Article

In Silico Mass Spectrometric Fragmentation and Liquid

Chromatography with Tandem Mass Spectrometry (LC-MS/MS)
Betalainic Fingerprinting: Identification of Betalains
in Red Pitaya
Jesús Alfredo Araujo-León 1 , Ivonne Sánchez-del Pino 2 , Ligia Guadalupe Brito-Argáez 1 ,
Sergio R. Peraza-Sánchez 3 , Rolffy Ortiz-Andrade 4 and Victor Aguilar-Hernández 1, *

1 Unidad de Biología Integrativa, Centro de Investigación Científica de Yucatán, A.C.,

Mérida 97205, Yucatán, Mexico; jalfredoaraujo@gmail.com (J.A.A.-L.); lbrito@cicy.mx (L.G.B.-A.)
2 Unidad de Recursos Naturales, Centro de Investigación Científica de Yucatán, A.C.,
Mérida 97205, Yucatán, Mexico; isanchez@cicy.mx
3 Unidad de Biotecnología, Centro de Investigación Científica de Yucatán, A.C., Mérida 97205, Yucatán, Mexico;
speraza@cicy.mx
4 Facultad de Química, Universidad Autónoma de Yucatán, Mérida 97069, Yucatán, Mexico;
rolffy@correo.uady.mx
* Correspondence: victor.aguilar@cicy.mx

Abstract: Betalains, which contain nitrogen and are water soluble, are the pigments responsible for
many traits of plants and biological activities in different organisms that do not produce them. To
better annotate and identify betalains using a spectral library and fingerprint, a database catalog
Citation: Araujo-León, J.A.;
of 140 known betalains (112 betacyanins and 28 betaxanthins) was made in this work to simplify
Sánchez-del Pino, I.;
Brito-Argáez, L.G.;
betalain identification in mass spectrometry analysis. Fragmented peaks obtained using MassFrontier,
Peraza-Sánchez, S.R.; along with chemical structures and protonated precursor ions for each betalain, were added to the
Ortiz-Andrade, R.; database, which is available as Supplementary Materials in the manuscript. Product ions made in
Aguilar-Hernández, V. In Silico Mass MS/MS and multistage MS analyses of betanin, beetroot extract, and red pitaya extract revealed the
Spectrometric Fragmentation and fingerprint of betalains, distinctive ions of betacyanin, betacyanin derivatives such as decarboxylated
Liquid Chromatography with Tandem and dehydrogenated betacyanins, and betaxanthins. A distinctive ion with m/z 211.07 was found
Mass Spectrometry (LC-MS/MS) in betaxanthins. By using the fingerprint of betalains in the analysis of red pitaya extracts, the
Betalainic Fingerprinting: catalog of betalains in red pitaya was expanded to 86 (31 betacyanins, 36 betacyanin derivatives, and
Identification of Betalains in Red
19 betaxanthins). Four unknown betalains were detected to have the fingerprint of betalains, but
Pitaya. Molecules 2024, 29, 5485.
further research will aid in revealing the complete structure. Taken together, we envisage that the
https://doi.org/10.3390/
further use of the fingerprint of betalains will increase the annotation coverage of identified molecules
molecules29225485
in studies related to revealing the biological function of betalains or making technologies based on
Academic Editors: Marilena Dasenaki these natural colorants.
and Niki Maragou

Received: 24 September 2024 Keywords: betacyanins; betacyanin derivatives; betaxanthins; LC-MS/MS; fingerprint betalains;
Revised: 6 November 2024 red pitaya
Accepted: 12 November 2024
Published: 20 November 2024

1. Introduction
Betalains are water-soluble natural pigments that contain nitrogen and have interesting
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
absorption and emission properties; they are backed by conjugated double bonds and
This article is an open access article
at least one heterocyclic nitrogen. Studies on the properties of betalains have revealed
distributed under the terms and that, besides their spectroscopic and fluorescent properties, betalains are essential for
conditions of the Creative Commons attracting pollinators and dispersers by providing color to plant tissues; contribute to
Attribution (CC BY) license (https:// drought tolerance and excessive light stress [1]; and are important for preserving good
creativecommons.org/licenses/by/ health through their diverse biological activities, such as anti-inflammatory, antioxidant,
4.0/). anti-hypertensive, antidiabetic, and immunomodulatory activities, as well as through

Molecules 2024, 29, 5485. https://doi.org/10.3390/molecules29225485 https://www.mdpi.com/journal/molecules

Molecules 2024, 29, 5485 2 of 24

their cancer chemoprotective properties [2,3]. Another health benefit of betalains has been
seen when betalains are used as a natural food colorant, which has been approved by the
Food and Drug Administration (FDA) and by the National Food Safety Standard in China
(National Food Safety Standard Use for Food Additive, GB2760-2011) [4]. However, the
different biological activity tests and the color of the plant tissues correlate with the betalains
contained in the extracts. The assertion that specific betalains are present points out the
value of diverse sources of betalains, suggesting that analytical methods are crucial for
accurately identifying betalains with high sensitivity. Sources of betalains include most
families of plants in the order Caryophyllales, fungi of the genus Amanita, and the bacteria
Gluconacetobacter diazotrophicus [5–7]. Biochemical, genetic, and evolutionary studies have
revealed that the lineage-specific production of betalains in the order Caryophyllales lies in
the conservation and duplication events of genes encoding enzymes in the biosynthetic
pathway of betalains [8].
Two main classes of betalains exist: red-violet betacyanins and yellow-orange be-
taxanthins. In plants, during the biosynthesis of betalains, betalamic acid is condensed
with either cyclo-DOPA or glycosylated cyclo-DOPA. The sugar moieties can be acylated
with aromatic organic acids, such as ferulic, p-coumaric, caffeic, sinapic, and malonic acid,
to further prevent the cleavage of betalains by β-glucosidases. Betaxanthins result from
condensed betalamic acid with an imino or amino group of amino acids. Extra expansion
of the chemical diversity of betalains is due to epimerization at the chiral center C15 [9].
By contrast, the degradation of betalains involves decarboxylation by removing carboxyl
groups and oxidative dehydrogenation. More factors that impact the stability of betalains
have been noted to result in damaging effects on the pigment. These causes include the
temperature of food storage, pH, and light, among other causes, which are extensively
reviewed in Sadowska-Bartosz and Bartosz 2021 [10]. Plant edibles of the order Caryophyl-
lales that produce betalains include roots of Beta vulgaris (beetroot), tubers such as Ullucus
tuberosus Caldas (Ullucos), leaf of B. vulgaris L. ssp. Cicla (Chard), grain and vegetable
amaranth classes (Amaranthus cruentus L., A. caudatus L. and A. hybridus L., Amaranthus
tricolor), and fruits of the genus Opuntia, Eulychnia, and Selenicereus (Hylocereus) [10].
Fruit of the genus Selenicereus exist in three different colors assigned to betalains [11,12]:
red-skinned fruit with white flesh (Selenicereus undatus), red-skinned fruit with red flesh (S.
costaricensis and S. monacanthus), and pink-skinned fruit with white flesh or yellow-skinned
fruit with white flesh (S. megalanthus). Among those species, S. undatus is the most widely
spread and cultivated around the world, including countries such as Vietnam, China,
Indonesia, the United States, and Mexico, among others. In Selenicereus spp., as in other
Caryophyllales plants, the lack of analytical standards for characterizing betalains has been
managed by the chemical synthesis of betalains and purifying and separating individual
endogenous betalains to reveal the biological activities supported by them [13–17]. How-
ever, key biological activities have been revealed for betalains in Selenicereus spp., such as
antioxidant activity [18–20]; the thermal stability of betacyanins from juice [21–24]; the sta-
bility of betalains in pure form, in spray-dried pitaya peel powders, or as a natural colorant
in food [25–27]; antibacterial activity [28–30]; and antiviral activity against IAV (influenza A
virus)-infected A549 cells [31]. As multiple betalains may exist, as in other Caryophyllales
plants, identifying betalains needs extensive coverage to assess the influence of the diverse
array of betalains.
Structural characterization and massive surveying of betalains through either Nuclear
Magnetic Resonance (NMR) or mass spectrometry (MS) not only provides opportunities to
understand the biology of betalains but also helps to distinguish unrevealed betalains. For
example, the preparative fractionation of betalains from Phytolacca americana has resulted
in the discovery of structural features of 15S-betanin and 15R-isobetanin and an expansion
of the betalains catalog to 17 betalains [16,32]. The catalog of betalains has been further
expanded by liquid chromatography (LC) coupled with MS and the fortunate discovery of
distinct retention times of betalain epimers during chromatography analysis, which typi-
cally display different retention times. Although candidates for betalains were identified
Molecules 2024, 29, 5485 3 of 24

at all five confidence levels [33,34], most of them were identified at level 4 with molecu-
lar formulas by means of charge state determination, adduct ion determination, isotope
abundance distribution, and UV–Vis absorption [35–37]. The catalog of betalains has been
expanded to 22 betalains (15 betaxanthins and 7 betacyanins) in beetroot [38], 24 betalains
(18 betaxanthins and 6 betacyanins) in 10 Mexican prickly pear cultivars [39], 28 betalains
(19 betaxanthins and 9 betacyanins) in Swiss chard [40], 43 betalains (30 betaxanthins and
13 betacyanins) in Amaranthus cruentus [37], 48 betalains (22 betaxanthins and 26 beta-
cyanins) in three beetroot cultivars [41], 68 betalains (34 betaxanthins and 34 betacyanins)
in Djulis [36], and 146 betacyanins, most of which have a high molecular weight of over
1000 Da, in Bougainvillea glabra [42]. Given that some betacyanins are specific to certain
species, the combined number of betacyanins from different species exceeds 200 [42,43],
which highlights the wide variety of these pigments.
This work aims to examine the fingerprint of betalains in samples that are compatible
with mass spectrometry by means of MSPD (Matrix Solid-Phase Dispersion)-based prepa-
ration samples, LC-MS/MS analysis, and spectra interpretation aided by in silico spectral
libraries of betalains.

