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MB Sample ID: SA154497
| Local Sample ID: | Th17_D2 |
| Subject ID: | SU001752 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Gender: | Not applicable |
Select appropriate tab below to view additional metadata details:
Combined analysis:
| Analysis ID | AN002733 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Waters Acquity UHPLC UNSPSC 41115709 |
| Column | Waters BEH C18 (00mm x 2.1mm,1.7um ) |
| MS Type | ESI |
| MS instrument type | Triple quadrupole |
| MS instrument name | ABI Sciex 5500 QTrap |
| Ion Mode | POSITIVE |
| Units | ng/ml |
MS:
| MS ID: | MS002530 |
| Analysis ID: | AN002733 |
| Instrument Name: | ABI Sciex 5500 QTrap |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | The ceramides were quantified using a targeted multiple reaction monitoring (MRM) method using UHPLC as a separation technique. The LC separation was based on the global lipidomics method previously described 71. Briefly, the UHPLC was a Exion AD (Sciex) integrated system. The samples were held in a cool box at 15 °C prior to the analysis. The needle was washed with both a 10% DCM in MeOH and ACN: MeOH: IPA: H2O (1:1:1:1 v/v/v/v) with 1% HCOOH for a total of 7.5 seconds each. The solvents were delivered using a quaternary solvent and a column oven (set to 50 °C). The separation was performed on an ACQUITY UHPLC BEH C18 column (2.1 mm × 100 mm, particle size 1.7 µm, Waters, Milford, MA, USA). The flow rate was set at 0.4 ml/min throughout the run with an injection volume of 1 µL. The following solvents were used for the gradient elution: Solvent A was H2O with 1% NH4Ac (1M) and HCOOH (0.1%) added. Solvent B was a mixture of ACN: IPA (1:1 v/v) with 1% NH4Ac (1M) and HCOOH (0.1%) added. The gradient was programmed as follows: 0 to 2 min 35-80% B, 2 to 7 min 80-100 % B, 7 to 14 min 100% B. The column was equilibrated with a 7min period of 35 % B prior to the next run. The mass spectrometer was a Sciex 5500 QTRAP (Sciex) set in scheduled MRM mode. All lipids were identified for their fatty acid composition by MS/MS to confirm their exact identification, there was also a linear relationship between the increasing number of carbons in the lipid chain and its corresponding retention time. Due to the isobaric nature of sugars we were unable to differentiate Glc and Glc head groups. All data were integrated using the quantitation tool in MultiQuant (3.0.3), all peaks were manually checked. Any analytes which were over the concentration of the standard curve were diluted (1:25) with the same extraction solvent minus the internal standards. The quantification was performed using class-based internal standards and in the case of those ceramide species without an authentic standard in the standard curve mix, we used the closest related structure. The standard curve mixture contained: Glucosyl (beta) C12 ceramide, Lactosyl (beta)) C12 ceramide, C18 ceramide (D18:1/18:1), C18:1 dihydroceramide (d18:0/18:1(9Z)) and was run at the following levels (all in ppb): 100, 80, 60, 50, 40, 30, 20, 10 for the C12 standards and 10, 8, 6, 5, 4, 3, 2,1 for all C18 standards. |
| Ion Mode: | POSITIVE |
