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Open Chem., 2018; 16: 87–94
Research Article Open Access
O Fayos, GF Barbero, M Savirón, J Orduna, AG Durán, M Palma, JMG Molinillo, FA Macías,
CG Barroso, C Mallor, A Garcés-Claver*
Synthesis of (±)-3,4-dimethoxybenzyl-4-
methyloctanoate as a novel internal standard for
capsinoid determination by HPLC-ESI-MS/MS(QTOF)
https://doi.org/10.1515/chem-2018-0007
received October 19, 2017; accepted December 15, 2017.
Abstract: Capsinoids exhibit health-promoting properties
and are therefore compounds of interest for medical and
food sciences. They are minor compounds present in
relatively high concentrations in only a few number of
pepper cultivars. It is desirable to quantify capsinoids to
provide selected cultivars with high capsinoid contents,
which can then be employed as health food product.
Quantifying low concentrations of capsinoids from
pepper fruit requires a precise and selective analytical
technique such as HPLC coupled to electrospray ionization
- mass spectrometry, with development of an internal
standard essential. In this work, the synthesis method
of a novel compound analogue of capsinoids, the (±)-3,4-
dimethoxybenzyl-4-methyloctanoate, which could be a
suitable internal standard for capsinoid determination by
electrospray ionization - mass spectrometry is described.
(±)-3,4-dimethoxybenzyl-4-methyloctanoate was stable
under the analysis conditions and exerted chemical and
physical properties similar to those of capsinoids. This
internal standard will provide an accurate capsinoid
determination by electrospray ionization - mass
spectrometry, thus facilitating the pepper breeding
programs, screening pepper cultivars and a better
understanding of capsinoid biosynthetic pathway.
Keywords: Capsiate; dihydrocapsiate, DMBO,
fragmentation pattern, HPLC-MS/MS, internal standard,
synthesis method
Introduction 1
Capsinoids are analogues to capsaicinoids, these are the
main compounds responsible for pungency in pepper fruits
(Capsicum spp.), and both are exclusively produced by fruits
of this genus [1-7]. Although capsinoids also contribute to
this eect, they are approximately 1,000 times less pungent
than capsaicinoids [7]. Capsinoids are similar in structure
to capsaicinoids, with the exception of an ester bond in
the place of an amide bond found in capsaicinoids [8].
Until now, three capsinoids have been identied in nature:
capsiate, dihydrocapsiate, and nordihydrocapsiate, with
the rst two the most common [8,9]. Both, capsaicinoids
and capsinoids are powerful biocompounds with
health-promoting properties, such as anti-oxidant, anti-
inammatory, anti-obesity, and analgesic activities [10-
13]. They are also used in arthritis treatments, digestive
disorders or in some diseases like cancer, among others
[14-17]. The advantage of capsinoids over capsaicinoids
is that these do not present side eects like irritancy or a
burning feeling [18,19]. Therefore, capsinoids could have a
greater application range both in medical eld and in food
sciences, in the development of new drugs for long-term
treatments or functional foods. In this regard, identifying
pepper cultivars with high capsinoid content becomes
*Corresponding author: A Garcés-Claver: Departamento
de Hortofruticultura. Centro de Investigación y Tecnología
Agroalimentaria de Aragón. Instituto Agroalimentario de Aragón -
IA2 (CITA-Universidad de Zaragoza), Avda. Montañana 930, 50059,
Zaragoza, Spain, E-mail: agarces@cita-aragon.es
O Fayos, C Mallor: Departamento de Hortofruticultura. Centro de
Investigación y Tecnología Agroalimentaria de Aragón. Instituto
Agroalimentario de Aragón - IA2 (CITA-Universidad de Zaragoza),
Avda. Montañana 930, 50059, Zaragoza, Spain
GF Barbero, M Palma, CG Barroso: Departamento de Química Analítica,
Instituto Universitario de Investigación Vitivinícola y Agroalimentaria
(IVAGRO), Facultad de Ciencias, Universidad de Cádiz, Campus de
Excelencia Internacional Agroalimentario (CeiA3). Campus Universitario
del Río San Pedro, 11510, Puerto Real (Cádiz), Spain
M Savirón, J Orduna: Instituto de Ciencia de Materiales de Aragón
(ICMA-CEQMA). Facultad de Ciencias, CSIC-Universidad de Zaragoza,
C/ Pedro Cerbuna 12, 50009, Zaragoza, Spain
AG Durán, Molinillo, FA Macías: Grupo de Alelopatía, Departamento
de Química Orgánica, Instituto de Biomoléculas (INBIO), Facultad de
Ciencias, Universidad de Cádiz, Campus de Excelencia Internacional
Agroalimentario (CeiA3), Campus Universitario del Río San Pedro,
11510, Puerto Real (Cádiz), Spain
The First Decade (1964-1972)
What Is So Different About
Neuroenhancement?