2. Results and Discussion

2.1. In Silico Fragmentation Library of Betalains Using MassFrontier Software
To better understand the natural diversity of betalains, we exploited spectral libraries
to achieve structural characterization, where the fragment peaks and precursor mass work
together to reveal the features of each pigment. We began by conducting a survey of all
known betalains [15,36,42,43], then created chemical structures and precursor masses for
in silico fragmentation using MassFrontier software 7.0 [44]. In the database, there were
140 betalains. Betalains can then be grouped into betacyanins and betaxanthins (Figure 1).
The database has more betacyanins than betaxanthins, with 112 betacyanins and 28 betax-
anthins, which correspond to 80% and 20%, respectively. Then, when the betacyanins were
classified into betacyanin classes as reported by Xie et al., 2021 and Kumorkiewicz-Jamro
et al., 2021 [36,43], the dominant betacyanin class was betanin-type, with 39.4% of the beta-
cyanins, followed by amaranthin-type, melocactin-type, apiocactin-type, gomphrenin-type,
and glabranin-type. When betaxanthin classes were reported by Esteves et al., 2022 [15], the
dominant class was the hydrophobic-type with 39.3%, followed by special cases that
include r-aminobutyric acid-bx(betaxanthin), dopamine-bx, L-DOPA-bx, tyramine-bx,
3-methoxy-tyramine-bx, 5-hydroxynorvaline-bx, methionine sulfoxide-bx, the positively
charged-type, the polar uncharged-type, and the negatively charged-type.
When the molecular mass distribution of betalains was analyzed, the molecular ion
[M+H]+ masses of betalains ranged from 250 to 1350. However, the molecular ion [M+H]+
masses of betacyanins and betaxanthins do not overlap (Figure 2). Betaxanthins with
smaller molecular ion [M+H]+ masses map up to 400, while betacyanins not only have larger
molecular masses but also display isomeric forms at C-15 (Figure 3A). For instance, betanin
and isobetanin, or the aglycone of almost all betacyanins known as betanidin, with its
isomeric form being isobetanidin. Betacyanin’s molecular ion [M+H]+ masses being larger
than betaxanthins is explained by the attached glycosyl groups, while betaxanthins contain
an amino group of amino acids. The database displayed in Table S3 also shows the chemical
structure, chemical formula, monoisotopic mass, [M+H]+ ion, and the product ion mass list
of [M+H]+ for each betalain. This library compiles a list of both shared and specific ion mass
lists for betacyanins and betaxanthins. They share the molecular frame of betalamic acid,
and the structure of aglycone has the frame of all betacyanins, with m/z 389.0979, except for
decarboxylated or dehydrogenated betacyanins. The specific framework for betacyanin-
types are as follows: betanin-type with m/z 551.1508, amaranthin-type with m/z 727.1829,
melocactin-type with m/z 713.2036, apiocactin-type with m/z 683.1930, gomphrenin-type
with m/z 551.1508, glabranin-type with m/z 713.2036, and specific ions for betaxanthin
classes. In betacyanins, extra shifts of m/z are associated with the addition of substituents
or the loss of an H2 or carboxylic group. For instance, Figure 2 shows the 16 amaranthin-
Molecules 2024, 29, 5485 4 of 24

Molecules 2024, 29, x FOR PEER REVIEW 4 of 24

type betacyanins, which are heavier and lighter than amaranthin. Currently, this database
is both new and has the largest collection of betalains available, as far as we know.

Molecules 2024, 29, x FOR PEER REVIEW 5 of 24

Figure 1. An outline of the classes of betalains used in the spectral library. (A) Betalain-types;
(B)Figure 1. An outline and
betacyanin-types; of the classes
(C) of betalains used in the spectral library. (A) Betalain-types; (B)
betaxanthin-types.
betacyanin-types; and (C) betaxanthin-types.

When the molecular mass distribution of betalains was analyzed, the molecular ion
[M+H]+ masses of betalains ranged from 250 to 1350. However, the molecular ion [M+H]+
masses of betacyanins and betaxanthins do not overlap (Figure 2). Betaxanthins with
smaller molecular ion [M+H]+ masses map up to 400, while betacyanins not only have
larger molecular masses but also display isomeric forms at C-15 (Figure 3A). For instance,
betanin and isobetanin, or the aglycone of almost all betacyanins known as betanidin, with
its isomeric form being isobetanidin. Betacyanin’s molecular ion [M+H]+ masses being
larger than betaxanthins is explained by the attached glycosyl groups, while betaxanthins
contain an amino group of amino acids. The database displayed in Table S3 also shows
the chemical structure, chemical formula, monoisotopic mass, [M+H]+ ion, and the prod-
uct ion mass list of [M+H]+ for each betalain. This library compiles a list of both shared
and specific ion mass lists for betacyanins and betaxanthins. They share the molecular
frame of betalamic acid, and the structure of aglycone has the frame of all betacyanins,
with m/z 389.0979, except for decarboxylated or dehydrogenated betacyanins. The specific
framework for betacyanin-types are as follows: betanin-type with m/z 551.1508, amaran-
thin-type with m/z 727.1829, melocactin-type with m/z 713.2036, apiocactin-type with m/z
683.1930, gomphrenin-type with m/z 551.1508, glabranin-type with m/z 713.2036, and spe-
cific ions for betaxanthin classes. In betacyanins, extra shifts of m/z are associated with the
addition of substituents or the loss of an H2 or carboxylic group. For instance, Figure 2
shows the 16 amaranthin-type betacyanins, which are heavier and lighter than amaran-
thin. Currently, this database is both new and has the largest collection of betalains avail-
able, as far as we know.
Figure 2.
Figure 2. Distribution
Distributionof the parent
of the ion masses
parent of betaxanthin
ion masses and betacyanin
of betaxanthin classes. There
and betacyanin are 140There are
classes.
betalains displayed, but many of them are isomeric.
140 betalains displayed, but many of them are isomeric.
Molecules 2024, 29, 5485
Figure 2. Distribution of the parent ion masses of betaxanthin and betacyanin classes. There are 140 5 of 24
betalains displayed, but many of them are isomeric.

Figure
Figure3.3.Identification of of
Identification betanin andand
betanin its isomer isobetanin.
its isomer (A) The
isobetanin. (A) chemical structure
The chemical of betanin
structure of betanin
and
andisobetanin.
isobetanin.(B)(B)
Extract ionion
Extract current chromatogram
current chromatogramobtained for the
obtained forion
them/z
ion551.1495.
m/z 551.1495.

2.2. Fingerprint Mass Spectrometry of Betalains Using Betanin and Betanidin

To highlight and support the main fragments in the made-in-silico fragmentation
library of betalains that can aid in the definition of the betalain fingerprint, we used a
previously reported untargeted metabolomics platform [37] that identified the betanin and
its isomer in a beetroot extract from a SIGMA vendor (Cat. 901266; Figure 3). Betanin differs
from isobetanin at the chiral center C15, and their retention times were 3.46 and 6.25 min,
as displayed in an extract ion current chromatogram (XIC) with m/z 551.1495 (Figure 3B).
The chromatographic resolution of enantiomers in betalains is not surprising; it did not
require the use of columns with a chiral stationary phase. For instance, longer retention for
isobetanin in C18-based reversed-phase chromatography than betanin has been reported in
numerous studies [36,37,45,46]. The difference in chromatographic retention time between
betanin and isobetanin has been attributed to their unique stereochemistry at the C-15
position, which affects their interaction with the chromatographic column and, thus, their
elution times [9]. Taking advantage of the link between structural features and fragments
of betanin in the in silico library, the peak with m/z 389.09 in the betanin spectrum was
annotated as the aglycone of betanin, also known as betanidin/isobetanidin.
Next, to explore the fragmentation of the betanidin in more detail, which is the agly-
cone of almost all betacyanins with m/z 389.09, our acquisition of MS/MS via fragmentation
with high-energy HCD at 50 eV in a data-dependent acquisition experiment was compared
with an analysis in multistage MS with collision-induced dissociation (CID) (Figure 4). Key
fragmentation features were discovered in the analysis, including common product ions
such as m/z 345.10 due to the loss of a carboxyl group and m/z 343.09 due to the loss of a
carboxyl group plus H2 , and the ions with m/z 150.05, 194.04, 178.05, 132.04, and 106.06.
Decarboxylated betanidin followed 5,6-dihydroxy-indole, and two carbonyls attached
to the pyridine ring were prominent feature assignments. Further, to reveal other ions
of betalains for the straightforward identification of betalains, the betanin MS2 spectra
Molecules 2024, 29, 5485 6 of 24

were surveyed for ions with relative abundance higher than 5% and matching structure
(Table S2). Up to 23 ions with m/z between 389.09 and 106.06 were discovered. Thirteen
ions with m/z greater or equal to 253.09 and ions with m/z 176.07 and 150.05 were matched
with exclusive structural sections of betacyanins, whereas eight distinct ions with m/z
211.07, 194.04, 179.08, 178.05, 166.05, 138.05, 132.04, and 106.06 were matched with the core
Molecules 2024, 29, x FOR PEER REVIEW 7 of 24 for
structure of betalains containing the pyridine ring. The structural information deduced
the ions found here suggests that they are essential fragments for identifying betalains.

