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Characterization of triacylglycerols in
unfermented cocoa beans by HPLC-ESI mass
spectrometry
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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
A R T I C L E I N F O A B S T R A C T
Keywords: The nutritional value of cocoa butter is mainly determined by the composition of triacylglycerols (TAGs). In this
Triacylglycerol paper we have developed a non-aqueous reversed-phase liquid chromatographic method, using ethanol as the
High-performance liquid chromatography mobile phase, coupled to electrospray ionization (ESI) tandem mass spectrometry to identify TAGs in raw cocoa
Tandem mass spectrometry beans from six different origins. Tandem mass spectrometry was adopted to facilitate the identification of TAGs
Raw cocoa beans
using unique diacylglycerol product ions and neutral losses. Additionally, two-dimensional m/z retention time
Homologous series
maps aided the identification of entire homologous series of TAGs. A total of 83 different TAGs were identified in
2D map
Neutral loss unfermented cocoa beans, 58 of which were not previously reported in cocoa. Thirty-one of these compounds
represent a new class of TAGs characterized by the presence of one to three hydroxyl groups on the unsaturated
fatty acid chain. To date, this represents the largest number of TAGs identified in cocoa.
1. Introduction substances but they play an indispensable role in energy production and
storage in a living organism (Carrasco-Pancorbo, Navas-Iglesias, &
Theobroma cacao is a small evergreen tree in the family Cuadros-Rodríguez, 2009). TAGs also have an important value in nu-
Sterculiaceae, native to Central and South America. Cacao trees need trition and health, being the main suppliers of essential fatty acids in-
rich, well-drained soils; they naturally grow within 20° latitude of the volved in maintaining life activities (Visioli et al., 2009). In seed plants,
equator, as they need about 2000 mm of rainfall a year and tempera- carbohydrates are accumulated as storage energy, and then trans-
tures in the range of 21–32 °C (Young, 2007). The seeds of Theobroma formed in TAGs during the growth stages. TAGs are then reconverted
cacao, cocoa beans, contain alongside proteins, polyphenols, carbohy- back into carbohydrates during germination, with the incorporation of
drates, alkaloids and minerals, lipids as their main chemical con- a high amount of water. One gram of oil is converted into approxi-
stituents, accounting for 45–60% of their dry mass (Visioli et al., 2009). mately 2 g of carbohydrates (Murphy, 1994).
About 98% of cocoa lipid mass is represented by triacylglycerols The analytical procedure for the characterization of individual TAG
(TAGs), which are hydrophobic molecular species formed by ester- compounds involves three elementary steps: extraction, chromato-
ification of three fatty acids (FAs) with a glycerol backbone. Their FAs graphic separation, and identification. TAG profiling and analysis is
composition varies depending on the geographical origins, varieties of performed by gas chromatography (GC) (Ulberth & Buchgraber, 2003)
Theobroma cacao, growing seasons, and cultivation method (Lipp & and high-performance liquid chromatography (HPLC) using normal-
Anklam, 1998a). Two saturated FAs with oleic acid in the middle phase HPLC and non-aqueous reversed-phase (NARP) HPLC (Christie
characterize a typical pattern of distribution of FAs on the TAG body. and Han, 2010; Jakab, Héberger, & Forgács, 2002; Lísa and Holčapek,
The main TAGs are 1,3-distearoyl-2-oleyl-sn-glycerol (SOS), 1-palmi- 2008). NARP-HPLC has been widely applied for the separation of the
toyl-2-oleoyl-3-stearoyl-sn-glycerol (POS) and 1,3-dipalmitoyl-2-oleyl- intricate acylglycerol class because it offers, under optimized condi-
sn-glycerol (POP); they are known to be major components in cocoa tions, better resolution of the peaks and separation of TAGs according
butter (CB) and discriminatory marker compounds allowing identifi- to the acyl chain lengths and the number of double bonds (DBs). The
cation of adulteration. Furthermore, this distribution of the FAs within retention is ruled by equivalent carbon number (ECN) defined as
the glycerol backbone has an essential nutritional and functional value ECN = CN-2DB, where CN is the number of carbon atoms (Holčapek,
highly appreciated in food, cosmetic and pharmaceutical industries Jandera, Zderadička, & Hrubá, 2003; Lísa & Holčapek, 2008). Due to
(Ulberth & Buchgraber, 2003). TAGs are relatively simple lipid lack of chromophores, characterization of TAGs is carried out mainly
⁎
Corresponding author at: Analytical Chemistry Laboratory, Jacobs University, Campus Ring 1, D-28759 Bremen, Germany.
