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MB Sample ID: SA094687
| Local Sample ID: | HFV2 |
| Subject ID: | SU001387 |
| Subject Type: | Mammal |
| Subject Species: | Rattus norvegicus |
| Taxonomy ID: | 10116 |
| Genotype Strain: | Sprague Dawley |
| Age Or Age Range: | 240 days |
| Gender: | Male |
| Animal Animal Supplier: | Harlan |
| Animal Housing: | polycarbonate-free caging |
| Animal Light Cycle: | 14-hr light and 10-hr dark |
| Animal Feed: | Phytoestrogen Reduced II 18-5 (Ziegler Bros, Inc) or D09100301 (Research Diets, Inc) |
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Combined analysis:
| Analysis ID | AN002186 | AN002187 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | Reversed phase | Reversed phase |
| Chromatography system | Shimadzu Nexera-x2 | Shimadzu Nexera-x2 |
| Column | Acquity HSS UPLC T3 (50 x 2.1mm,1.8um) | Acquity HSS UPLC T3 (50 x 2.1mm,1.8um) |
| MS Type | ESI | ESI |
| MS instrument type | QTOF | QTOF |
| MS instrument name | ABI Sciex 5600 TripleTOF | ABI Sciex 5600 TripleTOF |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | peak intensity | peak intensity |
MS:
| MS ID: | MS002033 |
| Analysis ID: | AN002186 |
| Instrument Name: | ABI Sciex 5600 TripleTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | For data acquisition through LC/MS analysis, we used a Shimadzu CTO-20A Nexera X2 UHPLC system equipped with a degasser, binary pump, thermostatted auto sampler, and a column oven for chromatographic separation. The column heater temperature was set at 55°C. For lipid separation, the 5 uL of the lipid extract was injected into a 1.8 μm particle 50 × 2.1 mm Acquity HSS UPLC T3 column (Waters, Milford, MA) which heats to 55°C. Acetonitrile/water (40:60, v/v) with 10 mM ammonium acetate was solvent A and acetonitrile/water/isopropanol (10:5:85 v/v) with 10 mM ammonium acetate was solvent B. For chromatographic elution we used a linear gradient over a 20 min total run time, with 60% solvent A and 40% solvent B gradient in the first 10 minutes, then the gradient was ramped in a linear fashion to 100% solvent B which was maintained for 7 minutes. After that the system was switched back to 60% solvent B and 40% solvent A for 3 minutes. The flow rate used for these experiments was 0.4 mL/min and the injection volume was 5μL. The column was equilibrated for 3 min before the next injection and run at a flow rate of 0.4 mL/min for a total run time of 20 min. The data acquisition of each sample was performed in both positive and negative ionization modes using a TripleTOF 5600 equipped with a Turbo VTM ion source (AB Sciex, Concord, Canada). The column effluent was directed to the electrospray ionization source. The voltage of source was set to 5500 V for positive ionization and 4500 V for negative ionization mode, the declustering potential was set to 60 V, and the source temperature to 450oC for both modes. The curtain gas flow, nebulizer, and heater gas were set to 30, 40, and 45 units, respectively. The instrument performed one TOF MS survey scan (150 ms) and 15 MS/MS scans with a total duty cycle time of 2.4 s. The mass range in both modes was 50-1200 m/z. We controlled the acquisition of MS/MS spectra by data-dependent acquisition (DDA) function of the Analyst TF software (AB Sciex, Concord, Canada) with the following parameters: dynamic background subtraction, charge monitoring to exclude multiply charged ions and isotopes, and dynamic exclusion of former target ions for 9 s. Rolling collision energy spread was set whereby the software calculated the collision energy value to be applied as a function of m/z. Mass accuracy was maintained by the use of an automated calibrant delivery system interfaced to the second inlet of the DuoSpray source. |
| Ion Mode: | POSITIVE |
| MS ID: | MS002034 |
| Analysis ID: | AN002187 |
| Instrument Name: | ABI Sciex 5600 TripleTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | For data acquisition through LC/MS analysis, we used a Shimadzu CTO-20A Nexera X2 UHPLC system equipped with a degasser, binary pump, thermostatted auto sampler, and a column oven for chromatographic separation. The column heater temperature was set at 55°C. For lipid separation, the 5 uL of the lipid extract was injected into a 1.8 μm particle 50 × 2.1 mm Acquity HSS UPLC T3 column (Waters, Milford, MA) which heats to 55°C. Acetonitrile/water (40:60, v/v) with 10 mM ammonium acetate was solvent A and acetonitrile/water/isopropanol (10:5:85 v/v) with 10 mM ammonium acetate was solvent B. For chromatographic elution we used a linear gradient over a 20 min total run time, with 60% solvent A and 40% solvent B gradient in the first 10 minutes, then the gradient was ramped in a linear fashion to 100% solvent B which was maintained for 7 minutes. After that the system was switched back to 60% solvent B and 40% solvent A for 3 minutes. The flow rate used for these experiments was 0.4 mL/min and the injection volume was 5μL. The column was equilibrated for 3 min before the next injection and run at a flow rate of 0.4 mL/min for a total run time of 20 min. The data acquisition of each sample was performed in both positive and negative ionization modes using a TripleTOF 5600 equipped with a Turbo VTM ion source (AB Sciex, Concord, Canada). The column effluent was directed to the electrospray ionization source. The voltage of source was set to 5500 V for positive ionization and 4500 V for negative ionization mode, the declustering potential was set to 60 V, and the source temperature to 450oC for both modes. The curtain gas flow, nebulizer, and heater gas were set to 30, 40, and 45 units, respectively. The instrument performed one TOF MS survey scan (150 ms) and 15 MS/MS scans with a total duty cycle time of 2.4 s. The mass range in both modes was 50-1200 m/z. We controlled the acquisition of MS/MS spectra by data-dependent acquisition (DDA) function of the Analyst TF software (AB Sciex, Concord, Canada) with the following parameters: dynamic background subtraction, charge monitoring to exclude multiply charged ions and isotopes, and dynamic exclusion of former target ions for 9 s. Rolling collision energy spread was set whereby the software calculated the collision energy value to be applied as a function of m/z. Mass accuracy was maintained by the use of an automated calibrant delivery system interfaced to the second inlet of the DuoSpray source. |
| Ion Mode: | NEGATIVE |