Figure 4. Fragmentation spectrum of betanin. (A) MS/MS spectrum of the ion m/z 551.1495 frag-
Figure 4. Fragmentation spectrum of betanin. (A)3MS/MS spectrum of the ion m/z 551.1495 frag-
mented with high-energy HCD at 50 eV. (B) MS spectrum obtained with the ion with m/z 389.09
mented with high-energy HCD at 50 eV. (B) MS3 spectrum obtained with the ion with m/z 389.09
fragmented with CID at 35 eV (551.1495 CID35 → 389 ).→).
CID35
fragmented with CID at 35 eV (551.1495 CID35  389 CID35

2.3. Identification of Betalains in Beetroot Extract

2.3. Identification of Betalains in Beetroot Extract
We used a previously reported method compatible with mass spectrometry sample
We used a previously reported method compatible with mass spectrometry sample
preparation based on Matrix Solid-Phase Dispersion (MSPD) [37] that extracted betalains
preparation based on Matrix Solid-Phase Dispersion (MSPD) [37] that extracted betalains
from
from beetroot extract.
beetroot Theanalysis
extract. The analysisofofhomemade
homemade beetroot
beetroot extract
extract waswas chosen
chosen as itas it would
would
survey
survey a large population of betalains. The mass spectrometry data, derived from data-data-
a large population of betalains. The mass spectrometry data, derived from
dependent
dependent acquisition withhigh-energy
acquisition with high-energyHCDHCD fragmentation,
fragmentation, waswas interrogated
interrogated by XIC,
by XIC,
selecting
selecting features harboring the core ions of betalains as product ions, including the ionsions
features harboring the core ions of betalains as product ions, including the
withm/z
with m/z 211.07, 194.04,179.08,
211.07, 194.04, 179.08,178.05,
178.05, 166.05,
166.05, 138.05,
138.05, 132.04,
132.04, and and
106.06. 106.06. Aof
A total total of 36 be-
36 beta-
talains were identified (Table S3). Furthermore, 26 betacyanins in the collected
lains were identified (Table S3). Furthermore, 26 betacyanins in the collected betalains betalains
were
wereidentified
identified using the
theions
ionswith m/z345.10,
withm/z 345.10, 343.09,
343.09, 299.10,
299.10, 297.08,
297.08, 281.09,
281.09, 269.09,
269.09, 255.11,
255.11,
253.09,
253.09, and
and 389.09, correspondingtotothe
389.09, corresponding theaglycone
aglycone of of almost
almost all all betacyanins,
betacyanins, except
except for for
betacyanin
betacyanin derivatives thatare
derivatives that aredecarboxylated
decarboxylated and
and dehydrogenated
dehydrogenated betacyanins.
betacyanins. The The
ions ions
with m/z 389.09 and 150.05 have been seen in the analysis of betalains from Chenopodium
formosanum and Amaranthus cruentus [36,37].
The betacyanin derivatives do not produce a product ion with m/z 389.09 because the
aglycone frame has lost at least one carboxylic group or has been dehydrogenated. The
identification of betacyanin derivatives was markedly straightforward with the finger-
print ions of betalains, but the confirmation of the carbons undergoing decarboxylation
Molecules 2024, 29, 5485 7 of 24

with m/z 389.09 and 150.05 have been seen in the analysis of betalains from Chenopodium
formosanum and Amaranthus cruentus [36,37].
The betacyanin derivatives do not produce a product ion with m/z 389.09 because the
Molecules 2024, 29, x FOR PEER REVIEW
aglycone frame has lost at least one carboxylic group or has been dehydrogenated. 8 of 24
The
identification of betacyanin derivatives was markedly straightforward with the fingerprint
ions of betalains, but the confirmation of the carbons undergoing decarboxylation will need
further investigation
will need since masssince
further investigation spectrometry is not able
mass spectrometry to specify
is not carbons
able to specify undergoing
carbons un-
decarboxylation in these conditions.
dergoing decarboxylation Based on the
in these conditions. data
Based onfor
thedecarboxylated betacyanins,
data for decarboxylated beta-for
which the decarboxylated
cyanins, betacyanins atbetacyanins
for which the decarboxylated either C2, C15, or C17
at either wereor
C2, C15, resolved
C17 werein resolved
C18-phase
in C18-phase chromatography
chromatography [32–34], the decarboxylated
[32–34], the decarboxylated betacyanins
betacyanins identified identified
here here
were mapped
were mapped
relative relative
to betanin. to betanin.
Betacyanin Betacyanin
derivatives derivatives
in the beetrootin the beetroot
extract extract were
were composed com-be-
of nine
posed
tanin of nine betanin
derivatives and fourderivatives
neobetanin andderivatives.
four neobetanin derivatives. Decarboxylated
Decarboxylated beta-
betacyanin derivatives
cyanin derivatives
displayed the loss ofdisplayed the group
a carboxylic loss of in
a carboxylic group in
the framework; the the
thus, framework;
precursor thus,
ion the
mass
precursor ion mass was smaller than their counterpart, unmodified betacyanin.
was smaller than their counterpart, unmodified betacyanin. For instance, the mass of the For in-
stance,
betanin the massion
precursor of was
the betanin precursor
m/z 551.1483, andionthe was
massm/z 551.1483,
of the and the mass of precursor
17-decarboxy-betanin the 17-
decarboxy-betanin precursor ion was m/z 507.1594. Decarboxylated
ion was m/z 507.1594. Decarboxylated betacyanin derivatives showed the typical loss of betacyanin deriva-
tives showed
a glucosyl the with
moiety typical
anloss
m/zofofa 162.053
glucosyl[M moiety
+ H with an m/z of
−162.053], 162.053
which [M + H for
explains, −162.053],
instance,
which explains, for instance, the product ion with m/z 345.10 or m/z
the product ion with m/z 345.10 or m/z 343.09 in decarboxylated betanins (2-decarboxy- 343.09 in decarbox-
ylated betanins (2-decarboxy-betanin, 15-decarboxy-betanin, and 17-decarboxy-betanin)
betanin, 15-decarboxy-betanin, and 17-decarboxy-betanin) and decarboxylated neobetanin
and decarboxylated neobetanin (for example, 2-decarboxy-neobetanin), respectively (Fig-
(for example, 2-decarboxy-neobetanin), respectively (Figure 5). Detecting those ions was
ure 5). Detecting those ions was in accordance with previous reports of betalain analysis
in accordance with previous reports of betalain analysis [22,36,47,48]. As decarboxylated
[22,36,47,48]. As decarboxylated betacyanin derivatives, dehydrogenated betacyanin de-
betacyanin derivatives, dehydrogenated betacyanin derivatives showed a loss of H2 in their
rivatives showed a loss of H2 in their framework; thus, the precursor ion mass was smaller
framework; thus, the precursor ion mass was smaller than the counterpart unmodified
than the counterpart unmodified betacyanin, as previously described [49]. Dehydrogen-
betacyanin, as previously
ated betacyanins described
and betacyanin [49]. Dehydrogenated
derivatives include neobetanin betacyanins and betacyanin
with m/z 549.13, 2-decar-
derivatives include neobetanin with m/z 549.13, 2-decarboxy-2,3-dehydro-betanin
boxy-2,3-dehydro-betanin with m/z 505.1435, 2-decarboxy-2,3-dehydro-isobetanin with with
m/zm/z
505.1435, 2-decarboxy-2,3-dehydro-isobetanin with m/z 505.1429,
505.1429, 2,17-bidecarboxy-2,3-dehydro-betanin with m/z 461.1538, 2-Decarboxy-2,3- 2,17-bidecarboxy-
2,3-dehydro-betanin
dehydro-neobetaninwith withm/z
m/z461.1538,
503.1281, 2-Decarboxy-2,3-dehydro-neobetanin
and 2,17-Bidecarboxy-2,3-dehydro-neobetanin with m/z
503.1281, and 2,17-Bidecarboxy-2,3-dehydro-neobetanin
with m/z 459.134. with m/z 459.134.

