Research Article
Open Access
Lipid Profiling of the Carob Fruit (Ceratonia Siliqua L.) Using
GC/LC/QTOF Accurate Mass Spectrometry
Thao Nguyen1, Mario Aparicio2 and Mahmoud A. Saleh1*
1Department of Chemistry, Texas Southern University, 3100 Cleburne Street, Houston, TX 77004, USA
2Agilent Technologies, Inc, 3750 Brookside Parkway, Suite 100, Alpharetta, GA 30022, USA
*Corresponding author: Mahmoud A. Saleh, PhD, Professor of chemistry, College of Science, Engineering and Technology, Texas Southern University, 3100 Cleburne Street, Houston, Texas 77004 USA; Tel: 713 313 1912 (office), 713 851 5023 (cell);
Fax: 713313 7824; E-mail:
@
Received: October 13, 2016; Accepted: December 16, 2016; Published: January 03, 2017
Citation: Nguyen T, Aparicio M, Saleh MA (2017) Lipid Profiling of the Carob Fruit (Ceratonia Siliqua L.) Using GC/LC/QTOF Accurate Mass Spectrometry. SOJ Chromatograph Sci 3(1): 6. DOI:
http://dx.doi.org/10.15226/2471-3627/2/1/00108
Abstract
The carob tree (Ceratonia siliqua L.) is a native of the
Mediterranean basin with several applications in the food, textile
and cosmetics industries and the pods of the carob fruit have long
been used as a feed for livestock and in human nutrition, including
sweets, biscuits and processed drinks, because of its high sugar
content and low price. This work serves as the first step towards
a comprehensive metabolomics characterization of Ceratonia
siliqua L. Gas chromatography and liquid chromatography coupled
with quadrupole time of flight mass spectrometry were used to
profile and quantify lipids in the whole pod/pericarp of the carob
fruits. Crude lipids were extracted from the carob powder using
CO2 supercritical fluid extraction in a yield of 2.0%. A total of 14
fatty acids, 5 phospholipids, 10 triacylglycerol, 8 steroids as well as
hydrocarbons and 16 head space volatile compounds were identified
and quantified.
Keywords: Fatty acids; Head space; Triglycerides; Phospholipids;
Unsaponifiable matters; Leguminosae
Introduction
The carob tree (Ceratonia siliqua L.) is a native of the
Mediterranean basin with several applications in the food, textile
and cosmetics industries [1]. It belongs to the Caesalpinaceae
sub-family of the family Leguminoseae [2].The pods of the carob
fruit have long been used as a feed for livestock and in human
nutrition, including sweets, biscuits and processed drinks,
because of its high sugar content and low price [3]. The world
production of carob pods is 374,800 to 441,000 tones/year, the
leading producer countries are Spain followed by Italy, Portugal,
Morocco, Greece, Cyprus, Turkey and Algeria [4,5]. Carob pods
are rich source of natural antioxidants which may, by different
mechanisms, act as an effective defense against reactive oxygen
species including free radicals such as superoxide anion and
hydroxyl radicals, and non-free radical species such as hydrogen
peroxide. The two principal components of the carob fruit are
the pulp and seed. The seeds represent 10% of the weight of the
fruit and the pulp represent the other 90% of the fruit, crushed
pods may be used to make a beverage, liqueur and syrup [6].
Image of the carob pods are shown in Figure 1.
Lipids are essential metabolites in cells and they fulfill a
variety of functions which include structural components of
cellular membranes, energy storage, cell signaling, and membrane
trafficking [7]. In plants, alterations in the composition of lipids
have been observed during cold/heat stress, nutrient deficiencies,
aging and response to pathogen attack [8]. Lipids are divided
into several classes based on their chemical structures: fatty
acyls, glycerol lipids, glycerol phospholipids, sphingolipids, sterol
lipids, prenol lipids, saccharolipids and polyketides [9]. In order
to understand the function of lipids in biological systems, it is
important to profile the diverse lipid species present in any given
organism and quantify their levels. Recent technological advances
in tandom chromatography and mass spectrometry have enabled
high-throughput, systematic profiling of lipid species known as
lipidomics [7,10]. This study aims to use accurate mass GCQTOF
and LCQTOF to profile and quantify levels of lipid classes in the
pods of the carob fruits as a first step towards understanding its
lipidome.
