thawed at 4°C overnight before the preparation. Samples, including study samples, study pool samples, HHEAR reference urine, CHEAR reference urine, NIST reference urine, and blanks were mixed with 400 µL methanol containing 500 ng/mL tryptophan-d5 as internal standard and vortexed by a multiple tube vortex mixer for 2 min at 5000 rpm at room temperature. All samples were centrifuged at 16,000 rcf for 10 min at 4°C. The supernatant (350 µL) was transferred into a pre-labeled 2.0 mL Lo-bind Eppendorf tube, dried by a SpeedVac overnight, and stored at -80° C. For immediate analysis, 100 µL of water-methanol solution (95:5, v/v) was used to reconstitute the dried extracts. Samples were thoroughly mixed on a multiple tube vortex mixer for 10 min at 5000 rpm at room temperature and then centrifuged at 4°C for 10 min at 16,000 rcf. The supernatant was transferred to pre-labeled autosampler vials for data acquisition by LC-MS. |
| 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 |
Combined analysis:
| Analysis ID | AN004110 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Thermo Vanquish |
| Column | Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive HF-X Orbitrap |
| Ion Mode | POSITIVE |
| Units | m/z |
Chromatography:
| Chromatography ID: | CH003045 |
| Chromatography Summary: | Reverse phase |
| Instrument Name: | Thermo Vanquish |
| Column Name: | Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um) |
| Column Pressure: | 6000-10000 |
| Column Temperature: | 50 oC |
| Flow Gradient: | Time(min) Flow Rate %A %B Curve 1. 0 0.4 99.0 1.0 5 2. 1.00 0.4 99.0 1.0 5 3. 16.00 0.4 1.0 99.0 5 4. 19.00 0.4 1.0 99.0 5 5. 19.50 0.4 99.0 1.0 5 6. 22.00 0.4 99.0 1.0 5 |
| Flow Rate: | 0.4 mL/min |
| Injection Temperature: | 8oC |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 100% methanol; 0.1% formic acid |
| Analytical Time: | 22 min |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS003857 |
| Analysis ID: | AN004110 |
| Instrument Name: | Thermo Q Exactive HF-X Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Instrument: Thermo Q Exactive HFx Software: Xcalibur 4.1.31.9 for data acquisition; Progenesis QI 2.4 for data preprocessing |
| Ion Mode: | POSITIVE |
| Capillary Temperature: | 320 °C |
| Capillary Voltage: | 3.5 KV |
| Collision Energy: | 20-45, ramp |
| Collision Gas: | N2 |
| Dry Gas Flow: | 55 |
| Dry Gas Temp: | 400°C |
| Fragment Voltage: | ES+ |
| Fragmentation Method: | CID |
| Ion Spray Voltage: | 3.5kV |
| Ionization: | ES+ |
| Mass Accuracy: | 5 ppm |
| Dataformat: | Profile |
| Desolvation Gas Flow: | 55 |
| Desolvation Temperature: | 400 oC |
