2.6 Measurements of melatonin, soluble sugars, and amino acids
First, 0.3 g of the sample was ground with liquid N and placed into 2 mL
of pre-cooled extraction solution (concentrated hydrochloric acid:water:
isopropanol, v:v:v=0.002:1:2), then vortexed for 1 min. After 24 h at
-20°C in the dark, the sample was centrifuged at 4 °C and 4000 rpm for
10 min and the supernatant was transferred to a 10 mL centrifuge tube.
After adding 1 mL of dichloromethane, samples were left at -20 °C for
0.5 h and centrifuged again at 4000 rpm for 10 min. After the
supernatant was dried with N gas, 200 μL of chromatographic grade
methanol was added, mixed well, and filtered through a 0.22 μm organic
membrane before injection. Detection was performed using LC-MS (SCIEX,
QTRAP5500) with an injection volume of 2 μL.
Soluble sugars content was measured according to Gao et al. (2020) and
amino acids levels were assessed as outlined by Huo et al. (2020)
described.