8. Filter the extract into an amber glass vial through a nylon
syringe filter by gently pressing down on the plunger of a
disposable syringe (see Note 7).
9. Place the open vial containing the extract briefly under a gentle
stream of N 2 gas (see Note 8) to blow away the air (O 2 ) above
the extract. Close the vial with a cap under the N 2 stream.
3.2 Obtaining Mass
Spectra with LC-PDAMS
1. Optimize the ionization parameters of MS by infusing
0.3–0.4 μg/mL solutions of β-carotene and lutein (representatively for carotenes and xanthophylls) dissolved in 1 mM
NH 4 Ac in MeOH/water (99.8, 0.2). The optimal parameters
will be different for different instruments. In the case of Xevo
TQ-S (Waters) with ESI in the positive mode, the capillary
voltage is set to 2.5 kV, cone voltage 30 V, desolvation temperature and source temperature to 500
C and 150
C, respectively. The desolvation gas flow is 1000 L/h and the cone gas
flow 150 L/h, both using N 2 .
2. Chromatographic separation is achieved with a C30 column
using the following LC parameters: column temperature
25
C, injection volume 50 μL and flow rate 0.5 mL/min.
Start a gradient program with a linear gradient from 85 to
70% solvent A over 12 min, isocratic separation at 70% A for
6 min, then a linear gradient to 15% A over 5 min, isocratic at
15% A for 7 min, return to the initial condition of 85% A over
5 min followed by equilibration at 85% A for 5 min (total run
time: 40 min).
3. Measure absorption spectra with PDA detector to identify
carotenoid species. To estimate the retention times and determine the concentrations of different carotenoids, set the PDA
detector to 450 nm and calibrate the system using the pigment
standards (see Note 9).
4. Subsequent to the PDA detection, mass spectra are obtained
for individual carotenoid species in the full scan mode in the
range of mass-to-charge ratio (m/z) between 350 and 1000 (see
Note 10). Adjust the dwell time and gain of the MS detection
to minimize background signal (see Note 11).
5. Process data using MassLynx software version 4.1 (Waters) or
equivalent.
3.3 Confirmation
of
13
C Isotopologs by
LC-DAD-FTICR-MS
1. Optimize the ionization parameters of the MS in the same way
as described for LC-MS in Subheading 3.2, step 1. The optimal parameters will be different for different instruments. We
use LTQ FT Ultra (Thermo Fisher Scientific) with APCI operated in the positive mode at 4.0 μA ion spray current. Nitrogen
serves as both the sheath gas (40 arb) and the auxiliary gas
(10 arb). The transfer capillary temperature is set at 350
C and
Carotenoid Isotopologue Profiling
267
syringe filter by gently pressing down on the plunger of a
disposable syringe (see Note 7).
9. Place the open vial containing the extract briefly under a gentle
stream of N 2 gas (see Note 8) to blow away the air (O 2 ) above
the extract. Close the vial with a cap under the N 2 stream.
3.2 Obtaining Mass
Spectra with LC-PDAMS
1. Optimize the ionization parameters of MS by infusing
0.3–0.4 μg/mL solutions of β-carotene and lutein (representatively for carotenes and xanthophylls) dissolved in 1 mM
NH 4 Ac in MeOH/water (99.8, 0.2). The optimal parameters
will be different for different instruments. In the case of Xevo
TQ-S (Waters) with ESI in the positive mode, the capillary
voltage is set to 2.5 kV, cone voltage 30 V, desolvation temperature and source temperature to 500
C and 150
C, respectively. The desolvation gas flow is 1000 L/h and the cone gas
flow 150 L/h, both using N 2 .
2. Chromatographic separation is achieved with a C30 column
using the following LC parameters: column temperature
25
C, injection volume 50 μL and flow rate 0.5 mL/min.
Start a gradient program with a linear gradient from 85 to
70% solvent A over 12 min, isocratic separation at 70% A for
6 min, then a linear gradient to 15% A over 5 min, isocratic at
15% A for 7 min, return to the initial condition of 85% A over
5 min followed by equilibration at 85% A for 5 min (total run
time: 40 min).
3. Measure absorption spectra with PDA detector to identify
carotenoid species. To estimate the retention times and determine the concentrations of different carotenoids, set the PDA
detector to 450 nm and calibrate the system using the pigment
standards (see Note 9).
4. Subsequent to the PDA detection, mass spectra are obtained
for individual carotenoid species in the full scan mode in the
range of mass-to-charge ratio (m/z) between 350 and 1000 (see
Note 10). Adjust the dwell time and gain of the MS detection
to minimize background signal (see Note 11).
5. Process data using MassLynx software version 4.1 (Waters) or
equivalent.
3.3 Confirmation
of
13
C Isotopologs by
LC-DAD-FTICR-MS
1. Optimize the ionization parameters of the MS in the same way
as described for LC-MS in Subheading 3.2, step 1. The optimal parameters will be different for different instruments. We
use LTQ FT Ultra (Thermo Fisher Scientific) with APCI operated in the positive mode at 4.0 μA ion spray current. Nitrogen
serves as both the sheath gas (40 arb) and the auxiliary gas
(10 arb). The transfer capillary temperature is set at 350
C and
Carotenoid Isotopologue Profiling
267