E-mail address: n.kuhnert@jacobs-university.de (N. Kuhnert).
https://doi.org/10.1016/j.foodchem.2018.01.194
Received 14 November 2017; Received in revised form 25 January 2018; Accepted 31 January 2018
Available online 02 February 2018
0308-8146/ © 2018 Published by Elsevier Ltd.
D. Sirbu et al. Food Chemistry 254 (2018) 232–240
using mass spectrometry (MS), using either atmospheric pressure che- evaporation to dryness in a rotary evaporator. The dry residue was then
mical ionization (APCI) or electrospray ionization (ESI) techniques. stored at −20 °C until further analysis. For HPLC analysis a con-
TAGs show a unique fragmentation pattern in tandem MS, since the centration of 0.045 mg/mL in chloroform/ethanol (50/50) of cocoa
most abundant fragment ions are formed by loss of one acyl moiety lipid extract was prepared.
with consequent formation of diacylglycerol product ions. In spite of
this fact, they provide a challenge for mass spectrometric analysis be-
2.3. HPLC chromatographic conditions
cause of the intricacy of naturally occurring TAGs, the formation of
multiple adduct ions and co-elution in chromatographic separation.
TAG molecular species were separated using HPLC (1100 Series;
Combinations of different fatty acyl substituents in TAGs, different
Agilent, Waldbronn, Germany). The column used was a Pursuit XRs C18
stereochemical positions sn-1, 2 or 3 on the glycerol backbone (re-
(250 mm × 3 mm i.d., 5 μm particles). The temperature of the column
gioisomers) or R/S optical configuration of TAGs esterified in sn-1 and
oven was set to 35 °C; 3 µL of sample were injected. Solvent A consisted
sn-3 positions by two different FAs (optical isomers) result in a large
of acetonitrile with 0.01% formic acid and solvent B consisted of
number of molecular species having identical elemental composition.
ethanol with 5 mmol/L ammonium formate and 0.01% formic acid. The
Ammonium adducts of TAGs, [M+NH4]+, generated by electrospray
mobile phase was pumped through the column at a flow rate of 0.6 mL/
ionization, enable the molecular weight of each TAG molecular species
min. The gradient elution program consisted of an isocratic step (A/
to be determined. This allows tandem mass spectroscopy to be used for
B = 40/60) for 3 min; followed by a linear gradient to 100% solvent B
the elucidation of TAGs (Cvacka, Krafková, Jiros, & Valterová, 2006;
over 33 min, and ending with isocratic elution with solvent B (100%)
Holčapek et al., 2003; Hsu and Turk, 2010; McAnoy, Wu, & Murphy,
for 10 min. The column was equilibrated at 40/60 A/B for 5 min before
2005; Murphy et al., 2007). Kuhnert et al. have recently shown, by
reuse.
using Fourier-transform ion cyclotron resonance (FTICR) mass spec-
trometry technique, that fermented cocoa beans constitute an ex-
tremely complex food matrix, containing more than 20 000 chemical 2.4. High-resolution mass spectrometry conditions
entities (Kuhnert, Dairpoosh, Yassin, Golon, & Jaiswal, 2013; Milev,
Patras, Dittmar, Vrancken, & Kuhnert, 2014). It is, therefore, a chal- High-resolution mass spectra were acquired using a time-of-flight
lenge to detect and differentiate different types of TAGs in cocoa beans (TOF) MicrOTOF Focus mass spectrometer (Bruker Daltonics, Bremen,
and various tools have to be applied. Germany) fitted with an ESI source as the detector with the following
Despite the many studies of the chemical composition of TAGs in parameter settings: capillary voltage of 4.5 kV; nebulizing gas pressure
cocoa butter previously published, there is no full characterization of of 2 bar; drying gas flow rate of 10 L/min; drying gas temperature of
the TAGs in unfermented cocoa beans, which are also known as raw or 220 °C. ESI mass spectra were measured in the range of m/z 200–1200
wet cocoa beans (Lipp & Anklam, 1998b; Lipp et al., 2001; Segall, Artz, in positive ion mode. Internal calibration was achieved with 10 mL of
Raslan, Ferraz, & Takahashi, 2005; Simoneau, Hannaert, & Anklam, 0.1 M sodium formate solution injected through a six-port valve prior to
1999; Ulberth & Buchgraber, 2003). This contribution presents a new each chromatographic run. Calibration was carried out using the en-
approach to the analysis of TAGs in different origin raw cocoa beans hanced quadratic mode. Molecular formulae suggestions were accepted
using NARP HPLC with ethanol as mobile phase. ESI tandem mass if the mass error was below 5 ppm.