Experimental
Plant material
Carob pods were collected from the seed department of
Figure 1: Image of the carob pods that was used in this investigation
the agricultural research center, Ministry of Agriculture, Giza,
Egypt in the summer of 2016 and kindly authenticated by the
agricultural botany department of the faculty of agriculture,
university of Cairo. The pods were dried in the dark smashed and
seeds were mechanically removed and the pulps were grounded
using a grinder (Sunbeam Osterizer blender, Boca Raton, USA) to
fine powder.
Extraction of crude lipids
CO2 supercritical fluid extraction was done using the SFT-10
Constant Flow /Constant Pressure
Dual Piston Pump SFE unit (Proras, Rome, Italy). The
extraction basket utilized in this study had an internal volume of
0.5 L and, for each test, batches of about 65 g of carob pods were
placed in the basket and utilized for the extraction. Pressure and
temperature were kept constant at 500 bar and a temperature of
50 C yielding a light yellow oil in 2% by weight.
Preparation of fatty acids methyl esters (FAMEs)
The transesterification was carried as we described before
[11] by adding 10 mg of the lipid extract to 2 mL of 2% BF3 in
methanol, mixed by vortex and placed on the heating block at
75 °C for 1 h. After cooling down to room temperature, 1 mL
of saturated sodium chloride solution was added to step the
reaction and FAMES were extracted in 2 mL of iso-octane, passed
through anhydrous sodium sulfate and transferred to GC vials for
GCMS analysis.
Accurate mass GC-QTOF
Agilent 7200 accurate mass GCQTOF System was used for
analyzing FAMES samples. GC separation was done using a BPX90
SGE Analytical Science column 30m x 0.25 mm x 0.25μm column
(SGE Analytical Science, Texas) run under average velocity of 63.9
cm/sec with a hold up time of 1 min. Temperature programming
started at 75 °C held for 2 min and heated up to 200 °C at a rate
of 5 °C/min, held at 200 °C for 3 min. Total run time was 30 min;
solvent delay 4 min; equilibration time 3 min. His collision gas
1.5 mL/min; injected volume 1μL; split mode 10:1. Mass spectral
acquisitions were separately performed in EI mode and CI mode
using methane as the reagent gas. The measurements and postrun
analyses were controlled by the software Mass Hunter
Qualitative Analysis B.07.00 SP1 [12].FAMES was identified
based on their retention times and their accurate mass data.
Their electron ionization fragmentation and mass spectral data
were also searched using Wiley10NIST mass spectral database.
LC-QTOF
Agilent 6530 accurate mass LC-QTOF system equipped
with 1290 binary pump, Phenomenexkinetex C18 100 mm x
3.0 mm 2.6 μm columns. Solvents A was made of isopropanol;
B acetonitrile run in a gradient protocol starting at 50% A, 50%
B with a flow 0.2 mL/min reaching 100% solvent A at 10.0 min
from the start, and held at 100% A for additional 5 min. Injection
volume was 2 μL; mass range from 100 m/z to 1500 m/z, with a
total run time of 30 min. The LC-QTOF instrument was operated
under the following conditions: Ion source ESI + Agilent Jet
Stream Technology in positive ionization mode. Instrument
parameters were set as follows: sheath gas temperature, 350ºC;
sheath gas flow, 8 L/min; nebulizer, 20 psi; dry gas temperature,
300 °C; dry gas flow, 5 L/min; and capillary entrance Voltage,
3500 V. Fragmentor and Skimmer1 were operated at 190 and 65
V, respectively. The MS scan data were collected at a rate of 1.02
spectra/s in the range of m/z 100–2000.All the MS data were
collected with Mass Hunter Data Acquisition B.06.00 (Agilent
Technologies), and Mass Hunter Qualitative Analysis B.06.00
SP1 (Agilent Technologies) was applied to identify lipid species.