Figure
Figure 5. Fragmentationspectra
5. Fragmentation spectraofofbetacyanin
betacyanin derivatives
derivatives with
withHCD
HCDatat5050eV.
eV.(A)
(A)MS/MS
MS/MS spectrum
spectrum
of 2-decarboxy-betanin.(B)
of 2-decarboxy-betanin. (B)MS/MS
MS/MS spectrum
spectrum of
of 2-decarboxy-neobetanin.
2-decarboxy-neobetanin.CoreCoreions areare
ions highlighted
highlighted
in black.
in black.

Next,
Next, betaxanthinswere
betaxanthins wereanalyzed
analyzed based
based on
on the
thehypothesis
hypothesisthat a fingerprint
that of beta-
a fingerprint of beta-
lains can be generated upon molecular ion fragmentation in a mass spectrometer
lains can be generated upon molecular ion fragmentation in a mass spectrometer since sincebe-
betacyanins
tacyanins and betaxanthins
and betaxanthins share
share the the framework
framework of betalamic
of betalamic acid.
acid. Indeed,
Indeed, 1313 betaxan-
betaxanthins
thins were identified in the beetroot extract, including 10 hydrophobic-type betaxanthins
were identified in the beetroot extract, including 10 hydrophobic-type betaxanthins and
and 3 polar uncharged-type betaxanthins. Remarkably, a product ion with m/z 211.07 was
3 polar uncharged-type betaxanthins. Remarkably, a product ion with m/z 211.07 was found
found in betaxanthins that structurally maps to betalamic acid, bearing a nitrogen at-
in betaxanthins that structurally maps to betalamic acid, bearing a nitrogen attached to the
tached to the corresponding amino acid of betaxanthins. Additionally, ions were structur-
corresponding amino acid of betaxanthins. Additionally, ions were structurally mapped
ally mapped to the heterocyclic pyrimidine ring, such as those with m/z 194.04, 166.05,
to 138.05,
the heterocyclic pyrimidine ring, such as those with m/z 194.04, 166.05, 138.05, 132.04,
132.04, 130.05, and 106.06. For instance, the generation of these ions was found in
the mass spectra of glutamine-bx and tryptophan-bx (Figure 6). Given the variety of con-
jugated amino acids in betaxanthins, defining distinct ions with m/z values that match to
a framework outside of the ion with m/z 211.07 was a challenge.
Molecules 2024, 29, 5485 8 of 24

130.05, and 106.06. For instance, the generation of these ions was found in the mass spectra
of glutamine-bx and tryptophan-bx (Figure 6). Given the variety of conjugated amino acids
Molecules 2024, 29, x FOR PEER REVIEW
in betaxanthins, defining distinct ions with m/z values that match to a framework9 outside
of 24
of the ion with m/z 211.07 was a challenge.

Figure
Figure 6. 6. Fragmentationspectra
Fragmentation spectraof
oftwo
two betaxanthins
betaxanthins with
withHCD
HCDatat5050eV.
eV.(A)
(A)MS/MS
MS/MS spectrum of of
spectrum
glutamine-bx. (B) MS/MS spectrum of tryptophan-bx. Core ions are highlighted in
glutamine-bx. (B) MS/MS spectrum of tryptophan-bx. Core ions are highlighted in black.black.

2.4.2.4. IdentificationofofBetalains
Identification Betalainsin
inRed
Red Pitaya
Pitaya Extract
Extract
Assumingthat
Assuming thatour
ouridentified
identified betalain
betalain features
featuresin inbeetroot,
beetroot,along
alongwithwiththethe
betalain
betalain
fingerprint ions determined in this study, can be used to survey betalains
fingerprint ions determined in this study, can be used to survey betalains in red pitaya, we in red pitaya,
we examined
examined the red thepitaya
red pitaya extract
extract mademade following
following thethe previously
previously reportedmethod
reported methodbased
based on
on MSPD that is compatible with mass spectrometry [37]. A total of
MSPD that is compatible with mass spectrometry [37]. A total of 86 betalains were identified86 betalains were
identified in the red pitaya extract distributed at 12 min chromatographic
in the red pitaya extract distributed at 12 min chromatographic resolution with different resolution with
different abundance (Table 1 and Figure 7). The betalains discovered
abundance (Table 1 and Figure 7). The betalains discovered in this study were previously in this study were
previously identified by others using chemical, LC-MS/MS, or NMR methods
identified by others using chemical, LC-MS/MS, or NMR methods [15,36,37,43,50–52], with
[15,36,37,43,50–52], with the exception of four unknown betalains. The abundance of each
the exception of four unknown betalains. The abundance of each betalain was determined
betalain was determined by measuring the area below the base peak of the ion m/z of
by measuring the area below the base peak of the ion m/z of betalain that was examined.
betalain that was examined. When the betalains in the extract were classified into two
When the betalains in the extract were classified into two main classes, it was notable that
main classes, it was notable that all 19 identified betaxanthins were of low abundance
all 19 identified betaxanthins were of low abundance compared to the total 67 betacyanins
compared to the total 67 betacyanins that map to m/z above 450. This work expanded the
that map to m/z above 450. This work expanded the catalog of betalains in pitaya to 86
catalog of betalains in pitaya to 86 compared with the previous number of betalains re-
compared with
ported. For the previous
instance, 8 betalainsnumber of betalains
in red-purple pitayareported.
HylocereusFor instance,
polyrhizus 8 betalains
(Weber) Britton in
red-purple Hylocereus polyrhizus
& Rose [53]; 11 in the red flesh and orange flesh varieties of Stenocereus pruinosus andred
pitaya (Weber) Britton & Rose [53]; 11 in the flesh
Sten‐
and flesh varieties of Stenocereus pruinosus and Stenocereus stellatus
ocereus stellatus [54]; 20 in the red flesh of Hylocereus costaricensis, white flesh of Hylocereusthe
orange [54]; 20 in
flesh of Hylocereus
redmegalanthus, costaricensis,
and Hylocereus undatuswhite flesh23ofinHylocereus
[55]; and megalanthus,
the red flesh of Hylocereus Hylocereus
andpolyrhizus
undatus [55]; andundatus
and Hylocereus 23 in the red flesh of Hylocereus polyrhizus and Hylocereus undatus [56].
[56].
Molecules 2024, 29, 5485 9 of 24

Table 1. Chromatographic and mass spectrometry data for betalains from Hylocereus costaricensis.

Relative Mass
Retention Chemical Theoretical Observed
# Compound A Rt from Accuracy Fragments Reference B
Time (Rt) Formula m/z [M+H]+ m/z [M+H]+
Betanin (ppm)
1 Betalamic acid 3.23 0.94 C9 H9 NO5 212.0553 212.0545 −3.77 194.04, 166.05, 148.04, 138.05, 120.04, 106.03 [15]
Betanin-type
389.09, 345.10, 343.09, 299.10, 297.08, 281.09, 269.09, 255.11,
2 Prebetanin 3.19 0.93 C24 H26 N2 O16 S 631.1076 631.1052 −3.8 253.09, 194.04, 166.05, 178.05, 176.07, 166.05, 150.05, 138.05, [57,58]
132.04, 106.6
389.09, 345.10, 343.09, 299.10, 297.08, 281.09, 269.09, 255.11,
3 Betanin 3.43 1 C24 H26 N2 O13 551.1508 551.1483 −4.54 253.09, 194.04, 166.05, 178.05, 176.07, 166.05, 150.05, 138.05, [9,59]
132.04, 106.6
389.09, 345.10, 343.09, 299.10, 297.08, 281.09, 269.09, 255.11,
4 Betanidin 3.44 1 C18 H16 N2 O8 389.0979 389.0964 −3.86 253.09, 194.04, 166.05, 178.05, 176.07, 166.05, 150.05, 138.05, [60]
132.04, 106.6
2-decarboxy- 343.09, 299.10, 297.08, 281.09, 269.09, 255.08, 253.09, 194.04,
5 3.62 1.06 C23 H24 N2 O11 505.1453 505.1429 −4.75 [61]
xanbetanin * 178.05, 176.07, 150.05, 138.05, 132.04, 106.06
17-decarboxy- 345.10, 299.10, 297.08, 281.09, 255.11, 253.09, 194.04, 178.05,
6 5.83 1.7 C23 H26 N2 O11 507.1609 507.1591 −3.55 [23,49,62]
betanin 176.07, 150.05, 138.05, 132.04, 106.06
389.09, 345.10, 343.09, 299.10, 297.08, 281.09, 269.09, 255.11,
7 Isoprebetanin 6.01 1.75 C24 H26 N2 O16 S 631.1076 631.1047 −4.6 253.09, 194.04, 166.05, 178.05, 176.07, 166.05, 150.05, 138.05,
132.04, 106.6
389.09, 345.10, 343.09, 299.10, 297.08, 281.09, 269.09, 255.11,
8 Isobetanin 6.59 1.92 C24 H26 N2 O13 551.1508 551.1481 −4.9 253.09, 194.04, 166.05, 178.05, 176.07, 166.05, 150.05, 138.05, [9]
132.04, 106.6
2-decarboxy- 343.09, 299.10, 297.08, 281.09, 269.09, 255.08, 253.09, 194.04,
9 6.6 1.92 C23 H24 N2 O11 505.1453 505.1433 −3.96 [22]
isoxanbetanin * 178.05, 176.07, 150.05, 138.05, 132.04, 106.06
2,17-
343.09, 299.10, 297.08, 281.09, 269.09, 255.08, 253.09, 194.04,
10 bidecarboxy- 7.61 2.22 C22 H24 N2 O9 461.1555 461.1534 −4.55 [22,51]
178.05, 176.07, 150.05, 138.05, 132.04, 106.06
xanbetanin *
17-decarboxy- 345.10, 299.10, 297.08, 281.09, 255.11, 253.09, 194.04, 178.05,
11 7.69 2.24 C23 H26 N2 O11 507.1609 507.1587 −4.34
isobetanin 176.07, 150.05, 138.05, 132.04, 106.06
389.09, 345.10, 343.09, 299.10, 297.08, 281.09, 269.09, 255.11,
12 Phyllocactin 7.71 2.25 C27 H28 N2 O16 637.1512 637.1497 −2.35 253.09, 194.04, 166.05, 178.05, 176.07, 166.05, 150.05, 138.05, [9,63]
132.04, 106.6
17-decarboxy- 345.10, 299.10, 297.08, 281.09, 255.11, 253.09, 194.04, 178.05,
13 8.01 2.34 C26 H28 N2 O14 593.1613 593.1591 −3.71 [22,23]
phyllocactin 176.07, 150.05, 138.05, 132.04, 106.06
Molecules 2024, 29, 5485 10 of 24