spectrometry and two dimensional (2D) map analysis revealed the true
complexity of cocoa butter and thus allowed structure elucidation of the
most abundant lipid molecular species in cocoa. 2.5. Tandem mass spectrometry conditions
2. Material and methods LC-tandem MS was carried out using an ion trap detector in positive
ion mode equipped with an ESI source (Bruker Daltonics UHT Ultra,
2.1. Chemicals and reagents Bremen, Germany). Full scan mass spectra were recorded in the range
m/z 200–1200, operating in positive ion mode. Capillary temperature
Ethanol (gradient grade) was purchased from Merck (Darmstadt, was set to 350 °C, drying gas flow rate of 10 L/min and nebulizer
Germany), isopropanol (Rotisolv® HPLC grade), acetonitrile (Rotisolv® pressure of 10 psi. Tandem mass spectra were acquired in Auto MSn
HPLC ultra gradient grade) and chloroform (Rotisolv® HPLC grade) (smart fragmentation), using a ramping of the collision energy.
were purchased from Carl Roth (Karlsruhe, Germany); dichloromethane
99,8% stabilized with amylene for synthesis was purchased from
2.6. Enrichment of polar TAG fraction
Panreac AppliChem (Darmstadt, Germany); ammonium formate LC-MS
Ultra and formic acid (puriss., ≥98% (T) for mass spectrometry) were
One gram of raw cocoa lipid extract was passed through a pre-
purchased from Sigma-Aldrich Chemie (Steinheim, Germany). Ethanol
parative silica gel column (15 cm × 3 cm i.d.), using a solvent gradient
was subjected to distillation prior to use. Standard TAGs used in this
from 0% ethyl acetate in heptane to 50% ethyl acetate in heptane, in
study: TAG (16:0/18:1/16:0), TAG (16:0/16:0/16:0), TAG (18:0/18:0/
order to prepare an enriched fraction of the TAGs eluting at lower re-
18:0), and TAG (18:1/18:1/18:1) all at ≥99% were purchased from
tention times (from 4 to 25 min) in HPLC-MS. This fraction showed a
Sigma-Aldrich Chemie (Steinheim, Germany).
more polar character and was used for further analysis by nuclear
magnetic resonance spectroscopy (NMR).
2.2. Sample preparation
Frozen cocoa bean samples from different origins were received in 2.7. Derivatization of hydroxylated fatty acids
several sets from Barry Callebaut (Lebbeke-Wieze, Belgium). A total of
12 samples coming from Ivory Coast, Indonesia, Ecuador, Tanzania, Derivatization of the hydroxyl group was carried out on 100 mg of
Malaysia, and Brazil were analyzed. Firstly, the seed material was the enriched fraction obtained from the preparative silica gel column
crushed using a Retsch Grindomix GM200 knife mill (Haan, Germany) using 0.5 mL benzoyl chloride with 200 µL of 0.1 M DMAP (4-(N,N-di-
with the purpose of making homogenous powder. Subsequently, an methylamino)pyridine) in pyridine. The reaction was performed in di-
overnight Soxhlet (Buchi Extraction System B811 instrument) method chloromethane and stirred at room temperature for 1 h. The benzoate
using dichloromethane as extraction solvent (150 mL with 5 g crushed obtained was diluted in chloroform/ethanol (50/50) and 3 µL were
beans) was used. Extracted lipids were quantified gravimetrically after injected in HPLC-MS.
233
D. Sirbu et al. Food Chemistry 254 (2018) 232–240
234
D. Sirbu et al. Food Chemistry 254 (2018) 232–240
Intens.
7
x10 POS
(a) 1.2
SOS TIC +
1.0
0.8
0.6 POP
0.4
SOO
0.2 ISTD PC18:2-O2H2S
PC18:1-OHP
SC18:1-OHS
PC18:2-OHP POO
PC18:1-OHS OOO SOA
PC18:2-O2H2P SPS
PC18:2-OHS PLP MaOP MaOS SSS SOB
PLO
0.0
5 10 15 20 25 30 35 40 45 Time [min]
Intens.
