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MB Sample ID: SA094330
| Local Sample ID: | 98 HZCD |
| Subject ID: | SU001377 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | c57bl6 |
| Age Or Age Range: | 16 weeks |
| Gender: | Male |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001377 |
| Subject Type: | Mammal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | c57bl6 |
| Age Or Age Range: | 16 weeks |
| Gender: | Male |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 98 HZCD | SA094330 | FL013479 | HZCD | Genotype |
Collection:
| Collection ID: | CO001372 |
| Collection Summary: | Kidney samples were powdered with mortar and pestle. Method used for extraction was previously validated for tissue |
| Sample Type: | Kidney |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR001392 |
| Treatment Summary: | Kidney homogenate was prepared by adding cold (−20 °C) methanol/water (1:1, v/v), (1:10 tissue/solvent). Tissue disruption was achieved with Tissue- Lyser LT homogenizer (Qiagen, Germany) for metabolite extraction. |
Sample Preparation:
| Sampleprep ID: | SP001385 |
| Sampleprep Summary: | 100 μL of kidney tissue homogenate was vortex-mixed with 320 μL of methanol for 2 min, followed by the addition of 80 μL of MTBE for the extraction of nonpolar compounds. Then, vials were rapidly capped and placed on a shaker for 1 h at room temperature. The extracted samples were centrifuged at 4000g for 20 min at 20 °C. For GC−MS analysis, 300 μL of supernatant was evaporated to dryness (SpeedVac Concentrator System, Thermo Fisher Scientific, Waltham, MA). Methoxymation was then performed with 20 μL of O-methoxyamine hydrochloride (15 mg/mL in pyridine) and vigorously vortex-mixed for 5 min. Vials were then incubated in darkness at room temperature for 16 h. For silylation, 20 μL of BSTFA/TMCS (99:1) was added and vortex-mixed for 5 min, and capped vials were placed in the oven at 70 °C for 1 h. Finally, 100 μL of heptane containing tricosane (20 ppm) as internal standard (IS) was added to each vial prior to injection. For LC−MS analysis, 90 μL of supernatant was transferred to an ultra-high-performance liquid chromatography−mass spectrometry. |
| Processing Storage Conditions: | On ice |
| Extract Storage: | -80℃ |
Combined analysis:
| Analysis ID | AN002169 | AN002170 | AN002171 |
|---|---|---|---|
| Analysis type | MS | MS | MS |
| Chromatography type | Reversed phase | Reversed phase | GC |
| Chromatography system | Agilent 1290 Infinity II | Agilent 1290 Infinity II | Agilent 7890B |
| Column | Poroshell 120 EC-C8 (100 x 2.1mm,2.5um) | Poroshell 120 EC-C8 (100 x 2.1mm,2.5um) | Agilent DB5-MS (30m x 0.25mm, 0.25um) |
| MS Type | ESI | ESI | EI |
| MS instrument type | QTOF | QTOF | GC QTOF |
| MS instrument name | Agilent 6545 | Agilent 6545 | Agilent 7890A |
| Ion Mode | POSITIVE | NEGATIVE | POSITIVE |
| Units | Area | Area | Area |
Chromatography:
| Chromatography ID: | CH001587 |
| Chromatography Summary: | LC-MS (+) |
| Instrument Name: | Agilent 1290 Infinity II |
| Column Name: | Poroshell 120 EC-C8 (100 x 2.1mm,2.5um) |
| Column Temperature: | 60 |
| Flow Gradient: | gradient started at 82% phase B, increasing to 90% B in 17 min. The gradient then increased to 100% B by minute 18 and was maintained for 2 minutes until 20 min. The starting condition was returned to by 21.5 min, followed by an 8.5 min reequilibration time, taking the total run time to 30 min |
| Flow Rate: | 0.5 mL/min |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 85% methanol/15% isopropanol; 0.1% formic acid |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH001588 |
| Chromatography Summary: | LC-MS (-) |
| Instrument Name: | Agilent 1290 Infinity II |
| Column Name: | Poroshell 120 EC-C8 (100 x 2.1mm,2.5um) |
| Column Temperature: | 60 |
| Flow Gradient: | gradient started at 82% phase B, increasing to 90% B in 17 min. The gradient then increased to 100% B by minute 18 and was maintained for 2 minutes until 20 min. The starting condition was returned to by 21.5 min, followed by an 8.5 min reequilibration time, taking the total run time to 30 min |
| Flow Rate: | 0.5 mL/min |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 85% methanol/15% isopropanol; 0.1% formic acid |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH001589 |
| Chromatography Summary: | GC-MS |
| Instrument Name: | Agilent 7890B |
| Column Name: | Agilent DB5-MS (30m x 0.25mm, 0.25um) |
| Chromatography Type: | GC |
MS:
| MS ID: | MS002018 |
| Analysis ID: | AN002169 |
