2.12 Analysis of non-volatile metabolite compounds by GC-MS
Analysis of non-volatile metabolites was carried out from flowers
harvested from plants grown at four different air temperatures in the
growth chamber. Extraction and derivatization of metabolites was
performed following the method adopted by Agarwal et al. (2018) with
minor modifications. Freeze-dried petals (10 mg) was powdered and
suspended in 300 μl ice-cold 80% (v/v) methanol. To this 6 μl ribitol
(0.4 mg/ml) was added as internal standard. The supernatant was mixed
with chloroform and deionized water. The water-methanol phase (polar
metabolite fraction) was used for determination of non-volatile
compounds. Methoximation of sample was carried out by addition of 20 μl
methoxyamine hydrochloride (20 mg/ml) followed by an incubation at 40°C
for 90 min. Trimethylsilylation of sample was performed by addition of
20 μl N ,O -Bis(trimethylsilyl)trifluoroacetamide (BSTFA)
containing 1% trimethylchlorosilane (TMCS) and subsequently incubated
at 80°C for 30 min.
The derivatized sample (1 μl) was analyzed on a
TraceTM 1300 Gas Chromatograph (Thermo Fisher
Scientific, Massachusetts, USA) coupled with an ISQ QD Single Quadrupole
Mass Spectrometer. The GC was equipped with a DB-5MS column (length 30
m, 0.25 mm i.d., 0.25 µm film thickness). Analysis of metabolites was
carried out by the method as described by Kutty and Mitra (2019). Helium
was used as the carrier gas where the flow rate of 1ml/min was
maintained. The GC inlet temperature was set at 280°C. Compounds were
separated with the following temperature method: initial oven
temperature was set at 70°C for 1 min, followed by an increase of
2°C/min to 135°C; hold for 10 min, then a linear increase of 4°C/min to
220°C; hold for 10 min and then a final increase to 270°C at 3.5°C/min
and a hold of 20 min. The temperature maintained for MS transfer line
and ion source was 290°C and 200°C respectively. Full scan mode at a
scanning range of m/z 40–700 a.m.u. was used for recording the mass
spectra of compounds. GC-MS raw files were deconvoluted by using
Automated Mass Spectral Deconvolution and Identification System (AMDIS)
software. Metabolites were identified by comparing the spectral data
from the NIST 14 (National Institute of Standards and Technology,
Gaithersburg, MD, USA) library. The compounds were confirmed by
calculating the retention indices relative to
C8-C40 n-alkanes. The relative abundance
of various metabolites was calculated manually in
XcaliberTM software by calculating the peak area
abundance with respect to the internal standard used.