m/z 6
(b) 10
1100
5
10
1000 4
10
3
900 10
2
10
800 1
10
0
700 10
5 10 15 20 25 30 35 40 45
Time [min]
m/z
(c) 1100
1000 SOC
C25:0
LgOO SOLg
S
SOC
C23:0
BOO SOB
SC
C SC
C A SOC
C21:0
21::0
AOO SOA SSA
SC
C S SC
C S 9:0OO
119:0 OSOCC19:
19:0
900 OOO MaOO
LS
OOS SLS SOS S
SSS
PC PC
C MaOS SMaS
PLO OOP PLS LS POS
P SPS
PC
C P PC
C P OC15:
15:0
5::0 MaOP MaPS
P
PLP POP
POP P PS
MOO
MC
C P POC
P C15:0 MaPP
MOP PPP
800
700
5 10 15 20 25 30 35 40 45
Time [min]
Fig. 1. (a) HPLC-MS profile of Ivory Coast raw unfermented cocoa TAGs in positive ionization mode; (b) Two-dimensional (2D) map of TAGs from raw cocoa beans, Ivory Coast origin, by
reverse-phase high resolution HPLC/ESI-MicroOTOF MS analysis. Colour rapresents the intensity of the peaks with red being the highest and blue the lowest: (c) 2D HPLC-MS feature map
displaying TAGs building blocks of homologous series.
235
D. Sirbu et al. Food Chemistry 254 (2018) 232–240
Table 2 produce a five membered oxonium ion (Hsu & Turk, 2010). In tandem
Abbreviations of fatty acids and characteristic fragment ion acyls and neutral losses. MS the first acyl substituent is believed to be lost from the sn-2 position,
therefore allowing unambiguous assignment of TAG regiochemistry.
common name mass symbol CN:DB [RCO]+ [RCOONH4]+
This assignment requires in our view a note of caution. It is applied
myristic acid 228.2089 M C14:0 211.2067 245.2338 throughout the literature; however based on intuition rather than on a
pentadecylic acid 242.2246 C15:0 C15:0 225.2224 259.2495 sufficient set of authentic standards. Therefore, we would like to add a
palmitic acid 256.2402 P C16:0 239.2380 273.2651
further argument. Migration from the sn-2 to the sn-1/3 position would
palmitoleic acid 254.2246 Po C16:1 237.2224 271.2495
margaric acid 270.2559 Ma C17:0 253.2537 287.2808 in terms of organic chemistry be defined as a “downhill migration” from
stearic acid 284.2715 S C18:0 267.2693 301.2964 a more substituted carbon to a less substituted carbon, whereas loss of
oleic acid 282.2559 O C18:1 265.2537 299.2808 the sn-2 acyl group would result in “uphill migration” towards the sn-1/
linoleic acid 280.2402 L C18:2 263.2380 297.2651 3 carbon atoms (Eames, Kuhnert, Sansbury, & Warren, 1999).
linolenic acid 278.2246 Ln C18:3 261.2224 295.2495
MS/MS data were used to identify each acyl group present for a
unusual 1 fatty 298.2351 C18:1-OH C18:1 281.2481 315.2773
acid given [M+NH4]+ precursor ion, and this information could be com-
unusual 2 fatty 296.2351 C18:2-OH C18:2 279.2324 313.2616 bined with high resolution MS data to identify possible TAG molecular
acid species present in cocoa bean extracts. Subsequent MS3 experiments on
unusual 3 fatty 312.2301 C18:2- C18:2 295.2274 329.2566
the resultant diacyl product ions, which gave rise to acylium (RCO+)
acid (OH)2
unusual 4 fatty 310.2144 C18:3- C18:3 293.2117 327.2410 and related ions, enabled unambiguous TAG molecular assignments.
acid (OH)2 MS3 experiments using the diacyl product ion as precursor ion allowed
unusual 5 fatty 328.2250 C18:3- C18:2 311.2223 345.2516 further assignment of the two remaining TAG fatty acid substituents.
acid (OH)3 Assignment of TAG structures with tandem MS data is given in the
nonadecylic acid 298.2872 C19:0 C19:0 281.2845 315.3138
supplementary information. In this section, two representative ex-
arachidic acid 312.3028 A C20:0 295.3006 329.3277
heneicosanoic acid 309.3163 C21:0 C21:0 325.3112 343.3434 amples are discussed in more detail. A compound with a pseudomole-
behenic acid 340.3341 B C22:0 323.3319 357.3590 cular ion of m/z 881 (C55H110NO6, [M + NH4]+) as precursor ion
tricosanoic acid 337.3476 C23:0 C23:0 353.3425 371.3747 yielded fragment ions at m/z 607.6 (C39H74O6, [M-P-NH4]+), 41.4%
lignoceric acid 368.3654 Lg C24:0 351.3632 385.3903
relative intensity, and 579.6 (C37H70O6, [M-S-NH4]+), 100% relative
intensity, corresponding to 1,3-distearoyl-sn-glycerol (SS) and 1-
stearoyl-2-palmytoyl-glycerol/2-palmytoyl-3-stearoyl-sn-glycerol (SP)
times. Series of TAGs lie within the plot on straight lines with a positive
diacyl product ions. SP m/z 579.6 was further fragmented and acylium
slope within an almost equidistant period, as indicated in Fig. 1(c).