All EICs were obtained with 10 ppm m/z expansion. Microsoft
Office Excel 2013(Microsoft, Redmond, WA, USA) were used for
statistical data analysis and data visualization [12].
LC-QTOF MS/MS experiment
MS/MS experiments were carried out as we described before
[12] at energies of 50 eV, run time 15 min; ion source: positive;
source gas temperature 300 °C; drying gas 3.0 L/min; nebulizer
15 psig; sheath gas temperature 125 °C;sheath gas flow 3.0 L/
min; dual AJS ESI; V cap 4000 V; capillary 0.063 μA; nozzle
Voltage 0 V; chamber 5.33 μA; ms/ms mass range 100 m/z to
1600 m/z; acquisition rate 1 spectrum/s; acquisition time1000
ms/spectrum; 13,503 transients/spectrum; auto recalibration
reference mass window, detection window 100 ppm; minimum
height 1000 counts; polarity type: positive; offset: 15; ms/ms
absolute threshold 5; reference threshold 0.01%.
Head space analysis
Head space analysis was carried out on the dry carob powder
(1g) using Agilent 7890B/5975C GCMS system equipped with
Agilent 7697A head space sampler. GCMS conditions were the
same as those described above.
Analysis of unsaponifiable matters
Unsaponifiable matters from the lipid extract were
analyzed using the standard AOCS method [13]. Oil samples
were saponified by refluxing with 0.5 (N) ethanolic potassium
hydroxide for 2 hours on a water bath the unsap matters were
extracted with petroleum ether, washed with water, dried over
anhydrous sodium sulfate and analyzed by GCMS at the same
condition described above.
Results and Discussion
Crude lipids extracts were obtained from dried carob powder
using CO2 SFE extraction as shown in the experimental sections
at a Yield (w/w) of 1.98 %.
Fatty acids composition
Fatty acid methyl esters (FAMES) relative percentage
composition is shown in Table 1. FAMES were identified based on
their retention times and their accurate mass data in the chemical
ionization mode and by their electron ionization fragmentation
mass spectral data by searched using Wiley10NIST mass spectral
database. GCMS analysis of the fatty acids methyl esters (FAMES)
was achieved with base line resolution as shown in Figure 2.
32% of the identified fatty acids were saturated fatty acid with
palmitic acid (16:0) as the most abundant fatty acid contributing
more than 21% of the total fatty acids, stearic acid (18:0) was
found at almost 4% of the total, all other saturated fatty acids
were found as minor components contributing only1-2% of the
total fatty acids. Relative concentration of unsaturated fatty acids
was 63% of the total and was made primarily of oleic acid Z-9-
octadecenoic acid (18:1)) with an overall concentration of 55%.
Z,Z-9,12-octadecadienoic acid (18:2) was found at concentration
of 6.5%.Other minor concentrations of fatty acids including Ω-3-
fatty acids C18:3, C22:2 and C24:3 as well as other unidentified
compounds were less than 4% of the total but no individual
compound exceeded 0.05%.
Identification of triacylglycerols (TAGs)
TAGs composition in the crude lipid extract was invigilated
using the accurate mass LC-QTOF system. Ten major TAGs were
identified as shown in Figure 3. Ions were identified as positive
ion sodium adducts with exact masses of 879.7426, 901.7251,
903.7418, 907.7743, 909.7891, 935.8055, 937.8205, 965.8523,
967.8679 and 993.8852. Although the ion [M+Na]+with the
accurate mass equivalent to 879.7426 represent 56 possible TAG
isomers this number can be reduced to 14,by eliminating TAGs
that have any fatty acid that was not detected in the oil (Table
2).Similarly, the accurate mass of 901.7256 represents 186
possible TAG isomers, this number can be reduced by eliminating
TAGs that have any fatty acid that was not detected in the oil
(Table 2).
