performed on a C30 analytical column (3 μm particle size,
25 Â 0.46 cm i.d.). Injection volume is 20 μL per sample. LC
separation is performed using the following gradient setting:
hold at 100% A for 10 min, then gradient from 100 to 50% A in
30 min, 50 to 100% B in 10 min, 100% A in 5 min, and finally
isocratic 100% A for 5 min for reequilibrating column.
2. Set the photodiode array detector to acquire the common
spectrum range for carotenoids (300–700 nm), although monitoring at 400–480 nm is commonly made during the run.
3. A split post-column of 0.4 mL min
À1 is directly introduced on
the APCI source, which is operated in positive mode.
4. A calibration routine of the MS instrument is made on each
HPLC run (start-and-end mode) with the calibration mix that
yields product ions in the entire mass range of the instrument.
Hence, it is not necessary to create different calibration files,
and any modification of both the ion source and the optics is
individually collected for each sample.
5. The MS scan modes are tailored depending on the mass analyzer available at the lab of the user (Q, IT, Q-IT, TOF,
Q-TOF). The strategy to follow at this point is whether the
acquisition of “only” protonated molecular ions is required
(full-scan mode, FS) or the choice of the user is to acquire
both protonated molecular ions and product ions (broad band
collision-induced dissociation mode, bbCID). Additionally, a
specific scan mode could be selected, the precursor ion scan
mode (PI) where some protonated molecular ions are designated for transmitting their corresponding product ions.
Depending on the MS scan mode, additional screening tasks
are subsequently required (see Note 5). The m/z scan in the
UHR-TOF is in the 50–1200 Da range. In our laboratory, MS
data are acquired in bbCID mode to obtain MS and tandem
MS spectra simultaneously. The conditions of the APCI source
are optimized for each LC-MS system. However, the user
should consider that the subsequent settings of the mass analyzer affect the precondition of the APCI source (see Note 6).
3.3 Peak Prediction
and Annotation
To get the effective information that finally drives the user to build
a scenario for the extract within a biological context, it is necessary
to apply a systematic workflow for peak annotation and identification. Figure 1 depicts the MS analysis of an extract from human
milk with the automatically annotated protonated molecular ions
to indicate the challenge of the study of MS data means. The
pipeline methodology is represented in Fig. 2, allowing the user
to take advantage of the complementary action of the inclusive
analytical approach, and the application of bioinformatics tools for
a comprehensive study of data [10].
138
Antonio Pe ´ rez-Ga ´ lvez et al.
25 Â 0.46 cm i.d.). Injection volume is 20 μL per sample. LC
separation is performed using the following gradient setting:
hold at 100% A for 10 min, then gradient from 100 to 50% A in
30 min, 50 to 100% B in 10 min, 100% A in 5 min, and finally
isocratic 100% A for 5 min for reequilibrating column.
2. Set the photodiode array detector to acquire the common
spectrum range for carotenoids (300–700 nm), although monitoring at 400–480 nm is commonly made during the run.
3. A split post-column of 0.4 mL min
À1 is directly introduced on
the APCI source, which is operated in positive mode.
4. A calibration routine of the MS instrument is made on each
HPLC run (start-and-end mode) with the calibration mix that
yields product ions in the entire mass range of the instrument.
Hence, it is not necessary to create different calibration files,
and any modification of both the ion source and the optics is
individually collected for each sample.
5. The MS scan modes are tailored depending on the mass analyzer available at the lab of the user (Q, IT, Q-IT, TOF,
Q-TOF). The strategy to follow at this point is whether the
acquisition of “only” protonated molecular ions is required
(full-scan mode, FS) or the choice of the user is to acquire
both protonated molecular ions and product ions (broad band
collision-induced dissociation mode, bbCID). Additionally, a
specific scan mode could be selected, the precursor ion scan
mode (PI) where some protonated molecular ions are designated for transmitting their corresponding product ions.
Depending on the MS scan mode, additional screening tasks
are subsequently required (see Note 5). The m/z scan in the
UHR-TOF is in the 50–1200 Da range. In our laboratory, MS
data are acquired in bbCID mode to obtain MS and tandem
MS spectra simultaneously. The conditions of the APCI source
are optimized for each LC-MS system. However, the user
should consider that the subsequent settings of the mass analyzer affect the precondition of the APCI source (see Note 6).
3.3 Peak Prediction
and Annotation
To get the effective information that finally drives the user to build
a scenario for the extract within a biological context, it is necessary
to apply a systematic workflow for peak annotation and identification. Figure 1 depicts the MS analysis of an extract from human
milk with the automatically annotated protonated molecular ions
to indicate the challenge of the study of MS data means. The
pipeline methodology is represented in Fig. 2, allowing the user
to take advantage of the complementary action of the inclusive
analytical approach, and the application of bioinformatics tools for
a comprehensive study of data [10].
138
Antonio Pe ´ rez-Ga ´ lvez et al.