ions of stearic [C17H35CO]+ m/z 276.2 and palmitic acid [C15H31CO]+
These series generate TAG homologues series. Homologous series are
m/z 239.1 could be observed. Hence, on the basis of MS2 and MS3
series of compounds, which differ in a defined mass increment corre-
fragments and their abundance, the overall structure was assigned as 1-
sponding to addition or subtraction of a fixed moiety and hence m/z
stearoyl-2-palmitoyl-3-stearoyl-glycerol SPS, TG(18:0/16:0/18:0). A
increment, e.g., −H2 for unsaturation of FAs or +C2H4 for chain
second example comprises the pseudomolecular ion at m/z 919
elongation of fatty acids. Therefore the information obtained allows
(C58H112NO6, [M+NH4]+) as precursor ion, which yielded two diacyl
identification of homologous series of fatty acids within the data set
product fragment ions at m/z ions at m/z 619.6 (C40H74O6, [M-O-
similar to the Kendrick formalism frequently used in petroleum analysis
NH4]+), 100% relative intensity, and 605.7 (C39H70O6, [M-C19:0-
or processed food analysis (Hughey, Hendrickson, Rodgers, Marshall, &
NH4]+), 41.7% relative intensity, corresponding to 1-oleoyl-2-non-
Qian, 2001).
adecanoyl-sn-glycerol (OC19:0) and 1,2-dioleoyl-sn-glycerol (OO)
The 2D map allows for the assignment of two types of homologous
diacyl product ions. Further fragmentation led to MS3 acylium ions of
series (Fig. 1(c)); the most common increments of 28 Da corresponding
oleic [C17H33CO]+ m/z 265.1 and nonadecylic acid [C19H39CO]+.
to +C2H4 and a second increment of 26 Da corresponding +C2H2. The
Therefore, the structure was assigned as C19:0OO, TAG (19:0/18:1/
retention times of TAGs are governed by ECN. Retention time typically
18:1). Fig. 2(a, b, c, d) illustrates the typical tandem mass spectra of SPS
increases with ECN number, whereas an increase in unsaturation
and of 3 other different TAGs with increasing unsaturation of the fatty
(measured by double bond equivalents, DB) leads to decrease in re-
acid chain in position 2 of the glycerol backbone. Interestingly we ob-
tention time. This trend can be clearly seen in the two-dimensional plot.
served TAGs with fatty acid substituents carrying an odd number of
The separation of most TAGs within one ECN group was feasible and
carbon atoms from pentadecylic acid C15H30O2 to pentacosylic acid
readily predicted by the elution regularity also described by Holčapek
C25H50O2. Such derivatives have been reported in other plants
et al. (2003). The molecular formulae for a total of 83 TAGs were as-
(Řezanka & Sigler, 2009); however, they are relatively rare.
signed, 58 of which were not previously reported in the literature.
As an additional tool for facilitating compound assignment, we used
Table 3 shows high-resolution MS data and molecular formulae as-
neutral loss analysis. The first fragmentation step in TAG fragmentation
signment of all cocoa TAGs identified in Ivory Coast raw unfermented
involves loss of the sn-1/3 acyl group followed by loss of the sn-2 acyl
cocoa beans, the sample showing the highest diversity.
group. Hence, neutral loss chromatograms with defined neutral losses
corresponding to fatty acids could be created. Such neutral loss chro-
3.2. Tandem mass spectrometry matograms show all compounds displaying loss of a defined acyl group.
Fig. 2(e, f, g, h, i) shows exemplary neutral loss chromatograms with
Assignment of individual TAG structures was based on ESI-tandem neutral loss of 273 Da (-C16H37O2N), 301 Da (-C18H41O2N), 299 Da
MS (MSn) measurements. For tandem mass spectrometry an ion trap (-C18H39O2N), 297 Da (-C18H37O2N) and 295 Da (-C18H35O2N) corre-
mass spectrometer was used, operating in positive ion mode using an sponding to ammonia adducts of palmitic acid, stearic acid, oleic acid,
auto MSn routine. The mechanism of TAG fragmentation of ammonia linoleic acid and linolenic acid (Table 1) (Murphy et al., 2007). Because
adducts of TAGs was first suggested by McAnoy et al. (McAnoy et al., one TAG was efficiently identified, another TAG is more easily re-
2005). Collision induced dissociation of [M+NH4]+ precursor ions cognized from the molecular related mass values, with the help of the
resulted in the neutral loss of NH3 and an acyl side-chain (lost as a assembled information from the retention time point, MS2 fragments,
carboxylic acid) to generate a diacyl product ion (Scheme S1 supple- and neutral loss scanning of the corresponding fatty acid moiety.