Pharmacological and Mental Self-transformation in Ethic
Comparison
Abstract: In the concept of the aesthetic formation of knowledge and its as soon
as possible and success-oriented application, insights and profits without the
reference to the arguments developed around 1900. The main investigation also
includes the period between the entry into force and the presentation in its current
version. Their function as part of the literary portrayal and narrative technique.
Keywords: Function, transmission, investigation, principal, period
Dedicated to
1 Studies and Investigations
The main investigation also includes the period between the entry into force and
the presentation in its current version. Their function as part of the literary por-
trayal and narrative technique.
*Max Musterman:
Paul Placeholder:
Open Access. © 2018 O Fayos et al., published by De Gruyter.
The First Decade (1964-1972)
What Is So Different About
Neuroenhancement?
Pharmacological and Mental Self-transformation in Ethic
Comparison
Abstract: In the concept of the aesthetic formation of knowledge and its as soon
as possible and success-oriented application, insights and profits without the
reference to the arguments developed around 1900. The main investigation also
includes the period between the entry into force and the presentation in its current
version. Their function as part of the literary portrayal and narrative technique.
Keywords: Function, transmission, investigation, principal, period
Dedicated to
1 Studies and Investigations
The main investigation also includes the period between the entry into force and
the presentation in its current version. Their function as part of the literary por-
trayal and narrative technique.
*Max Musterman:
Paul Placeholder:
This work is licensed under the Creative Commons Attribution-
NonCommercial-NoDerivatives 4.0 License.
The First Decade (1964-1972)
What Is So Different About
Neuroenhancement?
Pharmacological and Mental Self-transformation in Ethic
Comparison
Abstract: In the concept of the aesthetic formation of knowledge and its as soon
as possible and success-oriented application, insights and profits without the
reference to the arguments developed around 1900. The main investigation also
includes the period between the entry into force and the presentation in its current
version. Their function as part of the literary portrayal and narrative technique.
Keywords: Function, transmission, investigation, principal, period
Dedicated to
1 Studies and Investigations
The main investigation also includes the period between the entry into force and
the presentation in its current version. Their function as part of the literary por-
trayal and narrative technique.
*Max Musterman:
Paul Placeholder:
88 O Fayos et al.
really attractive for human consumption, pharmaceutical
purposes and pepper breeding programs.
The analytical techniques used, routinely in
laboratories, for capsinoid determination are high
performance liquid chromatography (HPLC) coupled to
UV or PDA/DAD detectors [2-4,20]. Since capsinoids and
capsaicinoids are measured at the same wavelength (280
nm), their concentration could be overestimated by these
techniques, which are based only on retention time and
UV spectra, and could assign erroneously a peak to a
given capsinoid. A promising high-resolution detection
technique such as electrospray ionization (ESI)-mass
spectrometry (MS) in combination with HPLC, would
enable a more accurate, precise, and sensitive analysis of
these compounds. This technique allows an unequivocal
identification of compounds by exact mass-to-charge
ratio (m/z), molecular formula and fragmentation pattern,
all of them distinctive features for each compound [21-24].