| Instrument Name: | Agilent 6545 |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | A UHPLC system (1290 Infinity UHPLC system, Agilent Technologies, Waldbronn, Germany), consisting of two degassers, two binary pumps, and a thermostated autosampler (maintained at 4°C) coupled with 6545 QTOF MS detector, was used in positive and negative ESI modes. In brief, 1 μL of each sample was injected into a reverse-phase Zorbax Eclipse Plus C8 column, 2.1 × 150 mm; 1.8 μm (Agilent Technologies) thermostated at 60°C. The gradient used for the analysis consisted of a mobile phase A (10 mM ammonium formate in Milli-Q water) and mobile phase B (10 mM ammonium formate in methanol:isopropanol, 85:15) pumped at 0.5 mL/min. The chromatography gradient started at 82% phase B, increasing to 90% B in 17 min. The gradient then increased to 100% B by minute 18 and was maintained for 2 minutes until 20 min. The starting condition was returned to by 21.5 min, followed by an 8.5 min reequilibration time, taking the total run time to 30 min. Data were collected in full scan mode from 100 to 1200 m/z, with a scan rate of 1.02 scans/s. The capillary voltage was set to 3500 V; the drying gas flow rate was 12 L/min at 290°C and gas nebulizer 45 psi, fragmentor voltage 175 V, and octopole radio frequency voltage (OCT RF Vpp) 750 V. Two reference masses were used over the course of the whole analysis: m/z 121.0509 (protonated purine) and m/z 922.0098 (protonated hexakis, (1H,1H,3H-tetrafluoropropoxy)phosphazine (HP-921)). These masses were continuously infused into the system to provide constant mass correction. Samples were randomly analyzed throughout the run. |
| Ion Mode: | POSITIVE |
| MS ID: | MS002019 |
| Analysis ID: | AN002170 |
| Instrument Name: | Agilent 6545 |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | A UHPLC system (1290 Infinity UHPLC system, Agilent Technologies, Waldbronn, Germany), consisting of two degassers, two binary pumps, and a thermostated autosampler (maintained at 4°C) coupled with 6545 QTOF MS detector, was used in positive and negative ESI modes. In brief, 1 μL of each sample was injected into a reverse-phase Zorbax Eclipse Plus C8 column, 2.1 × 150 mm; 1.8 μm (Agilent Technologies) thermostated at 60°C. The gradient used for the analysis consisted of a mobile phase A (0.1% formic acid in Milli-Q water) and mobile phase B (0.1% formic acid in methanol:isopropanol, 85:15) pumped at 0.5 mL/min. The chromatography gradient started at 82% phase B, increasing to 90% B in 17 min. The gradient then increased to 100% B by minute 18 and was maintained for 2 minutes until 20 min. The starting condition was returned to by 21.5 min, followed by an 8.5 min reequilibration time, taking the total run time to 30 min. Data were collected in full scan mode from 100 to 1000 m/z for the negative modes, with a scan rate of 1.02 scans/s. The capillary voltage was set to 3000 V; the drying gas flow rate was 12 L/min at 290°C and gas nebulizer 45 psi, fragmentor voltage 175 V, and octopole radio frequency voltage (OCT RF Vpp) 750 V. Two reference masses were used over the course of the whole analysis: m/z 112.9856 (proton-abstracted TFA anion) and m/z 966.0007 (formate adduct of HP921) for the negative mode. These masses were continuously infused into the system to provide constant mass correction. Samples were randomly analyzed throughout the run. |
| Ion Mode: | NEGATIVE |
| MS ID: | MS002020 |
| Analysis ID: | AN002171 |
| Instrument Name: | Agilent 7890A |
| Instrument Type: | GC QTOF |
| MS Type: | EI |
| MS Comments: | An Agilent GC instrument (7890A) coupled to an inert mass spectrometer with triple-Axis detector (5975C, Agilent Technologies) was used for kidney tissue fingerprinting. Briefly, 1 μL of derivatized samples were injected by an Agilent autosampler (7693). Samples were automatically injected in split mode (split ratio 1:12), into an Agilent ultra-inert deactivated glass wool split liner. Compound separation was achieved using a pre-column (10 m J&W integrated with Agilent 122-5532G) combined with a GC column DB5-MS (length, 30m; inner diameter, 0.25 mm; and 0.25 μm film of 95% dimethyl/5% diphenylpolysiloxane). The flow rate of helium carrier gas was constant at : 0.938 mL/min through the column. The lock of the retention time (RTL) relative to the internal standard (methyl stearate) peak at 19.66 minutes was performed. The column oven temperature was initially set at 60°C (maintained for 1 minute), then raised by 10°C/min until it reached 325°C, and then was held at this temperature for 10 minutes before cooling down. The injector and the transfer line temperatures were established at 250°C and 280°C, respectively. MS system: the electron impact ionization operating parameters were set as follows: filament source temperature, 230°C; electron ionization energy, 70 eV. Mass spectra were collected over a mass range of 50-600 m/z at a scan rate of 10 spectra/s. Data were acquired using the Agilent MSD ChemStation Software (Agilent Technologies). For retention index determination, a mixture of n-alkanes (C8-C28) dissolved in nhexane was run prior to the samples. Data were acquired using Agilent MSD ChemStation Software (Agilent Technologies). |
| Ion Mode: | POSITIVE |
