191
metabolites and can provide advanced, simple and real-time means of metabolome
analysis (Vaclavik et al. 2011). The objective of this study was to identify the metabolites in Z. muelleri using LC-Q-TOF (quadrupole time of flight)-MS and GC-MS.
9.2 Material and Methods
9.2.1 Chemicals
Methanol and acetonitrile (LC-MS grade) were obtained from Chem-Supply
(Gilman, SA, Australia) and Merck (French Forest, NSW, Australia). Milli-Q grade
water was used for all LC analysis. Formic acid, 2-amino anthracene and pentafluorobenzoic acid were obtained from Sigma Aldrich (Castle Hill, NSW, Australia).
9.2.2 Extraction and Sample Preparation
9.2.2.1 Gas Chromatography-Mass Spectrometry (GC-MS)
Metabolites were extracted using 500 μL extraction solution per 30 mg of lyophilized leaf powder (90% methanol with three internal standards, 100 μM labelled
valine, 10 μM labelled sorbitol, 10 μM ribitol). Samples were incubated 20 min at
65 °C and 1250 rpm incubation in a thermomixer and then centrifuged for 3 min at
20,800xg at 65 °C. An aliquot of 60 μL of supernatant was transferred into a glass
insert and evaporated to dryness in a vacuum evaporator for 3 h at ambient temperature. Glass inserts were transferred to a 2 mL GC vial and capped with magnetic
crimp top.
Samples were derivatized using a CTC autosampler: 20 μL of 20 mg/mL methoxylamine in pyridine were added. Samples were incubated at 37 °C for 2 h while
agitating at 750 rpm. Later, 20 μL N-methyl-N-(trimethylsilyl)trifluoroacetamide
(MSTFA) was added and incubated at 37 °C for 30 min, while agitating at 750 rpm.
Later, an aliquot of 1 μL was injected onto the GC-MS.
9.2.2.2 Liquid Chromatography-Mass Spectrometry (LC-MS)
Finely powdered freeze-dried material (20 mg) was weighed into a 1.5 mL
Eppendorf tube. 400 μL of 100% MeOH solution containing internal standards was
added and samples were vortexed for 3 min followed by incubation for 30 min at
25 °C. The samples were spun down in a microcentrifuge at 13,000 rpm for 10 min.
Supernatant was collected and remaining pellet was re-extracted twice using 100%
MeOH (400 μL) and 200 μL H 2 O (Milli-Q), respectively, using the protocol as
above. Combined extracts were stored at −80 °C until further analysis.
9 Gas and Liquid Chromatography-Mass Spectrometry-Based Metabolic Profiling…
metabolites and can provide advanced, simple and real-time means of metabolome
analysis (Vaclavik et al. 2011). The objective of this study was to identify the metabolites in Z. muelleri using LC-Q-TOF (quadrupole time of flight)-MS and GC-MS.
9.2 Material and Methods
9.2.1 Chemicals
Methanol and acetonitrile (LC-MS grade) were obtained from Chem-Supply
(Gilman, SA, Australia) and Merck (French Forest, NSW, Australia). Milli-Q grade
water was used for all LC analysis. Formic acid, 2-amino anthracene and pentafluorobenzoic acid were obtained from Sigma Aldrich (Castle Hill, NSW, Australia).
9.2.2 Extraction and Sample Preparation
9.2.2.1 Gas Chromatography-Mass Spectrometry (GC-MS)
Metabolites were extracted using 500 μL extraction solution per 30 mg of lyophilized leaf powder (90% methanol with three internal standards, 100 μM labelled
valine, 10 μM labelled sorbitol, 10 μM ribitol). Samples were incubated 20 min at
65 °C and 1250 rpm incubation in a thermomixer and then centrifuged for 3 min at
20,800xg at 65 °C. An aliquot of 60 μL of supernatant was transferred into a glass
insert and evaporated to dryness in a vacuum evaporator for 3 h at ambient temperature. Glass inserts were transferred to a 2 mL GC vial and capped with magnetic
crimp top.
Samples were derivatized using a CTC autosampler: 20 μL of 20 mg/mL methoxylamine in pyridine were added. Samples were incubated at 37 °C for 2 h while
agitating at 750 rpm. Later, 20 μL N-methyl-N-(trimethylsilyl)trifluoroacetamide
(MSTFA) was added and incubated at 37 °C for 30 min, while agitating at 750 rpm.
Later, an aliquot of 1 μL was injected onto the GC-MS.
9.2.2.2 Liquid Chromatography-Mass Spectrometry (LC-MS)
Finely powdered freeze-dried material (20 mg) was weighed into a 1.5 mL
Eppendorf tube. 400 μL of 100% MeOH solution containing internal standards was
added and samples were vortexed for 3 min followed by incubation for 30 min at
25 °C. The samples were spun down in a microcentrifuge at 13,000 rpm for 10 min.
Supernatant was collected and remaining pellet was re-extracted twice using 100%
MeOH (400 μL) and 200 μL H 2 O (Milli-Q), respectively, using the protocol as
above. Combined extracts were stored at −80 °C until further analysis.
9 Gas and Liquid Chromatography-Mass Spectrometry-Based Metabolic Profiling…