An aliquot of 20 μl of an internal standard mixture (5 reference compounds at concentration level 83–10 μg/ml), 50 μl of 0.15 M sodium chloride and chloroform: methanol (2:1) (200 μl) was added to the tissue sample (203–30 mg). The sample was homogenized, vortexed (2 min) let to stand (1 hour for liver) and centrifuged at 10000 RPM for 3 min. From the separated lower phase, an aliquot was mixed with 10 μl of a labelled standard mixture (3 stable isotope labelled reference compounds at concentration level 93–11 μg/ml) and 0.53–1.0 μl injection was used for LC/MS analysis.
Total 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 UPLCTM BEH C18 10 × 50 mm with 1.7 μm particles. The binary solvent system (flow rate 0.200 ml/min) 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 per sample, including a 5 min re-equilibration step, was 18 min. The temperature of the sample organizer was set at 10°C.
Mass spectrometry was carried out on Q-Tof Premier (Waters, Inc.) run in ESI+ mode. The data was collected over the mass range of m/z 3003–1600 with a scan duration of 0.08 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 and collision energy 5 V, respectively. Reserpine (50 μg/L) was used as the lock spray reference compound (10 μl/min; 10 sec scan frequency).
Data processing including peak detection, alignment, and de-isotoping, was performed using the MZmine software version 0.60 [17 (link)]. Identification was performed based on an internal reference database of lipid species. The implementation of normalization methods and data analysis were performed using Matlab version 7.2 (Mathworks, Inc.).
Total 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 UPLCTM BEH C18 10 × 50 mm with 1.7 μm particles. The binary solvent system (flow rate 0.200 ml/min) 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 per sample, including a 5 min re-equilibration step, was 18 min. The temperature of the sample organizer was set at 10°C.
Mass spectrometry was carried out on Q-Tof Premier (Waters, Inc.) run in ESI+ mode. The data was collected over the mass range of m/z 3003–1600 with a scan duration of 0.08 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 and collision energy 5 V, respectively. Reserpine (50 μg/L) was used as the lock spray reference compound (10 μl/min; 10 sec scan frequency).
Data processing including peak detection, alignment, and de-isotoping, was performed using the MZmine software version 0.60 [17 (link)]. Identification was performed based on an internal reference database of lipid species. The implementation of normalization methods and data analysis were performed using Matlab version 7.2 (Mathworks, Inc.).
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