3.3 DIALs
Derivatization
1. 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.
2. 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. Keep the derivatization regent on ice.
3. Add 50 μL of the derivatization regent to the DIAL extract and
transfer tubes to ultrasonic bath for the sonication of 30 s (see
Note 10). Than transfer tubes to a thermomixer for the incubation at 37
C for 15 min (Fig. 2).
4. Dilute the DIAL derivatization solution with 50 μL of 1%
Formic acid methanol stock solution.
3.4 UHPLC-QOrbitrap MS Detection
1. Prepare the DIAL standard solution by adding 10.25 μL of
DIAL10 standard stock solution to a volumetric flask and make
up to 1 mL with methanol (see Note 11). Keep the DIAL10
standard in dark.
2. Prepare the DIAL10 standard derivatization solution: Add
2 μL of DIAL10 standard solution and 50 μL of the derivatization regent in microcentrifuge tube (see Note 12). Transfer the
tube to a thermomixer at 37
C for 15 min. Then dilute the
solution with 50 μL of 1% formic acid methanol stock solution.
Fig. 2 Scheme of DIAL derivatization
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