mentary material). Loss of the first carboxylic acid moiety can occur Combined LC-tandem MS and neutral loss scanning have been applied
from the sn-1/3 position or the sn-2 position of the glycerol backbone. to the assignment of other lipid classes so far (Byrdwell & Neff, 2002;
The mechanism of fragmentation would involve a 1,2 acyl migration to Taguchi et al., 2005). Furthermore, the fragmentation pattern of some
236
D. Sirbu et al. Food Chemistry 254 (2018) 232–240
Table 3
TAG species in raw cocoa beans lipid extract subdivided into homologous series.
No. tR (min) experimental mass [M + NH4]+ theoretical mass [M + NH4]+ error (ppm) molecular formula common name short name
237
D. Sirbu et al. Food Chemistry 254 (2018) 232–240
Table 3 (continued)
No. tR (min) experimental mass [M + NH4]+ theoretical mass [M + NH4]+ error (ppm) molecular formula common name short name
*
Tentatively assigned.
available standards was cautiously studied and compared with cocoa epoxide functionality, as reported as oxidation products of oleic acid, or
TAGs. Their spectra are displayed in supplementary material. We con- an unsaturated fatty acid carrying an additional alcoholic moiety. The
sider that the above-described approach for the identification of TAGs tandem MS data of these polar lipids revealed in MS2 losses of 18 Da
will be a useful tool for the identification of TAGs in any plant material, and 36 Da, corresponding to the loss of respectively one or two mole-
especially seed lipids. cules of H2O, arguing in favour of OH-derivatives. In a second step both
in MS2 and MS3 neutral losses of 276 Da, 278 Da and 280 Da was ob-
served corresponding to a C18 fatty acid with respectively four, three
3.3. Characterization of TAGs with hydroxylated fatty acids substituents and two double bonds. This finding suggests that hydroxylation occurs
at an unsaturated fatty acid, producing a more polyunsaturated fatty
Next, to the TAGs containing saturated and unsaturated fatty acids, acid following fragmentation. An example is the compound assigned as
a second class of unusual TAGs was detected in unfermented beans. PC18:1-OHPS, pseudomolecular ion of m/z 895 (C55H108NO7,
These compounds eluted at earlier retention times (from 4 to 25 min), [M + NH4]+) as precursor ion, in which neutral losses of −273 Da,
indicating a more polar character. Assignment of molecular formulae −301 Da and −280 Da corresponding to stearic acid, palmitic acid,
from high resolution mass data indicated the presence of one, two or and linoleic acid were observed, defining all three fatty side chains of
three additional oxygen atoms in the TAGs: C55H112NO7, C55H110NO8 this polar TAG. The MS2 spectra of [M+NH4]+ and [M+Na]+ of one
and C57H108NO8. The compounds appeared as three sets of homologues, representative compound of each class are presented in Fig. 3(a–f). [M
each with three to four members, with mass increments of C2H4 cor- +Na]+ ions were found to be extremely useful for a better under-
responding to +28 Da. Taking into account the TAG abundancy, three standing of the hydroxyl fatty acid moieties, for the reason that during
main subclasses of these unusual TAGs could be observed (Fig. 3a–f). MS2 fragmentation these fatty acids do not lose their hydroxyl group
The observed molecular formulae suggest compounds with either an
Intensity
Intensity
Constant Neutral Loss: 273.0
x10
7 +MS x108
(a)
0.5 [SPS + NH4]+ 880.9 (e)
6 +MS2 (880.9146) 1.0
x10
[SP]+ 579.6
1 607.6 [SS]+
[C15H31CO]+ 0.5
0 267.1 [C17H35CO]+
4000 +MS3 (881.3604->579.9233)
239.1