Table 1. Cont.

Relative Mass
Retention Chemical Theoretical Observed
# Compound A Rt from Accuracy Fragments Reference B
Time (Rt) Formula m/z [M+H]+ m/z [M+H]+
Betanin (ppm)
15-decarboxy- 345.10, 299.10, 297.08, 281.09, 255.11, 253.09, 194.04, 178.05,
14 8.29 2.42 C23 H26 N2 O11 507.1609 507.1591 −3.55 [49,64]
betanin 176.07, 150.05, 138.05, 132.04, 106.06
387.07, 341.07, 313.08, 299.10, 299.06, 287.08, 281.09, 269.09,
15 Neobetanin 8.31 2.42 C24 H24 N2 O13 549.1351 549.1336 −2.73 255.08, 253.09, 194.04, 178.05, 176.07, 166.05, 150.05, 132.04, [51]
106.06
17-decarboxy- 343.09, 299.10, 297.08, 281.09, 269.09, 255.08, 253.09, 194.04,
16 8.39 2.45 C23 H24 N2 O11 505.1453 505.1438 −2.97 [22]
neobetanin * 178.05, 176.07, 150.05, 138.05, 132.04, 106.06
2-decarboxy- 343.09, 299.10, 297.08, 281.09, 269.09, 255.08, 253.09, 194.04,
17 8.4 2.45 C23 H24 N2 O11 505.1453 505.1432 −4.16 [47]
neobetanin 178.05, 176.07, 150.05, 138.05, 132.04, 106.06
389.09, 345.10, 343.09, 299.10, 297.08, 281.09, 269.09, 255.11,
18 Hylocerenin 8.39 2.45 C30 H34 N2 O17 695.193 695.1899 −4.46 253.09, 194.04, 166.05, 178.05, 176.07, 166.05, 150.05, 138.05, [9,65]
132.04, 106.6
389.09, 345.10, 343.09, 299.10, 297.08, 281.09, 269.09, 255.11,
19 Lampranthin-I * 8.41 2.45 C33 H32 N2 O15 697.1875 697.1861 −2.01 253.09, 194.04, 166.05, 178.05, 176.07, 166.05, 150.05, 138.05, [66]
132.04, 106.6
389.09, 345.10, 343.09, 299.10, 297.08, 281.09, 269.09, 255.11,
20 Isophyllocactin 8.46 2.47 C27 H28 N2 O16 637.1512 637.1491 −3.3 253.09, 194.04, 166.05, 178.05, 176.07, 166.05, 150.05, 138.05,
132.04, 106.6
17-decarboxy- 345.10, 299.10, 297.08, 281.09, 255.11, 253.09, 194.04, 178.05,
21 8.72 2.54 C29 H34 N2 O15 651.2032 651.2012 −3.07 [22,23]
hylocerenin * 176.07, 150.05, 138.05, 132.04, 106.06
2-decarboxy- 345.10, 299.10, 297.08, 281.09, 255.11, 253.09, 194.04, 178.05,
22 8.83 2.57 C23 H26 N2 O11 507.1609 507.1596 −2.56 [22,23]
betanin 176.07, 150.05, 138.05, 132.04, 106.06
389.09, 345.10, 343.09, 299.10, 297.08, 281.09, 269.09, 255.11,
Isolampranthin-
23 8.85 2.58 C33 H32 N2 O15 697.1875 697.1854 −3.01 253.09, 194.04, 166.05, 178.05, 176.07, 166.05, 150.05, 138.05,
I
132.04, 106.6
389.09, 345.10, 343.09, 299.10, 297.08, 281.09, 269.09, 255.11,
24 Isohylocerenin 8.88 2.59 C30 H34 N2 O17 695.193 695.1909 −3.02 253.09, 194.04, 166.05, 178.05, 176.07, 166.05, 150.05, 138.05, [22]
132.04, 106.6
17-decarboxy- 345.10, 299.10, 297.08, 281.09, 255.11, 253.09, 194.04, 178.05,
25 9.21 2.69 C26 H28 N2 O14 593.1613 593.1588 −4.21
isophyllocactin * 176.07, 150.05, 138.05, 132.04, 106.06
17-decarboxy- 345.10, 299.10, 297.08, 281.09, 255.11, 253.09, 194.04, 178.05,
26 9.26 2.7 C29 H34 N2 O15 651.2032 651.2009 −3.53
isohylocerenin * 176.07, 150.05, 138.05, 132.04, 106.06
2-descarboxy- 345.10, 299.10, 297.08, 281.09, 255.11, 253.09, 194.04, 178.05,
27 9.38 2.73 C26 H28 N2 O14 593.1613 593.1587 −4.38 [22,23]
phyllocactin 176.07, 150.05, 138.05, 132.04, 106.06
Molecules 2024, 29, 5485 11 of 24

Table 1. Cont.

Relative Mass
Retention Chemical Theoretical Observed
# Compound A Rt from Accuracy Fragments Reference B
Time (Rt) Formula m/z [M+H]+ m/z [M+H]+
Betanin (ppm)
2-descarboxy- 345.10, 299.10, 297.08, 281.09, 255.11, 253.09, 194.04, 178.05,
28 9.41 2.74 C26 H28 N2 O14 593.1613 593.1589 −4.05
isophyllocactin * 176.07, 150.05, 138.05, 132.04, 106.06
2-decarboxy- 341.07, 327.06, 313.08, 295.07, 277.07, 267.07, 253.06, 251.08,
29 10.06 2.93 C23 H22 N2 O11 503.1296 503.1277 −3.78 [51]
xanneobetanin * 221.07, 195.09, 132.04, 106.06
387.07, 341.07, 313.08, 299.10, 299.06, 287.08, 281.09, 269.09,
30 Neophyllocactin * 10.13 2.95 C27 H26 N2 O16 635.1355 635.1327 −4.41 255.08, 253.09, 194.04, 178.05, 176.07, 166.05, 150.05, 132.04, [22]
106.06
2,15,17-
31 tridecarboxy- 10.43 3.04 C21 H24 N2 O7 417.1656 417.1648 −1.92 417.17, 349.11, 271.09, 255.11, 159.04, 130.03 [47]
neobetanin *
17-decarboxy- 343.09, 299.10, 297.08, 281.09, 269.09, 255.08, 253.09, 194.04,
32 10.52 3.07 C26 H26 N2 O14 591.1457 591.1432 −4.23 [22]
neophyllocactin * 178.05, 176.07, 150.05, 138.05, 132.04, 106.06
387.07, 341.07, 313.08, 299.10, 299.06, 287.08, 281.09, 269.09,
33 Neohylocerenin 10.89 3.17 C30 H32 N2 O17 693.1774 693.1758 −2.31 255.08, 253.09, 194.04, 178.05, 176.07, 166.05, 150.05, 132.04,
106.06
17-decarboxy- 343.09, 299.10, 297.08, 281.09, 269.09, 255.08, 253.09, 194.04,
34 11.04 3.22 C29 H32 N2 O15 649.1875 649.1852 −3.54 [22]
neohylocerenin * 178.05, 176.07, 150.05, 138.05, 132.04, 106.06
2,17-
35 bidecarboxy- 11.14 3.25 C22 H22 N2 O9 459.1398 459.1383 −3.27 297.08, 269.09, 251.08, 223.08, 195.09, 133.08 [51]
xanneobetanin *
389.09, 345.10, 343.09, 299.10, 297.08, 281.09, 269.09, 255.11,
36 Lampranthin II 11.01 3.21 C34 H34 N2 O16 727.1981 727.1956 −3.44 253.09, 194.04, 166.05, 178.05, 176.07, 166.05, 150.05, 138.05, [59,66,67]
132.04, 106.6
389.09, 345.10, 343.09, 299.10, 297.08, 281.09, 269.09, 255.11,
Isolampranthin
37 11.34 3.31 C34 H34 N2 O16 727.1981 727.1948 −4.54 253.09, 194.04, 166.05, 178.05, 176.07, 166.05, 150.05, 138.05,
II
132.04, 106.6
2-decarboxy- 343.09, 299.10, 297.08, 281.09, 269.09, 255.08, 253.09, 194.04,
38 11.45 3.34 C29 H32 N2 O15 649.1875 649.1848 −4.16 [22]
neohylocerenin 178.05, 176.07, 150.05, 138.05, 132.04, 106.06
2-decarboxy- 343.09, 299.10, 297.08, 281.09, 269.09, 255.08, 253.09, 194.04,
39 11.62 3.39 C26 H26 N2 O14 591.1457 591.1438 −3.21 [22]
neophyllocactin * 178.05, 176.07, 150.05, 138.05, 132.04, 106.06
Molecules 2024, 29, 5485 12 of 24

Table 1. Cont.