Nevertheless, the addition of an internal standard (IS) to
the sample is of crucial importance in order to improve
the analytical parameters, avoid ionization fluctuations of
analytes, and correct errors in detection [21-27]. Currently,
there are not any commercially available internal standards
for capsinoid analysis by HPLC-ESI-MS. Isotope-labeled
analogues could be the internal standards. However, they
have disadvantages, such as their scarce availability and
high price. Thus, chemical synthesis is an interesting
alternative to isotopic labelling for obtaining new synthetic
analogues to capsinoids [22, 28].
In this report, we describe for first time a simple
synthesis approach of the (±)-3,4-dimethoxybenzyl-4-
methyloctanoate ((±)-DMBO), a novel synthetic analogue
to capsinoids, and their potential use as IS for capsiate and
dihydrocapsiate determination by HPLC-ESI-MS(QTOF).
Materials and methods2
Chemicals and reagents2.1
For (±)-DMBO synthesis: 3,4-dimethoxybenzaldehyde
(99%) (1), and (±)-4-methyloctanoic acid (98%) (3), and
di-isobutyl aluminium hydride (1 mol L-1 in toluene)
(DIBAL) were purchased from Sigma-Aldrich Chemical Co.
(St. Louis, MO, USA). Sodium sulphate anhydrous (99%),
dehydrated pyridine (99%), and tetrahydrofuran (THF)
(99.5%) were purchased from Panreac Química S.L.U.
(Barcelona, Spain). Ethyl acetate, methanol, and hexane,
all of them HPLC grade, were procured from Scharlab S.L.
(Barcelona, Spain). Thionyl chloride (SOCl2) (99%) was
purchased from Merck Schuchardt OHG (Hohenbrunn,
Germany). Capsiate and dihydrocapsiate standards were
supplied by the Department of Organic Chemistry at the
University of Cádiz, Spain. All standard solutions were
prepared with analytical grade type I water (Milli-Q
Synthesis, Millipore, Bedford, MA).
General experimental procedures2.2
The synthesis of (±)-DMBO (5) was accomplished in three
stages: reduction of the 3,4-dimethoxybenzaldehyde
(1) carbonyl group, chloride formation from
(±)-4-methyloctanoic acid (3) and esterication of 3,4-
dimethoxybenzyl alcohol (2) with (±)-4-methyloctanoyl
chloride (4).
The (±)-DMBO purity was determined by 1H NMR and
13C NMR analysis. 1H and 13C spectra were recorded using
deuterated chloroform (CDCl3) (99.8%, Sigma-Aldrich
Chemical Co. St. Louis, MO, USA) as the solvent, on an
Agilent INOVA spectrometer (Agilent Technologies, Santa
Clara, CA, USA) at 499.719 and 125.654 MHz, respectively.
The resonances of residual chloroform for 1H and 13C were
set to δH 7.25 ppm and δC 77.00 ppm, respectively, and used
as internal reference. UV-Vis spectra were obtained by
using a Varian Cary 50 BIO spectrophotometer (Agilent
Technologies) with chloroform as the solvent. General
IR spectra (KBr) were recorded on a Perkin-Elmer FT-IR
Spectrum 2 spectrophotometer (PerkinElmer, Waltham,
MA, USA). The reactions were monitored by thin-layer
chromatography (TLC) on silica gel (F245 Merck plates).
HPLC-ESI-MS and ESI-MS/MS(QTOF) 2.3
analysis
Individual standard solutions in 60% methanol (20
µmol L-1 for (±)-DMBO and 10 µmol L-1 for capsiate and
dihydrocapsiate) were introduced by direct injection with
a syringe pump (Cole-Parmer Instrument Co., Vernon
Hills, IL, USA) operating at 4 μL min-1, in a Quadrupole
Time-of-Flight (QTOF) mass spectrometer equipped with
an Electrospray Ionization Source (ESI) (MicrOTOF-Q,
Bruker Daltonics, Bremen, Germany). ESI-MS(QTOF) and
ESI-MS/MS(QTOF) analysis were carried out in positive-ion
mode, with capillary and endplate oset voltages of 4500
and -500 V, respectively, and using N2 as the collision gas.
