3. Microcentrifuge tubes of 2 mL capacity.
4. Freeze dry system (Labconco FreeZone 6 Liter Console, or
equivalent).
5. Bead beater (Biospec Products, INC. Mini bead beater, or
equivalent).
6. Analytical precision balance (Secura
® analytical balance, or
equivalent).
2.2 Crude DIALs
Extraction
1. Stock extraction solution: 10 mL acetonitrile with 0.1% butylated hydroxytoluene (BHT) (see Note 1) [55]. The stock
extraction solution is made to a volume of 10 mL and can be
stored for 1 month at 4
C.
2. Microcentrifuge tubes of 2 mL capacity.
3. Ultrasonic bath (Branson 5510EDTH, or equivalent).
4. Refrigerated microcentrifuge (Eppendorf centrifuge 5424R, or
equivalent).
5. Concentrator (Eppendorf concentrator plus, or equivalent).
2.3 DIAL
Derivatization
1. 1% Formic acid methanol stock solution (see Note 2). The
stock solution is made to a volume of 10 mL and can be stored
for 1 month at 4
C.
2. Stock derivatization solution: 1 mL of 10 mg/mL N
2 ,N
2 ,N
4 ,
N
4 -tetraethyl-6-hydrazineyl-1,3,5-triazine-2,4-diamine
(T3) methanol solution with 1% formic acid (see Note 3)
[56]. The stock derivatization solution is made to a volume
of 1 mL and can be stored for 1 month at 4
C.
3. Ultrasonic bath (Branson 5510 EDTH, or equivalent).
4. ThermoMixer (HLC Heating-ThermoMixer MHR 23, or
equivalent).
2.4 UHPLC-QOrbitrap MS Detection
1. DIAL standard stock solution: add 1.6 mg all-trans-2,7Dimethyl-2,4,6-octatrienedial (DIAL10) to a volumetric flask
and make up to 10 mL with methanol (see Note 4). The stock
DIAL solution is made to a volume of 10 mL and can be stored
for 3 months at À20
C.
2. Ultra-high performance liquid chromatography (UHPLC) solvents: 450 mL water, 50 mL acetonitrile, 1 mL formic acid
(solvent A); 450 mL acetonitrile, 50 mL isopropanol, 1 mL
formic acid (solvent B) (see Note 5).
3. 1 mL Syringe (BD 1 mL Syringe, or equivalent).
4. 0.22 μm Syringe filter (Thermo Scientific 4 mm PTFE syringe
filter, or equivalent).
5. 1.5 mL Amber glass vial (VWR, or equivalent).
180
Jianing Mi et al.