Relative Mass
Retention Chemical Theoretical Observed
# Compound A Rt from Accuracy Fragments Reference B
Time (Rt) Formula m/z [M+H]+ m/z [M+H]+
Betanin (ppm)
387.07, 341.07, 313.08, 299.10, 299.06, 287.08, 281.09, 269.09,
Neolampranthin
40 11.64 3.39 C34 H32 N2 O16 725.1825 725.1794 −4.27 255.08, 253.09, 194.04, 178.05, 176.07, 166.05, 150.05, 132.04,
II
106.06
2,15,17-
41 tridecarboxy- 11.94 3.48 C21 H22 N2 O7 415.1499 415.1497 −0.48 415.14, 355.12, 347.09, 285.09, 185.04, 143.02 [51]
xanneobetanin *
Melocactin-type
389.09, 345.10, 343.09, 299.10, 297.08, 281.09, 269.09, 255.11,
42 Melocactin 4.81 1.4 C30 H36 N2 O18 713.2036 713.2005 −4.35 253.09, 194.04, 166.05, 178.05, 176.07, 166.05, 150.05, 138.05, [68,69]
132.04, 106.6
389.09, 345.10, 343.09, 299.10, 297.08, 281.09, 269.09, 255.11,
43 Isomelocactin 7.18 2.09 C30 H36 N2 O18 713.2036 713.2009 −3.79 253.09, 194.04, 166.05, 178.05, 176.07, 166.05, 150.05, 138.05, [68]
132.04, 106.6
Apiocactin-type
389.09, 345.10, 343.09, 299.10, 297.08, 281.09, 269.09, 255.11,
44 Apiocactin 7.21 2.1 C29 H34 N2 O17 683.193 683.1904 −3.81 253.09, 194.04, 166.05, 178.05, 176.07, 166.05, 150.05, 138.05, [69]
132.04, 106.6
389.09, 345.10, 343.09, 299.10, 297.08, 281.09, 269.09, 255.11,
45 Isoapiocactin 7.91 2.31 C29 H34 N2 O17 683.193 683.1911 −2.78 253.09, 194.04, 166.05, 178.05, 176.07, 166.05, 150.05, 138.05,
132.04, 106.6
389.09, 345.10, 343.09, 299.10, 297.08, 281.09, 269.09, 255.11,
2′ -O-apiosyl-
46 8.31 2.42 C32 H36 N2 O20 769.1934 769.1912 −2.86 253.09, 194.04, 166.05, 178.05, 176.07, 166.05, 150.05, 138.05, [65,69]
phyllocactin *
132.04, 106.6
389.09, 345.10, 343.09, 299.10, 297.08, 281.09, 269.09, 255.11,
2′ -O-apiosyl-
47 8.76 2.55 C32 H36 N2 O20 769.1934 769.1903 −4.03 253.09, 194.04, 166.05, 178.05, 176.07, 166.05, 150.05, 138.05, [65]
isophyllocactin *
132.04, 106.6
17-decarboxy-2′ -
345.10, 299.10, 297.08, 281.09, 255.11, 253.09, 194.04, 178.05,
48 O-β-apiosyl- 9.89 2.88 C31 H36 N2 O18 725.2036 725.2011 −3.45
176.07, 150.05, 138.05, 132.04, 106.06
phyllocactin *
Molecules 2024, 29, 5485 13 of 24

Table 1. Cont.

Relative Mass
Retention Chemical Theoretical Observed
# Compound A Rt from Accuracy Fragments Reference B
Time (Rt) Formula m/z [M+H]+ m/z [M+H]+
Betanin (ppm)
17-decarboxy-2′ -
345.10, 299.10, 297.08, 281.09, 255.11, 253.09, 194.04, 178.05,
49 O-β-apiosyl- 10.19 2.97 C31 H36 N2 O18 725.2036 725.2007 −4.00 [65]
176.07, 150.05, 138.05, 132.04, 106.06
isophyllocactin *
Gomphrenin-type
389.09, 345.10, 343.09, 299.10, 297.08, 281.09, 269.09, 255.11,
50 Gomphrenin-I 4.06 1.18 C24 H26 N2 O13 551.1508 551.1481 −4.9 253.09, 194.04, 166.05, 178.05, 176.07, 166.05, 150.05, 138.05, [59,70]
132.04, 106.6
2-decarboxy- 343.09, 299.10, 297.08, 281.09, 269.09, 255.08, 253.09, 194.04,
51 6.71 1.96 C23 H24 N2 O11 505.1453 505.1431 −4.36 [71]
xangomphrenin * 178.05, 176.07, 150.05, 138.05, 132.04, 106.06
389.09, 345.10, 343.09, 299.10, 297.08, 281.09, 269.09, 255.11,
Isogomphrenin-
52 6.87 2 C24 H26 N2 O13 551.1508 551.1486 −3.99 253.09, 194.04, 166.05, 178.05, 176.07, 166.05, 150.05, 138.05, [72]
I
132.04, 106.6
387.07, 341.07, 313.08, 299.10, 299.06, 287.08, 281.09, 269.09,
53 Neogomphrenin 8.58 2.5 C24 H24 N2 O13 549.1351 549.1327 −4.37 255.08, 253.09, 194.04, 178.05, 176.07, 166.05, 150.05, 132.04,
106.06
389.09, 345.10, 343.09, 299.10, 297.08, 281.09, 269.09, 255.11,
54 Gomphrenin-IV 10.91 3.18 C35 H36 N2 O17 757.2087 757.2065 −2.91 253.09, 194.04, 166.05, 178.05, 176.07, 166.05, 150.05, 138.05, [50,73]
132.04, 106.6
389.09, 345.10, 343.09, 299.10, 297.08, 281.09, 269.09, 255.11,
Isogomphrenin-
55 11.16 3.25 C35 H36 N2 O17 757.2087 757.2071 −2.11 253.09, 194.04, 166.05, 178.05, 176.07, 166.05, 150.05, 138.05,
IV
132.04, 106.6
389.09, 345.10, 343.09, 299.10, 297.08, 281.09, 269.09, 255.11,
56 Gomphrenin-III 11.33 3.3 C34 H34 N2 O16 727.1981 727.1949 −4.4 253.09, 194.04, 166.05, 178.05, 176.07, 166.05, 150.05, 138.05, [50,72]
132.04, 106.6
389.09, 345.10, 343.09, 299.10, 297.08, 281.09, 269.09, 255.11,
Isogomphrenin-
57 11.62 3.39 C34 H34 N2 O16 727.1981 727.1957 −3.3 253.09, 194.04, 166.05, 178.05, 176.07, 166.05, 150.05, 138.05,
III
132.04, 106.6
387.07, 341.07, 313.08, 299.10, 299.06, 287.08, 281.09, 269.09,
Neogomphrenin
58 11.59 3.38 C35 H34 N2 O17 755.193 755.1914 −2.12 255.08, 253.09, 194.04, 178.05, 176.07, 166.05, 150.05, 132.04,
IV
106.06
Molecules 2024, 29, 5485 14 of 24

Table 1. Cont.