The collision cell energy was set to 10 eV, with an isolation
width for the precursor ion of 2 mass-to-charge ratio (m/z)
Synthesis of (±)-3,4-dimethoxybenzyl-4-methyloctanoate as a novel internal standard... 89
units. The nebulizer gas (N2) pressure, the drying gas (N2)
ow rate and the drying gas temperature were 1.6 bar, 8.0
L min-1 and 200 ºC, respectively. Spectra were acquired in
the m/z 50–800 range. The mass axis was calibrated by
using Na-formate adducts [10 mmol L-1 NaOH, 2.5% (v/v)
formic acid and 50% (v/v) 2-propanol] that were introduced
through a divert valve at the beginning of each direct
injection. For MS/MS spectra the [M+Na]+ ions with m/z
values of 329 and 331 m/z for capsiate and dihydrocapsiate
and (±)-DMBO, respectively, were chosen as precursors,
with an isolation width of 4 m/z units and an amplitude
voltage of 0.45 V. The structures of capsiate, (±)-DMBO,
and dihydrocapsiate are shown in Table 1. Bruker Daltonik
soware packages micrOTOF Control v.2.3 and HyStar
v.3.2 were used to control the system. Data Analysis
v.4.0 was used to process data. Capsiate, (±)-DMBO, and
dihydrocapsiate standards were detected by coupling
the HPLC instrument to the QTOF mass spectrometer.
Chromatographic separation was performed on an
Agilent 1100 series HPLC system (Agilent Technologies,
Waldbron, Germany) equipped with an isocratic pump, an
autosampler, and a thermostated column compartment.
The column used was an analytical HPLC C18 column
(Symmetry C18, 2.1 x 150 mm; 3.5 μm spherical particle
size, Waters, Milford, MA, USA) protected by a guard
column (Symmetry C
18, 2.1 mm i.d. x 10 mm length,
3.5 μm spherical particle size, Waters). The HPLC
conditions were methanol (solvent A) and Milli-Q water
acidied with 0.1% (v/v) acetic acid (solvent B) as eluents,
0.2 µL min-1 as ow rate, 25 ºC as column temperature,
and 5 μL as injection volume. The elution program was
set according to the following gradient: 0-7 min, 60% B;
7-15 min, 60-70% B; 15-52 min, 70-100% B.
Ethical approval: The conducted research is not
related to either human or animals use.
Results and discussion3
(±)-DMBO Synthesis procedure (5) 3.1
Reduction of the 3,4-dimethoxybenzaldehyde (1) 3.1.1
carbonyl group
The initial reaction stage involved the reduction of the
carbonyl group of 3,4-dimethoxybenzaldehyde (1) to its
corresponding alcohol (2) (Figure 1). The reduction of the
carbonyl group was performed by using DIBAL (1 mol L-1
in toluene). The compound (1) (3.0042 g, 0.0181 mol) was
dissolved in 40 mL of dry THF. Subsequently, 2 equivalents
of DIBAL (1 mol L-1 in toluene) were added slowly, with a
syringe, in an ice bath, and the reaction mixture was
stirred, under argon, at ambient temperature, for 2 h. The
reaction was monitored by TLC (eluent: 20% ethyl acetate,
80% hexane; chromogenic reagent: anisaldehyde). Then,
the reaction was stopped with water and aer purication
of the reaction mixture, by column chromatography
(eluent: 20% ethyl acetate, 80% hexane; chromogenic
reagent: anisaldehyde), and (2) was obtained with a yield
of 77.16%.
Table 1: Common name, chemical formula, molecular weight, and [M+Na]+ of capsiate, (±)-DMBO, and dihydrocapsiate.
