solution in a brown bottle for 3 month at À20
C as soon as
possible to minimize degradation.
5. UHPLC solvents were prepared with LC-MS grade regents
and water. Mix 450 mL water and 50 mL acetonitrile in mobile
phase A glass bottle, and 450 mL acetonitrile and 50 mL isopropanol in mobile phase B glass bottle, then degas them in
ultrasonic bath for 20 min. After that, add 1 mL formic acid in
mobile phase A glass bottle and mobile phase B glass bottle,
respectively. The expiry period of mobile phase A and mobile
phase B are 5 days and 2 weeks, respectively.
6. Collect fresh Arabidopsis seedlings in 2 mL microcentrifuge
tubes and keep them frozen in liquid nitrogen for 5 min. Work
quickly and quench the metabolism in plant materials as soon as
possible.
7. Make sure that the freeze-dry system is ready, and plant materials is in a frozen state. It is difficult to dry the plant material if it
is in a defrozen state.
8. Transfer tubes with dry plant materials to liquid nitrogen for
1 min. It makes it easier to homogenize plant material.
9. The drying time of the supernatant in a concentrator is not
fixed. It depends on the samples number and the liquid volume. It is better to test the drying time, as too long drying time
leads to degradation of DIALs.
10. The sonication of 30 s in ultrasonic bath is used to dissolve
DIALs and to mix DIALs with the derivatization regent.
11. Prepare the 10 pmol μL
À1 of DIAL standard solution by adding 10.25 μL of DIAL standard stock solution to a volumetric
flask and make up to 1 mL with methanol. It makes it easier to
take 20 pmol DIAL for the derivatization with a minimum
amount of solvent.
12. Based on its MS response signal in UHPLC-Q-Orbitrap MS,
we use 20 pmol DIAL standard (2 μL of DIAL standard
solution) to prepare the DIAL standard derivatization solution. The amount of DIAL standard also need to be optimized
for different LC-MS systems with different sensitivity.
13. To minimize the effect of free derivatization regent on MS, LC
system was switched to waste channel—disconnected from MS
system—in the first 6 min of the solvent gradient program.
After 6 min, LC system was switched to MS system for detecting DIAL derivatives.
Method for Identification of Carotenoid-Derived Dialdehydes
185
C as soon as
possible to minimize degradation.
5. UHPLC solvents were prepared with LC-MS grade regents
and water. Mix 450 mL water and 50 mL acetonitrile in mobile
phase A glass bottle, and 450 mL acetonitrile and 50 mL isopropanol in mobile phase B glass bottle, then degas them in
ultrasonic bath for 20 min. After that, add 1 mL formic acid in
mobile phase A glass bottle and mobile phase B glass bottle,
respectively. The expiry period of mobile phase A and mobile
phase B are 5 days and 2 weeks, respectively.
6. Collect fresh Arabidopsis seedlings in 2 mL microcentrifuge
tubes and keep them frozen in liquid nitrogen for 5 min. Work
quickly and quench the metabolism in plant materials as soon as
possible.
7. Make sure that the freeze-dry system is ready, and plant materials is in a frozen state. It is difficult to dry the plant material if it
is in a defrozen state.
8. Transfer tubes with dry plant materials to liquid nitrogen for
1 min. It makes it easier to homogenize plant material.
9. The drying time of the supernatant in a concentrator is not
fixed. It depends on the samples number and the liquid volume. It is better to test the drying time, as too long drying time
leads to degradation of DIALs.
10. The sonication of 30 s in ultrasonic bath is used to dissolve
DIALs and to mix DIALs with the derivatization regent.
11. Prepare the 10 pmol μL
À1 of DIAL standard solution by adding 10.25 μL of DIAL standard stock solution to a volumetric
flask and make up to 1 mL with methanol. It makes it easier to
take 20 pmol DIAL for the derivatization with a minimum
amount of solvent.
12. Based on its MS response signal in UHPLC-Q-Orbitrap MS,
we use 20 pmol DIAL standard (2 μL of DIAL standard
solution) to prepare the DIAL standard derivatization solution. The amount of DIAL standard also need to be optimized
for different LC-MS systems with different sensitivity.
13. To minimize the effect of free derivatization regent on MS, LC
system was switched to waste channel—disconnected from MS
system—in the first 6 min of the solvent gradient program.
After 6 min, LC system was switched to MS system for detecting DIAL derivatives.
Method for Identification of Carotenoid-Derived Dialdehydes
185