Relative Mass
Retention Chemical Theoretical Observed
# Compound A Rt from Accuracy Fragments Reference B
Time (Rt) Formula m/z [M+H]+ m/z [M+H]+
Betanin (ppm)
387.07, 341.07, 313.08, 299.10, 299.06, 287.08, 281.09, 269.09,
Neogomphrenin-
59 11.92 3.48 C34 H32 N2 O16 725.1825 725.1798 −3.72 255.08, 253.09, 194.04, 178.05, 176.07, 166.05, 150.05, 132.04,
III
106.06
Glabranin-type
389.09, 345.10, 343.09, 299.10, 297.08, 281.09, 269.09, 255.11,
60 Glabranin 3.15 0.92 C30 H36 N2 O18 713.2036 713.2015 −2.94 253.09, 194.04, 166.05, 178.05, 176.07, 166.05, 150.05, 138.05, [74]
132.04, 106.6
389.09, 345.10, 343.09, 299.10, 297.08, 281.09, 269.09, 255.11,
61 Coumglabranin * 10.59 3.09 C39 H42 N2 O20 859.2403 859.2377 −3.03 253.09, 194.04, 166.05, 178.05, 176.07, 166.05, 150.05, 138.05, [74]
132.04, 106.6
389.09, 345.10, 343.09, 299.10, 297.08, 281.09, 269.09, 255.11,
62 Isocoumglabranin * 10.85 3.16 C39 H42 N2 O20 859.2403 859.2371 −3.72 253.09, 194.04, 166.05, 178.05, 176.07, 166.05, 150.05, 138.05,
132.04, 106.6
387.07, 341.07, 313.08, 299.10, 299.06, 287.08, 281.09, 269.09,
63 Neocoumglabranin * 11.29 3.29 C39 H40 N2 O20 857.2247 857.2208 −4.55 255.08, 253.09, 194.04, 178.05, 176.07, 166.05, 150.05, 132.04,
106.06
Unknown type
389.09, 345.10, 343.09, 299.10, 297.08, 281.09, 269.09, 255.11,
64 Unknown-1 * 8.83 2.57 C35 H42 N2 O21 827.2353 827.2316 −4.47 253.09, 194.04, 166.05, 178.05, 176.07, 166.05, 150.05, 138.05,
132.04, 106.6
389.09, 345.10, 343.09, 299.10, 297.08, 281.09, 269.09, 255.11,
65 Unknown-2 * 9.23 2.69 C35 H42 N2 O21 827.2353 827.2328 −3.02 253.09, 194.04, 166.05, 178.05, 176.07, 166.05, 150.05, 138.05,
132.04, 106.6
345.10, 299.10, 297.08, 281.09, 255.11, 253.09, 194.04, 178.05,
66 Unknown-3 * 10.27 2.99 C34 H42 N2 O19 783.2455 783.2422 −4.21
176.07, 150.05, 138.05, 132.04, 106.06
345.10, 299.10, 297.08, 281.09, 255.11, 253.09, 194.04, 178.05,
67 Unknown-4 * 10.54 3.07 C34 H42 N2 O19 783.2455 783.2428 −3.45
176.07, 150.05, 138.05, 132.04, 106.06
Betaxanthin
Positively charged-type s
287.12, 261.13, 256.89, 211.07, 194.04, 166.05, 150.05, 138.05,
68 Histamine-bx * 9.04 2.64 C14 H16 N4 O4 305.1244 305.1232 −3.93
132.04, 130.05, 106.06
Molecules 2024, 29, 5485 15 of 24

Table 1. Cont.

Relative Mass
Retention Chemical Theoretical Observed
# Compound A Rt from Accuracy Fragments Reference B
Time (Rt) Formula m/z [M+H]+ m/z [M+H]+
Betanin (ppm)
Polar uncharged-type
323.08, 277.08, 249.08, 233.09, 231.07, 211.07, 194.04, 166.05,
69 Glutamine-bx * 1.2 0.35 C14 H17 N3 O7 340.1139 340.1129 −2.94 [15]
150.05, 138.05, 132.04, 130.05, 106.06
269.09, 267.09, 211.07, 194.04, 166.05, 150.05, 138.05, 132.04,
70 Threonine-bx * 1.52 0.44 C13 H16 N2 O7 313.103 313.1022 −2.56 [15]
130.05, 106.06
255.09, 253.09, 211.07, 194.04, 166.05, 150.05, 138.05, 132.04,
71 Serine-bx * 4.77 1.39 C12 H14 N2 O7 299.0874 299.0861 −4.35 [15]
130.05, 106.06
Hydrophobic-type
331.12, 239.11, 285.12, 283.10, 239.11, 211.07, 194.04, 166.05,
72 Glycine-bx * 6.49 1.89 C11 H12 N2 O6 269.0768 269.0758 −3.72 [15]
150.05, 138.05, 132.04, 130.05, 106.06
315.09, 269.09, 211.07, 194.04, 166.05, 150.05, 138.05, 132.04,
73 Methionine-bx * 7.3 2.13 C14H18N2O6S 343.0958 343.0943 −4.37 [15]
130.05, 106.06
267.13, 265.11, 237.12, 221.12, 219.11, 211.07, 194.04, 166.05,
74 Valine-bx * 7.97 2.32 C14H18N2O6 311.1238 311.1229 −2.89 [15]
150.05, 138.05, 132.04, 130.05, 106.06
237.08, 211.07, 194.04, 166.05, 150.05, 138.05, 132.04, 130.05,
75 Alanine-bx * 8.16 2.38 C12H14N2O6 283.0925 283.0915 −3.53 [15]
106.06
265.11, 263.10, 235.1, 219.11, 217.09, 211.07, 194.04, 166.05,
75 Proline-bx * 9.02 2.63 C14 H16 N2 O6 309.1081 309.1068 −4.21 [15]
150.05, 138.05, 132.04, 130.05, 106.06
279.13, 251.13, 235.14, 233.12, 211.07, 194.04, 166.05, 150.05,
77 Isoleucine-bx * 9.87 2.88 C15 H20 N2 O6 325.1394 325.1384 −3.08 [15]
138.05, 132.04, 130.05, 106.06
281.14, 279.13, 251.13, 235.14, 233.12, 211.07, 194.04, 166.05,
78 Leucine-bx * 10.01 2.92 C15 H20 N2 O6 325.1394 325.1385 −2.77 [15]
150.05, 138.05, 132.04, 130.05, 106.06
269.07, 223.07, 211.07, 194.04, 166.05, 150.05, 138.05, 132.04,
79 Tryptophan-bx * 10.12 2.95 C20 H19 N3 O6 398.1347 398.1331 −4.02 [15]
130.05, 106.06
Phenylalanine- 315.13, 313.11, 269.12, 267.11, 223.12, 211.07, 194.04, 166.05,
80 10.15 2.96 C18 H18 N2 O6 359.1238 359.1225 −3.62 [15]
bx * 150.05, 138.05, 132.04, 130.05, 106.06
g-aminobutyric 253.11, 251.10, 233.09, 211.07, 194.04, 166.05, 150.05, 138.05,
81 2.82 0.82 C13 H16 N2 O6 297.1081 297.1069 −4.04
acid-bx * 132.04, 130.05, 106.06
255.11, 211.07, 194.04, 166.05, 150.05, 138.05, 132.04, 130.05,
82 Dopamine-bx * 7.67 2.24 C17 H18 N2 O6 347.1238 347.1226 −3.46 [15]
106.06
287.13, 285.12, 239.11, 211.07, 194.04, 166.05, 150.05, 138.05,
83 Tyramine-bx * 8.64 2.52 C17 H18 N2 O5 331.1288 331.1274 −4.23
132.04, 130.05, 106.06
Molecules 2024, 29, 5485 16 of 24

Table 1. Cont.

Relative Mass
Retention Chemical Theoretical Observed
# Compound A Rt from Accuracy Fragments Reference B
Time (Rt) Formula m/z [M+H]+ m/z [M+H]+
Betanin (ppm)
3-methoxy- 315.13, 269.12, 211.07, 194.04, 166.05, 150.05, 138.05, 132.04,
84 8.98 2.62 C18 H20 N2 O6 361.1394 361.1383 −3.05
tyramine-bx * 130.05, 106.06
5-
283.15, 237.15, 211.07, 194.04, 166.05, 150.05, 138.05, 132.04,
85 hydroxynorvaline- 9.89 2.88 C14 H18 N2 O7 327.1187 327.1179 −2.45
130.05, 106.06
bx *
Methionine 267.11, 223.12, 211.07, 194.04, 166.05, 150.05, 138.05, 132.04,
86 10.09 2.94 C14 H18 N2 O7 S 359.0907 359.0893 −3.9
sulfoxide-bx * 130.05, 106.06
A The prefix “xan” is equivalent to 2,3-dehydro, while “neo” is equivalent to 14, 15-dehydro, as described by Starzak et al. [75]. * Tentatively identified. B A reference to the identified
betalain.
MoleculesMolecules
2024, 29,2024,
548529, x FOR PEER REVIEW 1717ofof24
24

Figure 7. Resolving the betalains discovered in red pitaya. (A) The color circle scale indicates the
Figure 7. Resolving the betalains discovered in red pitaya. (A) The color circle scale indicates the
change in the parent ion base peak measurement area for betalains. (B) Chromatogram of red pitaya
change in the parent ion base peak measurement area for betalains. (B) Chromatogram of red pitaya
at 540atnm.
540 The
nm. peak displays
The peak the the
displays retention time
retention and
time label
and number
label numberofofbetalain.
betalain. Table
Table 11 contains the
contains the
complete list oflist
complete identified betalains.
of identified betalains.