Common Name Chemical Structure Chemical Formula Molecular Weight Measured [M+Na]+
Capsiate
Common Name
Chemical Structure
Chemical
Formula
Molecular
Weight
Measured
[M+Na]+
Capsiate
C18H26O4
306.3966
329.1678
Dihydrocapsiate
C18H28O4
308.4125
331.1879
()-DMBO
C18H28O4
308.4125
331.1855
CHO. .
Dihydrocapsiate
Common Name
Chemical Structure
Chemical
Formula
Molecular
Weight
Measured
[M+Na]+
Capsiate
C18H26O4
306.3966
329.1678
Dihydrocapsiate
C18H28O4
308.4125
331.1879
()-DMBO
C18H28O4
308.4125
331.1855
CHO . .
(±)-DMBO
O
H
3
CO
O
H
3
CO
CHO. .
Figure 1: Step 1 of the synthesis procedure of (±)-DMBO. Reduction of
3,4-dimethoxybenzaldehyde (1) to 3,4-dimethoxybenzyl alcohol (2).
90 O Fayos et al.
Chloride formation from (±)-4-methyloctanoic acid 3.1.2
(3)
The (±)-4-methyloctanoic acid (3) was transformed into
(±)-4-methyloctanoyl chloride (4), in order to subsequently
perform the acylation of this compound (2) (Figure 2).
To do so, 2 equivalents of (3) (4.4307 g, 0.028 mol) were
placed in a 50-mL round-bottom ask into which argon
was introduced. An amount of SOCl2, sucient to dissolve
the acid, was added very slowly, dropwise from a syringe,
and stirred magnetically. Aer the addition of SOCl2, the
reaction mixture was heated at 60 oC for 1 h. Subsequently,
the excess of SOCl2 excess was eliminated in vacuo, and
the transparent oil of (4) was obtained. This compound
(4) was dissolved in dry THF for the subsequent acylation
with compound (2).
Esterification of 3,4-dimethoxybenzyl alcohol (2) 3.1.3
with (±)-4-methyloctanoyl chloride (4)
The (±)-3,4-dimethoxybenzyl-4-methyloctanoate
((±)-DMBO) (5) was prepared by the acylation of
the 3,4-dimethoxybenzyl alcohol ring (2) using a
(±)-4-methyloctanoyl chloride chain (4) (Figure 3).
The nal product (±)-DMBO (5) is a racemic mixture
since the synthesis has been started from the racemic
product (±)-4-methylocatanoic acid (3) and the proposed
synthesis is not stereoselective. Compound (2) (2.3321 g,
0.0140 mol) was dissolved in 20-25 mL of dry pyridine in a
50-mL round-bottom ask under a protective atmosphere
with argon. Then, 2 equivalents of (4) were added slowly
to this solution and agitated for 18 h. The reaction was
monitored by TLC (eluent: 20% ethyl acetate, 80% hexane;
chromogenic reagent: anisaldehyde) up to the reaction´s
completion.
The (±)-DMBO esterification step was simpler than
that reported for capsinoid and capsaicinoids standards
synthesis [29]. Through this synthesis procedure, (±)-DMBO
(5) was obtained, after purification of the reaction mixture,
with a yield of 95% and a purity ≥ 98%. The overall yield
obtained for the synthesis of (±)-DBMO starting from the
initial material has therefore been 73.15%. This yield was
higher than those obtained by other synthesis procedures
previously reported for capsinoid and capsaicinoid
standards [12,29-33]. Thus, this procedure would be an
efficient alternative to other synthesis methodologies
for capsinoid derivatives, such as enzymatic catalysis or
labeled compound synthesis, due to its lower cost and
higher yield.