2000 323.2 521.7 0.0
Constant Neutral Loss: 301.0
0
200 300 400 500 600 700 800 m/z x108
x10
8 (f)
[POP + NH4]+ 850.9 +MS 2
(b) 0.5
0.0 1
7 577.6 [PO]+ +MS2 (851.4->577.4)
x10 3
2 [PP]+ 551.6 0
0 Constant Neutral Loss: 299.0
5 265.1 [C17H33CO]+ +MS3 (850.9) x108
3
x10
2 247.1
503.5
0.8
(g)
0 0.6
100 200 300 400 500 600 700 800 900 m/z
0.4
7 +MS
x10
0.2
(c) 0.5
0.0
[PLP + NH4]+ 848.8
0.0
5 575.5 [PL]+ +MS2 (848.8) Constant Neutral Loss: 297.0
x10
[PP]+ 551.6 x106
2
0 (h)
+MS3 (849.2->575.8)
[C17H31CO]+ 313.2 1.0
4000
263.2 500.3
465.5
0
200 300 400 500 600 700 800 m/z 0.5
8 +MS
x10 0.0
[PLnP + NH4 ]+ 829.7 Constant Neutral Loss: 295.0
(d) 1 x105
0
x10 7 573.5 [PLn ]
+ +MS2 (829.7) 0.8 (i)
4 [PP ]+ 551.5 811.7 0.6
261.1 317.2
0 0.4
6 +MS3 (830.2->573.5)
x10 317.2
2 [C17H29CO]+ 0.2
261.1
499.4
0 0.0
100 200 300 400 500 600 700 800 m/z 5 10 15 20 25 30 35 40 45 Time [min]
Fig. 2. Positive ion mode tandem mass spectrum showing the fragmentation of triacylglycerols (a) SPS (m/z 881.9), (b) POP (m/z 851.9), (c) PLP (m/z 848.8) and (d) PLnP (m/z 829.7)
ammonium adduct ions. P, palmitic; O, oleic; S, stearic; L, linoleic; Ln, linolenic and n eutral loss chromatograms of (e) 273 Da, (f) 301 Da, (g) 299 Da, (h) 297 Da and (i) 295 Da (from up
to down) corresponding to ammonia adducts of palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid in Ivory Coast cocoa lipid extract.
238
D. Sirbu et al. Food Chemistry 254 (2018) 232–240
Intens.
[PC18:2-OHP + NH4]+ +MS2 (865.0), 12.9 min
x10 6
830.0
(a) 6
4 573.8 [P18C:2-OH - H2O]
+
(e)
2 [PP]+ 551.8
261.2 317.3 812.1
0
+MS2 (870.0), 12.9 min
x10 6 613.8 [PC18:2-OH + Na]
+
[PC18:2-OHP + Na]+
(b) 2.0
1.0
301.2 319.3 573.8
0.0
300 400 500 600 700 800 m/z
Intens.
[PC18:1-OH - H2O]+ [PC18:1-OHP + NH4]+ +MS2 (867.0), 18.2 min
x10 6
4 593.8 [PC18:1-OH]
+ 850.0
(c) 3
[PP]+ 551.8 575.8
2
1 313.3 467.6
0
+MS2 (872.0), 18.2 min
(f)
x10 6
+
615.8 [PC18:1-OH + Na] [PC18:1-OHP + Na]+
1.0
(d)
0.5
321.3
552.8573.7 853.0
0.0
300 400 500 600 700 800 900 m/z
Fig. 3. Positive ion mode tandem mass spectrum showing the fragmentation of triacylglycerols (a) PC18:2-OHP (m/z 865.0), (c) PC18:1-OHP (m/z 867.0) ammonium adduct ions and (b)
PC18:2-OHP (m/z 870.0) and (d) PC18:1-OHP (m/z 872.0) sodium adduct ions. (e) and (f) proposed possible structures of the unusual TAGs in raw cocoa beans.
and the value of the neutral loss matches to the absolute m/z of the compounds in castor oil were identified in cocoa. However, contrary to
corresponding fatty acid. In the case of Fig. 3(b), (d) and (f), it corre- the assignment of Lin et al. (2003) as SLR (1-stearoyl-2-linoleoyl-3-ri-
sponds to palmitic acid, m/z 256. cinoleoyl-glycerol) TAG, we could suggest from tandem MS data
The elemental composition suggests the presence of oxygen within (fragmentation of [M+Na+] spectra shown in supplementary material)
one of the fatty acid side chains. To answer the question of the nature of a molecular species with m/z 924 with an sn-2 position for the ricinoleic
oxygen functionality, an extract enriched in this TAG fraction was acid, therefore revising the initially suggested structures. We could af-
prepared using preparative column chromatography on silica gel, and firm that castor oil contains SRL and not SLR TAG moiety, eluting at
NMR spectra were recorded, as well as a derivatization reaction carried 0.6 min earlier than SC18:1(-OH)O (C57H110NO7) TAG moiety, with the
out using benzoyl chloride. The 1H NMR spectrum shows four olefinic same m/z ratio as in cocoa but having a different fragmentation pattern.
