until 18 min, return to initial conditions of 20% B at 19 min
and equilibrate at 20% B until 22 min (see Note 13).
6. Ionization was achieved using heated-electrospray ionization
with an atmospheric pressure ionization source operating
under positive ionization mode. The spray voltage was
4000 V, the sheath gas flow rate was 40 arbitrary units, the
auxiliary gas flow rate was 10 arbitrary units, the capillary
temperature was 350
C, the auxiliary gas heater temperature
was 400
C, and the S-lens RF was 50. For MS/MS, NCE was
20 eV. The ionization parameters also need to be optimized for
different LC-MS systems.
7. Xcalibur 2.2 software (Thermo Scientific) was used for data
acquisition and processing. Identify the DIAL derivatives produced by the DIAL derivatization reaction using highresolution MS and MS/MS data, confirmed by synthetic
DIAL10 standard (Fig. 3).
4 Notes
1. We usually make a 10 mL solution by dissolving 10 mg of BHT
in 10 mL acetonitrile in a volumetric flask. Then transfer solution to a new glass bottle and keep it at 4
C.
2. Prepare 1% formic acid methanol stock solution by adding
100 μL LC-MS grade formic acid to a volumetric flask and
make up to 10 mL with methanol. To decrease evaporation of
formic acid, we usually add 2 mL methanol to the volumetric
flask firstly, then add 100 μL LC-MS grade formic acid to the
volumetric flask with methanol using pipette. Lastly, make up
to 10 mL with methanol in the volumetric flask. 1% Formic acid
methanol stock solution should be kept at 4
C. It is helpful for
storage. Use protective clothing, gloves and protection glasses
when using formic acid.
3. Prepare the derivatization regent by adding 10 mg of T3 to a
volumetric flask and make up to 1 mL with 1% formic acid
methanol stock solution. The stock derivatization solution is
stored for 1 month at 4
C. To avoid degradation, do not
suggest keeping the stock derivatization solution for more
than 1 month.
4. Prepare DIAL standard stock solution by adding 1.6 mg all-trans-2,7-dimethyl-2,4,6-octatrienedial (Tokyo Chemical
Industry Co., Ltd.) to a brown volumetric flask and make up
to 10 mL with methanol. DIAL is unstable under light or
oxygen conditions. In the DIAL stock solution preparation,
methanol should be precooled on ice to reduce the amount of
oxygen. Work quickly and keep the DIAL standard stock
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Jianing Mi et al.
and equilibrate at 20% B until 22 min (see Note 13).
6. Ionization was achieved using heated-electrospray ionization
with an atmospheric pressure ionization source operating
under positive ionization mode. The spray voltage was
4000 V, the sheath gas flow rate was 40 arbitrary units, the
auxiliary gas flow rate was 10 arbitrary units, the capillary
temperature was 350
C, the auxiliary gas heater temperature
was 400
C, and the S-lens RF was 50. For MS/MS, NCE was
20 eV. The ionization parameters also need to be optimized for
different LC-MS systems.
7. Xcalibur 2.2 software (Thermo Scientific) was used for data
acquisition and processing. Identify the DIAL derivatives produced by the DIAL derivatization reaction using highresolution MS and MS/MS data, confirmed by synthetic
DIAL10 standard (Fig. 3).
4 Notes
1. We usually make a 10 mL solution by dissolving 10 mg of BHT
in 10 mL acetonitrile in a volumetric flask. Then transfer solution to a new glass bottle and keep it at 4
C.
2. Prepare 1% formic acid methanol stock solution by adding
100 μL LC-MS grade formic acid to a volumetric flask and
make up to 10 mL with methanol. To decrease evaporation of
formic acid, we usually add 2 mL methanol to the volumetric
flask firstly, then add 100 μL LC-MS grade formic acid to the
volumetric flask with methanol using pipette. Lastly, make up
to 10 mL with methanol in the volumetric flask. 1% Formic acid
methanol stock solution should be kept at 4
C. It is helpful for
storage. Use protective clothing, gloves and protection glasses
when using formic acid.
3. Prepare the derivatization regent by adding 10 mg of T3 to a
volumetric flask and make up to 1 mL with 1% formic acid
methanol stock solution. The stock derivatization solution is
stored for 1 month at 4
C. To avoid degradation, do not
suggest keeping the stock derivatization solution for more
than 1 month.
4. Prepare DIAL standard stock solution by adding 1.6 mg all-trans-2,7-dimethyl-2,4,6-octatrienedial (Tokyo Chemical
Industry Co., Ltd.) to a brown volumetric flask and make up
to 10 mL with methanol. DIAL is unstable under light or
oxygen conditions. In the DIAL stock solution preparation,
methanol should be precooled on ice to reduce the amount of
oxygen. Work quickly and keep the DIAL standard stock
184
Jianing Mi et al.