Chemical characterization of (±)-DMBO 3.2
(5)
The formation of (±)-DMBO was conrmed by 1H NMR
and 13C NMR analysis. Since (±)-DMBO (5) (Figure 4) has
never been previously described in the bibliography, its
chemical characterization is presented below:
Yellow oil. 1H NMR (500 MHz, CDCl3, δ, ppm)
(Assignments with identical superscripts are
interchangeable) (S1 _Figure): 6.91 (dd, 1H, J = 2.0 Hz, J =
8.1 Hz, 6), 6.87 (bd, 1H, J = 1.9 Hz, 2), 6.83 (bd, 1H, J = 8.1
Hz, 5), 5.03 (s, 2H, 8), 3.87 (sa, 3H, 7), 3.86 (sa, 3H, 7´), 2.38-
2.26 (m, 2H, 2”), 1.69-1.63 (m, 1H, 3a”), 1.47-1.41 (m, 1H, 3b”),
1.41-1.35 (m, 1H, 4”), 1.30-1.17 (m, 1H, 5a”), 1.30-1.17 (m, 2H,
7”), 1.30-1.17 (m, 2H, 6”), 1.12-1.07 (m, 1H, 5b”), 0.86 (t, 3H, J
= 6.4 Hz, 8”), 0.84 (d, 3H, J = 6.5 Hz, 9”); 13C NMR (125 MHz,
CDCl3, δ, ppm): 173.98 (1”), 148.99b (4), 148.91b (3), 128.59
(1), 121.17 (6), 111.70 (2), 110.94 (5), 66.14 (8), 55.85c (7),
55.81c (7´), 36.27 (5”), 32.31 (4”), 32.12 (2”), 31.85 (3”), 29.09
(6”), 22.87 (7”), 19.24 (9”), 14.05 (8”); IR (cm–1 ): 2956, 1735,
Figure 2: Step 2 of the synthesis procedure of (±)-DMBO.
Preparation of (±)-4-methyloctanoyl chloride (4) from
(±)-4-methyloctanoic acid (3).
Figure 3: Step 3 of the synthesis procedure of (±)-DMBO. Procedure for the preparation of (±)-DMBO (5) from the acylation of 3,4-
dimethoxybenzyl alcohol (2) with (±)-4-methyloctanoyl chloride (4).
Synthesis of (±)-3,4-dimethoxybenzyl-4-methyloctanoate as a novel internal standard... 91
1519, 1162; λmax (nm) 279.9; ε (L/mol·cm-1) 338; HRMS (ESI)
calculated for C18H28O4 [M+Na]+: 331.1879, found: 331.1884.
HPLC-ESI-MS(QTOF) and HPLC-ESI-MS/3.3
MS(QTOF) analysis of capsiate, (±)-DMBO,
and dihydrocapsiate standards
In order to evaluate the suitability of (±)-DMBO as an IS
for capsinoid determination by ESI-mass spectrometry, its
retention time and ionization behaviour were determined
by HPLC-ESI-MS(QTOF) and HPLC-ESI-MS/MS(QTOF)
and compared to those of capsiate and dihydrocapsiate
standards.
The MS spectra of capsiate, (±)-DMBO, and
dihydrocapsiate were acquired, in positive ion mode, by
direct injection of the standard solutions on the QTOF
mass analyser. The major peaks observed in the MS
spectra corresponding with the [M+Na]+ ions were at the
m/z 329.1678 for capsiate, 331.1879 for dihydrocapsiate
and 331.1855 for (±)-DMBO (Table 1). These molecular ions
were in agreement with the calculated m/z 329.1723 for
capsiate and 331.1880 for (±)-DMBO and dihydrocapsiate;
therefore, they were selected as precursors for ESI-MS/
MS experiments. Figure 5 shows the MS/MS spectra
obtained for capsiate, (±)-DMBO, and dihydrocapsiate.
For both capsinoid standards, the most intense peak
was observed at m/z 159.0375 for capsiate and 159.0418
for dihydrocapsiate, in accordance with the calculated
m/z 159.0417, corresponding to sodiated vanillyl ring.