Determination of regioisomers by MS/MS
There are large number of different triacylglycerols species
(TAGs), which differ in the total length of acyl chains and their
degree of unsaturation. Isobaric TAGs are designated by having
the same number of carbon atoms and double bonds possessing
acylchains with different length, position and configuration of
double bonds. The acylchain may be located in three different
Table 1: Fatty acid methyl esters (FAMES) composition and relative
percentage of lipid extract of the carob pods. Peak numbers are those
shown in Figure 2.
Peak # |
tR (min) |
Fatty acids as methyl esters (FAMES) |
Short names |
% |
1 |
9.42 |
Dodecanoic acid |
12:0 |
0.16 |
2 |
12.35 |
Tetradecanoic acid |
14:0 |
0.30 |
3 |
13.76 |
Pentadecanoic acid |
15:0 |
0.09 |
4 |
15.14 |
Hexadecanoic acid |
16:0 |
21.69 |
5 |
15.86 |
(Z)-9-Hexadecenoic acid |
16:1 |
1.05 |
6 |
16.37 |
Heptadecanoic acid |
17:0 |
1.82 |
7 |
17.73 |
(Z)-9-Octadecenoic acid |
18:1 |
54.93 |
8 |
18.33 |
Octadecanoic acid |
18:0 |
3.94 |
9 |
19.29 |
(Z,Z)-9,12-Octadecadienoic acid |
18:2 |
6.49 |
10 |
20.06 |
Eicosanoic acid |
20:0 |
1.06 |
11 |
20.52 |
(Z,Z,Z)-6,9,12-Octadecatrienoic acid |
18:3 |
2.08 |
12 |
22.29 |
Docosanoic acid |
22:0 |
0.85 |
13 |
23.31 |
11-Eicosenoic acid |
20:1 |
2.05 |
14 |
24.37 |
Tetracosenoic acid |
24:0 |
0.26 |
Figure 2: GCMS Total ions chromatogram (TIC) of carob FAMES, peak
numbers and identifications are shown in Table 1.
Figure 3: LCMS total ions chromatogram (TIC) of carob TAGs, peak
numbers and identifications are shown in Table 2.
Table 2: Triacylglycerol (TAGs) composition and relative percentage of
the carob pods, Peak numbers are those shown in Figure 2.
tR (min) |
Accurate Mass* |
Identified structures
(sn1, sn2, sn3) |
Loss of RCOOH from |
RCO+ |
% |
sn1 position |
sn3 positions |
17.58 |
901.7251 |
18:2,18:2,18:2 |
621.4806
18:2 |
621.4806
18:2 |
263
18:2 |
0.07 |
19.67 |
903.7418 |
18:1,18:2,18:2 |
623.5020
18:2 |
621.4806
18:1 |
265/263
18:1/18:2 |
0.27 |
21.60 |
879.7426 |
16:0,18:0,18:3 |
623.4707
16:0 |
601.9316
18:3 |
261/239
18:3/16:0 |
7.26 |
23.47 |
907.7743 |
18:1,18:1,18:1 |
625.5150
18:1 |
625.5150
18:1 |
265
18:1 |
34.57 |
24.95 |
935.8055 |
18:1,18:1,20:1 |
653.5457
18:1 |
625.5152
20:1 |
293/265
20:1/18:1 |
7.11 |
25.26 |
909.7891 |
18:0,18:1,18:1 |
627.5288
18:1 |
625.5151
18:0 |
267/265
18:0/18:1 |
13.85 |
26.88 |
937.8205 |
18:1,18:1,20:0 |
655.5611
18:1 |
625.5134
20:0 |
295/265
20:0/18:1 |
10.61 |
28.36 |
965.8523 |
18:1,18:1,22:0 |
683.5962
18:1 |
625.5150
22:0 |
323/265
22:0/18:1 |
14.07 |
29.76 |
993.8852 |
18:1,18:1,24:0 |
711.6209
18:1 |
625.5151
24:0 |
351/265
24:0/18:1 |
2.92 |
30.02 |
967.8679 |
16:0,20:1,22:0 |
711.6171
16:0 |
627.5321
22:0 |
323/239
22:0/16:0 |
077 |
positions of TAG, known as the sn1, sn2, or sn3 positions. The
region specific analysis is restricted to determine the acylchains
orientation as sn1 (3) and sn2 positions. We have shown previously
[13] that mass spectral fragmentation can provide information
about the stereochemistry and region specific identification of
the TAGs. Based on our finding on the fragmentation of standard
authentic TAGs and available published work [13-15] that ions
corresponding from the loss of fatty acids from the sn1and sn3
positions are much more abundance the ion corresponding to
the loss of fatty acid at the sn2 position. In addition, the fact that
each fatty acid chain produces an RCO+ ion, if two or three of the
chains are different, two or three different RCO+ are found but
more for the sn1 and sn3 positions. Applying that information to
the TAGs of carob Figure 3, the region isomers in the carob were
identified as shown in Table 2.
