Chromatographic Analysis for Targeted

Metabolomics of Antioxidant and Flavor-

Related
Metabolites in Tomatoes
 Group# 6

Naveera Khan L1F17BSBT0055

Muhammad Ibrahim L1F17BSBT0058

Areesha Fareed L1F17BSBT0075

Shifa Tariq L1F17BSBT0079

Outline
• Introduction
• Materials and Methods
• Results and Discussion
• Conclusion
 Introduction
• Tomato (Lycopersicon esculentum Mill.) is one of the superfoods.

• Health benefits include antioxidant, anti-inflammatory, anti-cancer etc

• Due to the benefits of tomatoes it became a highly popular fruit.

• Recently, metabolomics approaches are harnessed to improve our fundamental

understanding of metabolite composition in foods and plants.

• Most food metabolomics studies revealed a relation between metabolites and their
bioactive functions, which can be potentially contributed to human health care.
• Five different types were chosen.

• Non-targeted metabolomics analysis by Gas Chromatography Time-Of-

Flight Mass Spectrometry (GC-TOF-MS) and UltraHigh-Performance
Liquid Chromatography–Linear Trap Quadrupole-Orbitrap–tandem Mass
Spectrometry (UHPLC-LTQ-Orbitrap-MS/MS) platforms.

• Evaluation of metabolomics characteristics and other traits such as

antioxidant activities and morphological traits.
MATERIALS AND METHODS
Chemicals and Reagents:
6-hydroxy-2,5,7,8-
Analytical-grade
Acetonitrile Water. tetramethylchroman-2-
methanol
carboxylic acid (trolox)

2,2′-azinobis (3-
Hydrochloride, 2,4,6-
Hydrochloric acid Potassium persulfate ethylbenzothiazoline6- tris(2-pyridyl)-trizine
sulfonic acid)
(TPTZ)
diammonium salt (ABTS)

Iron(III) chloride
Sodium acetate Acetic acid Sodium carbonate
hexahydrate

Pyridine, and N-methyl-

Methoxyamine N-(trimethylsilyl)-
Sodium hydroxide Formic acid
hydrochloride trifluoroacetamide
(MSTFA)
 Sample Information and Preparation:
Five types of tomato
were selected.

Rinsed with distilled

water.

Stored at −80°C.

Each tomato was

lyophilized for 4 days.

Grounded into a powder

with a mortar and
pestle.

Stored at −80°C until

metabolite extraction.
Sample Extraction
The powdered sample was
extracted with 80%
aqueous methanol using a
MM 400 mixer mill.

Sonicated at 4°C for 5 min.

Centrifuged at 13,000 rpm

for 10 min at 4°C.
Supernatant filtered with
polytetrafluoroethylene
syringe filters.
Dried using a speed-vacuum
concentrator.

Re-dissolved with 80% methanol.

 Ultrahigh-Performance Liquid Chromatography–
Gas Chromatography Time-of-Flight
Linear Trap Quadrupole-Orbitrap–Tandem Mass
Mass Spectrometry Analysis
Spectrometry Analysis
Oximation
Oximation was
was performed
performed by
by
100
100 μl
μl of
of the
the supernatant
supernatant was
was
taken
taken in
in aa fresh
fresh e-tube
e-tube and
and
adding
adding methoxyamine
methoxyamine
hydrochloride to the dried
Dried extracts re-dissolved in 80% MeOH
hydrochloride to the dried
completely
completely dried.
dried. extract
extract for UHPLC-LTQ-Orbitrap-MS/MS analysis.

Chromatographic separation was

performed with a C18 column.
The
The silylation
silylation was
was performed
performed
by
by adding
adding MSTFA
MSTFA to
to the
the Incubated
Incubated at
at 30°C
30°C for
for 90
90 min.
min.
reaction
reaction mixture.
mixture.

Temperature was set at 40°C.

Daidzein
Daidzein (0.25
(0.25 mg/ml)
mg/ml) was
was
Incubated
Incubated at
at 37°C.
37°C. used
used as
as the
the added
added internal
internal
standard (IS).
standard (IS).

Flow rate was 0.3 ml/min.