Other minor product ions detected at m/z 137.0570 and
137.0605, in conformity with the calculated m/z 137.0597,
corresponding to the protonated vanillyl ring of capsiate
and dihydrocapsiate, respectively, and generated by
the cleavage of the C7-O8 bond of these molecules
(Figure 5, A and B). On (±)-DMBO MS/MS spectrum,
the most intense peak was observed at m/z 151.0750
(Figure 5C), in agreement with the calculated m/z
151.0754 for the expected vanillyl-type ring resulting from
fragmentation at the ester bond. The ionization behaviour
and break site of (±)-DMBO molecules were similar to those
Figure 4: Chemical structure of (±)-DMBO (5). Chemical
characterization of (±)-DMBO by 1H NMR and 13C NMR analysis.
Figure 5: ESI-MS/MS spectra in positive ion mode of capsiate,
dihydrocapsiate and (±)-DMBO. The individual standard solutions
in 60% methanol of 10 µmol L-1 capsiate (A) and dihydrocapsiate (B),
and 20 µmol L-1 (±)-DMBO (C) were acquired by direct injection on the
QTOF mass analyser.
92 O Fayos et al.
shared by capsinoid standards. MS/MS spectra obtained
for (±)-DMBO, capsiate, and dihydrocapsiate matched well
with the typical fragmentation pattern for capsinoids [34]
and capsaicinoids [35].
Ion chromatograms were extracted for the exact
m/z values corresponding to the [M+Na]+ species of
capsiate, (±)-DMBO, and dihydrocapsiate (Figure 6). The
peaks of the capsiate, (±)-DMBO, and dihydrocapsiate
standards showed their elution times at 31.0, 34.0, and
35.3 min, respectively. Since the chromatogram traces
corresponding to m/z 331 were extracted for (±)-DMBO
and dihydrocapsiate, the resolution (Rs) of these two
compounds was calculated in order to confirm the correct
separation of both compounds. The Rs of dihydrocapsiate
and (±)-DMBO (defined as 2 times the separation between
peaks divided by the sum of the peak base widths) was 1.2,
a value that can be considered as adequate.
The potentiality of using (±)-DMBO as IS in capsinoid
determination by ESI-mass spectrometry was supported
by the following facts i) it is a capsinoid analogue not
occurring naturally in Capsicum species, ii) the ionization
behaviour of (±)-DMBO in ESI mass is similar to that of
capsiate and dihydrocapsiate, and iii) its retention time is
similar to that of capsiate and dihydrocapsiate, (±)-DMBO
eluting between the two analytes but being able to be
separated from dihydrocapsiate.
Conclusion4
In this study, a (±)-DMBO synthesis procedure has been
described. The synthesis procedure developed is simple,
economical, and achievable in any basic chemistry
laboratory, and it also allows the obtaining of yields
greater than 95%.
(±)-DMBO could be a potential IS for capsinoid
determination because: (i) it is not present in pepper fruits;
(ii) it is stable under analysis conditions; (iii) it elutes
between capsiate and dihydrocapsiate; (iv) its chemical
structure is closely related to that of capsinoids; (v) and its
ionization behaviour and fragmentation pattern is shared
by capsiate and dihydrocapsiate.
As far as we know, there is not any other IS
available for capsinoid determination by HPLC-ESI-MS/
MS(QTOF) analysis. Synthetic analogues have already
been successfully used as IS for another compound
determination by ESI-MS techniques [36-37]. We have
described for first time a simple synthesis approach of
a capsinoid analogue suitable for IS. The development
of a new IS will allow an accurate quantification of
capsinoid by HPLC-ESI-MS, a useful tool for screening
and selecting of cultivars containing capsinoids in pepper
breeding programs and for gaining understanding of the
metabolism of capsinoids.
Acknowledgments: This study was supported by the
National Institute for Agriculture and Food Research
and Technology (INIA) and has been co-nanced by the
European Fund for Regional Development (FEDER) (grant
number RTA2011-00118-C02 and RTA2015-00042-C02-00).
Also, it was supported for Aragon Government-A16. Author
O. Fayos has received research grants from INIA (RTA2011-
00118-C02-01).
Conict of interest: Authors state no conict of interest.
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