2.3 UPLC-MS
Systems
1. Acquity Ultra Performance LC
™ (UPLC; Waters Corporation,
Wexford, Ireland).
2. Sample organizer for automatic sampling.
3. LC column: Acquity UPLC
® BEH C 18 1.7 μm, 2.1 Â 100 mm
column (Waters Corporation).
4. Solvent A: 1% 1 M NH 4 Ac and 0.1% HCOOH in water.
5. Solvent B: AcCN/2-propanol (1:1, v/v), 1% 1 M NH 4 Ac, and
0.1% HCOOH.
6. Quadrupole-time-of-flight (Q-Tof) Premier mass spectrometer (Waters Corporation).
7. LTQ-Orbitrap mass spectrometer (Thermo Scientific Corp.,
San Jose, CA).
2.4 Other
Instrumentation
1. Ultrasonication bath.
2. Mixer mill homogenizer.
3. Vortexer.
4. Centrifuge.
2.5 Data Processing
Software
MZmine 2 software for peak detection, alignment, and normalization is used to process raw MS data (http://mzmine.github.io/).
Supported data formats are mzML, mzXML, mzData, NetCDF,
Thermo RAW, Waters RAW, and Agilent CSV.
3 Methods
The UPLC-MS-based lipidomics platform consists of multiple
steps (Fig. 1), including sample preparation, separation and detection, and finally data analysis. Total lipid extracts are obtained using
a modified Folch extraction, and the extracts are analyzed by
UPLC-MS in positive mode (ESI+). The ionization mode is chosen
based on the studied lipids. If the sample set contains a large
number of samples, it is important to randomize the samples
prior to sample preparation and analysis in order to avoid potential
bias. Peaks are detected, aligned, and calibrated with internal standards in specialized software packages such as MZmine 2 [9, 10].
The identification of lipids may be performed automatically
based on an internal database of mass-to-charge ratio (m/z) and
retention time values. After the first data processing, the most
important components are further characterized by mass spectrometry (UPLC/MS/MS or UPLC/MS
n
) in order to confirm the
identification or to identify a so far unknown compound.
To minimize carry-over effects, relatively strong solvent is used
as the UPLC eluent. Blank samples are analyzed between the real
samples to detect possible carry-over. No significant carry-over was
Liquid Chromatography-Mass Spectrometry (LC-MS)-Based Analysis of Molecular. . .
217
Systems
1. Acquity Ultra Performance LC
™ (UPLC; Waters Corporation,
Wexford, Ireland).
2. Sample organizer for automatic sampling.
3. LC column: Acquity UPLC
® BEH C 18 1.7 μm, 2.1 Â 100 mm
column (Waters Corporation).
4. Solvent A: 1% 1 M NH 4 Ac and 0.1% HCOOH in water.
5. Solvent B: AcCN/2-propanol (1:1, v/v), 1% 1 M NH 4 Ac, and
0.1% HCOOH.
6. Quadrupole-time-of-flight (Q-Tof) Premier mass spectrometer (Waters Corporation).
7. LTQ-Orbitrap mass spectrometer (Thermo Scientific Corp.,
San Jose, CA).
2.4 Other
Instrumentation
1. Ultrasonication bath.
2. Mixer mill homogenizer.
3. Vortexer.
4. Centrifuge.
2.5 Data Processing
Software
MZmine 2 software for peak detection, alignment, and normalization is used to process raw MS data (http://mzmine.github.io/).
Supported data formats are mzML, mzXML, mzData, NetCDF,
Thermo RAW, Waters RAW, and Agilent CSV.
3 Methods
The UPLC-MS-based lipidomics platform consists of multiple
steps (Fig. 1), including sample preparation, separation and detection, and finally data analysis. Total lipid extracts are obtained using
a modified Folch extraction, and the extracts are analyzed by
UPLC-MS in positive mode (ESI+). The ionization mode is chosen
based on the studied lipids. If the sample set contains a large
number of samples, it is important to randomize the samples
prior to sample preparation and analysis in order to avoid potential
bias. Peaks are detected, aligned, and calibrated with internal standards in specialized software packages such as MZmine 2 [9, 10].
The identification of lipids may be performed automatically
based on an internal database of mass-to-charge ratio (m/z) and
retention time values. After the first data processing, the most
important components are further characterized by mass spectrometry (UPLC/MS/MS or UPLC/MS
n
) in order to confirm the
identification or to identify a so far unknown compound.
To minimize carry-over effects, relatively strong solvent is used
as the UPLC eluent. Blank samples are analyzed between the real
samples to detect possible carry-over. No significant carry-over was
Liquid Chromatography-Mass Spectrometry (LC-MS)-Based Analysis of Molecular. . .
217