Phospholipids
The LC–MS analysis of phospholipids of carob oil is presented
in the Table 3 and Figure 4. On the basis of the mass fragmentation
pattern of each component and the NIST library search, we found
that carob oil contains one major phosphatidylcholine phosphor
lipid (PC(O-18:1(9Z)/16:0)) representing 56.7% of the identified
phospholipids, two phosphatidylinositol phospholipids; PI-
18:0/0:0 (2.6%), PI-20:1,11Z/22:0 (13.5%),one phosphatidate
phospholipidPA-20:0/22:0 (10.9%)and one PG-16:0/21:0 (16.3),
their chemical structures are shown in Figure 5.
Unsaponifiable matters
The GCMS analyses of the unsaponifiable matters of carob
oil are presented in the Table 4 and Figure 6. On the basis of
the mass fragmentation pattern of each component, the NIST
library search, it was found that carob oil contains stigmasterol,
β-sitosterol, γ-sitosterol, 5α- cholestan-3-one, betulin, β-amyrin,
betulin free and squalene. Their structures are shown in Figure
7 and Figure 9.
Head space volatile compounds
The volatile emitted by fresh carob pod powder (Table 5) was
analyzed by headspace followed by capillary gas chromatography
and mass spectrometry Figure 8. The headspace of carob pods
is mainly constituted of monoterpenes and sesquiterpenes, and
minor amounts of nonterpenoid aldehydes and hydro carbons.
The identity of each compound was determined by comparison of
its retention index relative to C6–C24 n-alkanes, as well as of its
Table 3: Phospholipids composition and relative percentage of the
carob pods
tR
(min) |
Accurate Mass |
Identified structures |
Formula |
%* |
9.61 |
585.4859 |
PI(P-18:0/0:0) |
C27H54O11P |
0.09 (2.6) |
10.04 |
799.6055 |
PG(20:1(11Z)/18:3(6Z,9Z,12Z)) |
C44H79O10P |
0.57 (16.3) |
11.35 |
746.5966 |
PC(O-18:1(9Z)/16:0) |
C42H85NO7P |
1.98 (56.7) |
13.22 |
773.6264 |
PA(P-20:0/22:0) |
C45H90O7P |
0.38 (10.9) |
14.49 |
949.6962 |
PI(20:1(11Z)/22:0) |
C51H98O13P |
0.47 (13.5) |
*Value is expressed as % in the total lipid extract and values in prances
are relative % in phospholipids fraction.
Figure 4: LCMS total ions chromatogram (TIC) of carob phospholipids,
peak retention times (tR) and identifications are shown in Table 3.
