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.