signals, two of them at 5.33 and 5.25 ppm corresponding to a cis-ole- Oxygenated FAs, containing hydroxyl, epoxy, or oxo groups are an
finic moiety, probably in an n–9 oleic acid-like environment. Two fur- uncommon component of seed oils, but have widespread distribution
ther signals at 6.46 and 5.63 ppm show cross peaks in the 1H1H-COSY among higher plant families (Smith, Zhang, & Purves, 2014). Studies
spectrum and an additional cross peak to a triplet at 5.96 ppm, re- confirm that hydroxy fatty acids are plant self-defense substances (Hou
vealing the presence of an allylic alcohol functionality. These peaks & Forman, 2000). In addition, hydroxylated fatty acids have been
must be assigned to olefinic protons with a trans double bond geometry, suggested as precursors in jasmonic acid biosynthesis, a plant hormone
due to a large 3J-coupling of 16.0 Hz. In the 1H13C-HSQC spectrum, the involved in defense reaction that has been observed in fermented cocoa
allylic alcohol CHOH carbon can be assigned to a signal at 73.1 ppm. beans in high abundance (Patras, Milev, Vrancken, & Kuhnert, 2014).
The 1H13C-HMBC spectrum reveals from the CHOH hydrogen signal a When comparing different origins of beans, a large majority of the
2
JHCC interaction to the olefinic carbon at 129.1 ppm and two further TAGs were found in all samples. Indeed, unusual TAGs were found in all
interactions to a CH2 carbon at 30.3 ppm, as well as a 3JHCCC interaction samples analyzed. However, there were subtle differences in relative
to the sn-2 carbonyl carbon at 173.2 ppm. These interactions clearly concentrations when comparing samples within the raw unfermented
place the OH substituent in the β-position of the carbonyl. A char- beans. A detailed analysis of such differences is outside the scope of this
acteristic pair of high field-shifted carbons, as expected for an epoxide, contribution, and accurate quantification will be reported elsewhere.
is clearly absent (Xia, Budge, & Lumsden, 2016). Taking into account
the tandem MS data placing the hydroxy-fatty acid substituent into the
sn-2 position, we propose the structures shown in Fig. 3. All NMR data 4. Conclusions
and key 2D NMR correlations are given in the supplementary material.
Following derivatization with benzoyl chloride the intensities of LC- A new method for TAG analysis, using reversed-phase HPLC coupled
MS signals of all polar TAGs were significantly reduced, indicating high to ESI mass spectrometry, was developed, which allowed the identifi-
yielding derivatization. Additional peaks were observed. In particular, a cation not only of the most abundant TAGs but also of the low abun-
compound with MS data reminiscent of PLnP was observed with a fatty dance TAGs, showing weak signals buried in the noise. Lipid extracts
acid with three double bonds in the sn-2 position, probably obtained by from raw cocoa beans from six different origins were profiled for the
facile elimination from the allylic alcohol functionality by β-elimina- very first time for their TAG content. A total of 83 TAGs could be
tion. EIC signals corresponding to compounds with m/z values of 971.7, characterized in unfermented cocoa beans, 77 of which were positively
999.7 or 1027.8 could be observed. Tandem MS data reveal neutral identified. Fifty-eight of these compounds are reported for the first time
losses corresponding to loss of 139 Da (–C7H6O2 + NH4+) from the in cocoa. Among the newly identified compounds, TAGs carrying a
precursor ions. These findings indicate the presence of benzoyl deri- hydroxyl side-chain were characterized. The reason why these com-
vatives, but further tandem MS data are inconclusive. pounds were not reported before may lie in the fact that these com-
Chromatographic, MS data and 2D map for the derivatives are provided pounds possibly undergo transformation or degradation during cocoa
in the supplementary material. production. These unusual constituents may offer the promise to blend
In nature hydroxylated fatty acids occur prominently in castor oil cocoa butter with unfermented cocoa lipids to produce CB with special
with the TAG containing ricinoleic and dihydroxystearic acid (Lin, physical and chemical properties in new dietary or cosmetic products.
Turner, Liao, & McKeon, 2003). We have used castor oil as surrogate However, further research needs to be carried out to validate this
standard (Clifford & Madala, 2017) in a direct comparison with raw possibility and to elucidate their function.
cocoa bean lipid extract. LC-MS data showed that none of the
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D. Sirbu et al. Food Chemistry 254 (2018) 232–240
240