Figure 5: Phospholipids chemical structures that were identified in the
carob pods
Table 4: Composition and relative percentages of the unsaponifiable
matters of carob oil identified astrimethylsilyl ethers
tR (min) |
Chemical Name |
% |
tR (min) |
Chemical Name |
% |
8.31 |
Stigmasterol |
8.39 |
9.67 |
Betulin |
8.12 |
8.92 |
β-Sitosterol |
48.34 |
9.79 |
β-Amyriner |
11.26 |
9.25 |
γ-Sitosterol |
1.71 |
10.25 |
Betulin free |
1.36 |
9.48 |
5α-Cholestan-3-one |
3.12 |
10.97 |
Squalene |
16.48 |
spectral data with the Wiley’s library spectral data bank (G1035B;
Rev D.02.00; Agilent Technologies). For semi quantification
purposes, the normalized peak area of each compound was used
without any correction factors to establish abundances. The
sesquiterpene compounds were present in higher abundant than
monoterpenes.
Conclusion
This study has revealed qualitatively and quantitatively the
particular compounds that make up the bulk of lipids in the carob
fruit particularly the composition of the minor fractions, i.e. the
profiles of phytosterols and other compounds more distinctly
define the genuineness of individual oils and fats along with their
respective fatty acid compositions. Carob pods are rich source of
nutritional and health components, to extract those components,
Figure 6: GCMS total ions chromatogram of the unsaponifiable matters
of carob oil.
Figure 7: Chemical structures of the identified unsaponifiable matters
of carob oil
Figure 8: GCMS total ions chromatogram of the head space volatile
components of the fresh carob pods powder.
Figure 9: Chemical structures of the identified volatile compounds
found in the head space of the fresh carob pods powder.
Table 5: Composition and relative percentages of the head space
volatile compounds of the fresh carob pods powder.
# |
tR
(min) |
Chemical names |
% |
# |
tR
(min) |
Chemical names |
% |
1 |
10.07 |
cis-3-methyl-2-pentene |
4.0 |
9 |
14.15 |
2-undecanone |
2.5 |
2 |
10.45 |
3-methyl-6-hepten-1-ol |
1.5 |
10 |
15.55 |
3-cadinene |
1.7 |
3 |
11.40 |
terpinen-4-ol |
1.8 |
11 |
15.80 |
(-)-elema-1,3,11(13)-trien-12-ol |
1.2 |
4 |
11.78 |
α-terpineol |
5.1 |
12 |
16.84 |
(+)-aromadendrene |
1.2 |
5 |
12.97 |
isopropylbenzaldehyde |
21.9 |
13 |
17.05 |
aromadendrene |
1.0 |
6 |
13.08 |
(-)-carvone |
14.9 |
14 |
17.14 |
α-curcumene |
7.3 |
7 |
13.30 |
piperitone |
1.8 |
15 |
17.34 |
isoledene |
13.8 |
8 |
13.91 |
2-caren-10-al |
6.0 |
16 |
17.69 |
cis-β-farnesene |
3.5 |
both a solubility and diffusivity of the extracting agent should
be investigated. In order to facilitate the use of the extracts, it is
important to carry out the extraction that allows for maximum
amount of a particular component while minimizing or limiting
the presence of other components. For that purpose, we used
supercritical fluid extraction (SCFE-CO2) as a green technology
to extract lipids from carob pods. Oils extracted by SCFE-CO2
showed similar quality, in terms of fatty acid composition
than those obtained by traditional extraction techniques such
soxhlet extraction using hazardous solvents like chloroform and
methanol. Our study could be considered as the first detailed
document on lipids profiling of carob pods. It showed that carob
pods are very rich in oleic acid, but does not contain sufficient
amounts of Ω-3 polyunsaturated fatty acids to be considered as
good nutritional oil.
Fatty acids, triacyclycerol, phospholipids, terpenoides,
hydrocarbons and steroids pattern can be helpful for
understanding of biosynthesis of TAG and establishing lipid
profiling of plants is fundamentally important to gain insights of
its lipid composition or lipidome.
Acknowledgement
This publication was made possible in part by research
infrastructure support from grant number 2G12MD007605 from
NIMHD/NIH.
Disclaimers
The views expressed in the submitted article are the authors
own conclusion and not an official position of the institution or
funder.
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