An aliquot (10 µl) of an internal standard mixture containing 11 lipid classes, and 0.05 M sodium chloride (10 µl) was added to plasma samples (10 µl) and the lipids were extracted with chloroform/methanol (2∶1, 100 µl). After vortexing (2 min), standing (1 hour) and centrifugation (10000 RPM, 3 min) the lower layer was separated and a standard mixture containing 3 labeled standard lipids was added (10 µl) to the extracts. The sample order for LC/MS analysis was determined by randomization.
Lipid extracts were analysed on a Waters Q-Tof Premier mass spectrometer combined with an Acquity Ultra Performance LC™ (UPLC). The column, which was kept at 50°C, was an Acquity UPLC™ BEH C18 10×50 mm with 1.7 µm particles. The binary solvent system included A. water (1% 1 M NH4Ac, 0.1% HCOOH) and B. LC/MS grade (Rathburn) acetonitrile/isopropanol (5 2, 1% 1 M NH4Ac, 0.1% HCOOH). The gradient started from 65% A/35% B, reached 100% B in 6 min and remained there for the next 7 min. The total run time including a 5 min re-equilibration step was 18 min. The flow rate was 0.200 ml/min and the injected amount 0.75 µl. The temperature of the sample organizer was set at 10°C.
The lipid profiling was carried out on Waters Q-Tof Premier mass spectrometer using ESI+ mode. The data was collected at mass range of m/z 300–1200 with a scan duration of 0.2 sec. The source temperature was set at 120°C and nitrogen was used as desolvation gas (800 L/h) at 250°C. The voltages of the sampling cone and capillary were 39 V and 3.2 kV, respectively. Reserpine (50 µg/L) was used as the lock spray reference compound (5 µl/min; 10 sec scan frequency).
Data was processed using MZmine software version 0.60 [14] (link). Lipids were identified using internal spectral library. The normalization was performed using multiple internal standards as described in the Supporting InformationText S1 . Only the identified lipid molecular species were included in further data analyses.
The Supporting InformationText S1 , Figures S6 –23 and Tables S8 –11 also include general lipidomics platform characteristics such as internal and external standards used, calibration curves, dynamic ranges, recovery, variability, identification and quality control workflow, as well as illustrative spectra (MS and MS/MS) demonstrating how the specific species can be identified.
Lipid extracts were analysed on a Waters Q-Tof Premier mass spectrometer combined with an Acquity Ultra Performance LC™ (UPLC). The column, which was kept at 50°C, was an Acquity UPLC™ BEH C18 10×50 mm with 1.7 µm particles. The binary solvent system included A. water (1% 1 M NH4Ac, 0.1% HCOOH) and B. LC/MS grade (Rathburn) acetonitrile/isopropanol (5 2, 1% 1 M NH4Ac, 0.1% HCOOH). The gradient started from 65% A/35% B, reached 100% B in 6 min and remained there for the next 7 min. The total run time including a 5 min re-equilibration step was 18 min. The flow rate was 0.200 ml/min and the injected amount 0.75 µl. The temperature of the sample organizer was set at 10°C.
The lipid profiling was carried out on Waters Q-Tof Premier mass spectrometer using ESI+ mode. The data was collected at mass range of m/z 300–1200 with a scan duration of 0.2 sec. The source temperature was set at 120°C and nitrogen was used as desolvation gas (800 L/h) at 250°C. The voltages of the sampling cone and capillary were 39 V and 3.2 kV, respectively. Reserpine (50 µg/L) was used as the lock spray reference compound (5 µl/min; 10 sec scan frequency).
Data was processed using MZmine software version 0.60 [14] (link). Lipids were identified using internal spectral library. The normalization was performed using multiple internal standards as described in the Supporting Information
The Supporting Information
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