GC-TOF-MS
GC-TOF-MS analysis
analysis was
was
The
The chromatographic
chromatographic performed
performed using
using an
an Agilent
separation
separation was
was conducted
conducted by by 7890A
7890A GC
GC system
Agilent
system coupled
coupled
The MS data were collected using an
an
an Rtx-5MS
Rtx-5MS column
column with
with aa
helium
helium as
as carrier
carrier gas
gas at
at aa
with
with an
an Agilent
autosampler
Agilent 7693
7693 Orbitrap Velos Pro™ system.
constant autosampler and
and aa Pegasus
Pegasus
constant flow.
flow. HT
HT TOF-MS.
TOF-MS.
 Data Processing and Multivariate
Total Soluble Solids and Titratable Acidity
Statistical Analysis
• Different metabolites among five • Total Soluble Solids (TSSs) and Titratable Acidity
different tomato types were selected by (TA) were measured.
variable importance projection values • The TSS contents in fresh juice extract were
based on partial least squares- measured using a portable refractometer for sugar
discriminant analysis (PLS-DA) score measurements.
plot.
• The TA was determined using the formal titration
• The significance test (p-value < 0.05) method.
between experimental groups was tested
by analysis of variance (ANOVA).
• The selected metabolites were tentatively
identified by comparing their retention
time, mass fragment patterns, and
elemental compositions and mass
spectrum of analysis data with standard
compounds.
 Determination of Antioxidant
Activities by ABTS and FRAP test.
• For ABTS (2, 2'-Azino-Bis-3-
Ethylbenzothiazoline-6-Sulfonic Acid)
assay, sample extract was added with the
diluted ABTS solution in 96-well plates;
and the mixture was incubated under dark
condition for 7 min.
• The absorbance was measured at 750 nm
using a microplate reader.
• For FRAP (Ferric Reducing Antioxidant
Power) assay sample extract was added
with FRAP reagent; and the reaction
mixture was incubated in the 96-well
microtiter plates.
• The absorbance was measured at 570 nm
using a microplate reader.
Determination of Total Phenolic and
Flavonoid Contents
• 20 μl of sample extracts mixed with phenol
reagent in a 96-well plate.
• mixture was incubated at room temperature
in the dark.
• After incubating, 80 μl of 7.5% NaCO3 was
added and incubated at room temperature.
• Finally, the absorbance was measured using
a spectrophotometer at 750 nm.
• For Total Flavonoid Contents measurement,
20 μl of sample extracts was added to 90%
diethylene glycol with NaOH solution.
• Incubated for 60 min at room temperature
in the dark.
• The absorbance was measured at 405 nm.
 Analysis of Carotenoids
Carotenoids were extracted by adding ethanol containing 0.1% ascorbic acid.

Mixtures vortexed for 20 s.

Placed in a water bath 5 min.

The carotenoid extract saponicated with potassium hydroxide.

Samples were placed immediately on ice.

Carotenoids extracted twice with hexane by centrifugation to separate the layers.

Aliquots of the extracts dried and redissolved in 50:50 (v/v) dichloromethane/methanol.

The content of carotenoid analyzed by liquid chromatography–diode-array detection (LC-DAD) system. The absorbance was measured at 450 nm.
RESULTS AND DISCUSSION
Differences in Morphology,
Physicochemical Characteristics, and
Antioxidant Activities in Five Types of
Tomatoes:
• The weight of fruit ranged from 10.97
(JT) to 150.08 g (CT).

• The length was within 2.33 (JT) and 6.48

cm (CT).

• The width of fruit was within 2.48 (CH)

and 5.94 cm (ST).
• The TSS average of JT and ST was the highest and lowest, respectively.
• CH, CT, and KT were insignificantly different.
• The highest TA was observed in CT, while the lowest in ST.
• JT showed the highest TI, representing flavor intensity of fruits, while the lowest value was
observed with ST.
• TPC and TFC levels of CH and JT were higher than those of other types.
• Also, CH and JT showed higher antioxidant activities than did other tomatoes.
• JT presented the highest level of lycopene (77.27 mg/100 g
DW) while KT was the lowest (25.78 mg/100 g DW)
among all tomatoes.
• α-Carotene and β-carotene were the highest in JT, whereas
lutein was the highest in KT.
Non-targeted Metabolite Profiling of Five Types of Tomato:

• The differences among tomato samples were observed mostly depending on

their types, based on the PCA score plot.
• A total of 58 distinguished metabolites were identified.
• 32 and 26 metabolites were identified by GCTOF-MS and UHPLC-LTQ-
Orbitrap-MS/MS, respectively.
• These metabolites include 15 amino acids, 7 organic acids, 8 carbohydrates,
12 phenylpropanoids, 10 lipids, 3 polyamines, and 2 glycoalkaloids.
• The common tomatoes showed higher contents of primary metabolites such
as amino acids, organic acids, and lipids, than did cultivars of cherry tomato.
• Amino acids, organic acids, and lipids were observed higher in CT and ST.
CONCLUSION
It can be predicted that various tomatoes with different appearances may contain different nutrients
and have different effects on human.

Non-targeted metabolite profiling was performed of five representative tomato types to evaluate the
metabolite differences.

Quality (Titratable Acidity, Total Soluble Solids, size, and weight) and functional properties (Total
Phenolic Content, Total Flavonoid Cintent, ABTS, and FRAP) were also determined.

It was observed the higher levels of TSS, TI, TPC, TFC, and antioxidant activity in cherry
tomatoes.

Common tomatoes (CT and ST) showed higher levels of morphological traits.

This study of comparing functional properties among the five tomato types can provide useful
information to improve tomato cultivars.

In the future, multi-omics data such as genome and transcriptome of different types should be
integrated with metabolome data.
Thank You