2.5. Annotating Unknowns with the Fingerprint of Betalains

2.5. Annotating Unknowns with the Fingerprint of Betalains
Surprisingly, we saw
Surprisingly, we two
saw unknown betalains
two unknown in red
betalains inpitaya during
red pitaya the analysis
during of mass
the analysis of
spectrometry data made in this study. Those betalains, referred to here as unknown-1
mass spectrometry data made in this study. Those betalains, referred to here as unknown-
and unknown-2, werewere
1 and unknown-2, not contained in our
not contained database
in our databaseoror
previously
previouslyreported,
reported,as
as far as we
far as we
know. They were characterized by the molecular formula C35 H42 N2 O21 and by MS/MS
spectra containing the distinctive ions of betalain with m/z 389.09, 345.10, 343.09, 297.08,
194.04, 150.05, and 106.06. Unknown-1 had an m/z of 827.2316 and eluted at 8.83 min,
 Molecules 2024, 29, x FOR PEER REVIEW 18 of 24

Molecules 2024, 29, 5485 18 of 24

know. They were characterized by the molecular formula C35H42N2O21 and by MS/MS
spectra containing the distinctive ions of betalain with m/z 389.09, 345.10, 343.09, 297.08,
194.04,
while 150.05, andhad
unknown-2 106.06. Unknown-1
an m/z of 827.2328 hadand
an m/z of 827.2316
eluted at 9.23 and
min.eluted
Givenat that
8.83 betalains
min, whilehave
unknown-2
isomeric hadwe
forms, an suspected
m/z of 827.2328 and eluted atwas
that unknown-1 9.23the
min. Given that
isomeric formbetalains have iso-Next,
of unknown-2.
meric
we forms,towe
decided usesuspected thatMS
multistage unknown-1
with CIDwas the isomeric
to reveal form ofofunknown-2.
the identity neutral losses Next,
in the
we decided
unknown to use multistage
betalains and compare MSitwith CID
to the to reveal
MS/MS theHCD
with identity of neutral losses
fragmentation in the
(Figure 8). The
unknownobtained
spectrum betalains and
withcompare it to the MS/MS
HCD displayed with HCD
the typical ions fragmentation
with m/z 389.09,(Figure 8). The
194.04, 150.05,
spectrum
and 106.06obtained with HCD
of betalains. Next,displayed
fragmenting the typical ions with
the parent m/z 389.09,
ion with 194.04, 150.05,
m/z 827.2316 using CID
and 106.06
revealed theofneutral
betalains.
lossNext, fragmenting
of 134.0423 the parent ion with
Da, corresponding to a m/z 827.2316
pentoside orusing CIDacid.
glutaric
revealed the neutral loss of 134.0423 Da, corresponding to a pentoside
Then, fragmenting the product ion with m/z 695.1930 using CID revealed a neutral lossor glutaric acid.
ofThen, fragmenting
162.0528 the product
Da, matching to aion with m/z 695.1930
glucoside. using CID revealed
Also, fragmenting a neutral
the product ionloss
withof m/z
162.0528 Da, matching to a glucoside. Also, fragmenting the product ion with m/z 551.1508
551.1508 revealed a neutral loss of 162.0528 Da, corresponding to a glucoside. Although
revealed a neutral loss of 162.0528 Da, corresponding to a glucoside. Although these data
these data reveal the decorating groups on the framework of betalain, further research will
reveal the decorating groups on the framework of betalain, further research will aid in
aid in revealing the complete structure of unknown-1 and unknown-2. Interestingly, both
revealing the complete structure of unknown-1 and unknown-2. Interestingly, both un-
unknowns had decarboxylated forms in the extract that eluted at 10.27 and 10.54 min with
knowns had decarboxylated forms in the extract that eluted at 10.27 and 10.54 min with
an m/z of 783.2455.
an m/z of 783.2455.

Figure8.8.Spectra
Figure Spectra of
of unknown-2
unknown-2 betacyanin.
betacyanin.(A)
(A)MS/MS
MS/MS spectrum obtained
spectrum withwith
obtained fragmentation of of
fragmentation
ion m/z 827.23 with HCD. (B) MS/MS spectrum obtained with the ion m/z 827.23 fragmented with
ion m/z 827.23 with HCD. (B) MS/MS spectrum obtained with the ion m/z 827.23 fragmented with
CID at 35 eV. (C) MS 3 spectrum obtained with the ion m/z 695.19 fragmented with CID at 35 eV
3 spectrum obtained with the ion m/z 695.19 fragmented with CID at 35 eV (827.23
CID at 35 eV. (C) MS
(827.23 CID35  695.19 CID35 ). (D) MS4 spectrum obtained with fragmentation of the ion m/z
4 spectrum obtained with fragmentation of the ion m/z 551.15 with
551.15→
CID35 695.19
with CIDCID35
at 35 eV →).(827.23
(D) MSCID35  695.19 CID35  551.15 CID35). Core ions are high-
CID at 35ineV
lighted (827.23 CID35 → 695.19 CID35 → 551.15 CID35→). Core ions are highlighted in black.
black.

3. Materials and Methods

3.1. Chemicals and Reagents
Analytical-grade methanol (MeOH) and water (H2 O) were purchased from J.T. Baker
(Phillipsburg, NJ, USA). Analytical-grade formic acid and acetic acid were obtained from
Merck (Sigma Aldrich, St. Louis, MI, USA). As an analytical standard, a betanin red beet
extract diluted with dextrin was used (Sigma Aldrich, St. Louis, MI, USA). Methanol and
water (mass spectrometry-MS grade) were purchased from TEDIA (Fairfield, OH, USA).
Molecules 2024, 29, 5485 19 of 24

Ultrapure Helium gas (>99.999%) was obtained from a local supplier (INFRA, Merida,
Mexico). N2 was generated in-house using an NM32LA PEAK Generator (PEAK Scientific
System, Inchinnan, Scotland, UK).

3.2. Fruit Juice Extraction and Matrix Solid-Phase Dispersion Extraction (MSPD)
Dragon fruit (Hylocereus costaricensis) juice was obtained using an electronic mill.
Subsequently, the tissue debris was separated from the juice and then discharged. After
collection, the juice was frozen and kept at −80 ◦ C until it was lyophilized for 48 h (chamber
temperature −50 ◦ C and vacuum pressure −460 mmHg). The dry material was kept in
darkness at −20 ◦ C until it was used.
Total betalains were extracted from 100 mg of dry material using the Matrix Solid-
Phase Dispersion (MSPD) method as described by Araujo-Leon et al., 2023 [37]. A homoge-
nous mix of 400 mg of BondElut-C18 (Agilent Technologies, San Jose, CA, USA) and 100 mg
of dry material was packed in an empty SPE cartridge with frits to minimize diffusion.
Bound betalains were eluted with 9 mL of H2 O with 0.1% acetic acid and then with 9 mL
of MeOH:H2 O (1:1, v/v) with 0.1% acetic acid using a vacuum manifold system (Visiprep
SPE Vacuum Manifold, Sigma-Aldrich, Burlington, MA, USA). Separately, the eluents were
collected and evaporated. The residues were resuspended in 0.6 mL of H2 O with 0.1%
acetic acid and transferred to an amber vial (MS-grade) for LC-MS analyses.

3.3. UHPLC-UV-VIS-MS/MS Orbitrap

The red pitaya extracts were analyzed by means of a UHPLC Ultimate 3000 system
(Thermo Scientific, Waltham, MA, USA) equipped with a quaternary solvent manager,
autosampler, column heater, and UV–Vis detector and coupled to a LTQ-Orbitrap Elite
mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) equipped with a Heated
Electrospray Ionization interface (HESI-II, Thermo Fisher Scientific, Waltham, MA, USA).
The mobile phase consisted of H2 O with 0.1% acetic acid (solvent A) and MeOH with 0.1%
acetic acid (solvent B) delivered according to the following gradient profile: 95% A kept
for 2 min; 5% B to 100% B in 18 min; 100% B to 5% and isocratic for 10 min for column
reconditioning. The sample was eluted from the column at a flow rate of 300 µL/min. The
red pitaya extracts (20 µL) were injected into the UHPLC system equipped with a Hypersil
GOLD C18 (Thermo Scientific, Waltham, MA, USA) reversed-phase column with 100 mm
length, 2.1 mm internal diameter, and 1.9 µm particle size. The chromatographic column
was kept at 45 ◦ C ± 0.5 ◦ C. UV/Vis spectra data were collected at 480 and 540 nm.
The electrospray ionization was optimized as follows: spray voltage, +4.5 kV; capillary
temperature, 300 ◦ C; heater temperature, 180 ◦ C; sheath gas flow rate, 40; auxiliary gas,
15. MS spectra data were acquired in positive mode. Multidimensional MS (MSn ) data
were acquired to support the proposed structure hypothesis. For data acquisition under
data-dependent acquisition (DDA) mode: MS1 resolution 60,000, scan range 100–1500 m/z;
and MS2 resolution 60,000, scan range 100–600 m/z. Higher-energy collisional dissociation
(HCD) was used as a fragmentation method by applying 50 eV. N2 and helium (He) were
applied as cone and collision gases, respectively. Peak areas of each compound based
in MS1 mode using the extracted ion chromatograms were determined with Xcalibur 4.1
software (Thermo Scientific, Waltham, MA, USA). Graphs were obtained using GraphPad
Prism version 9.0 (GraphPad, San Diego, CA, USA).

3.4. MassFrontier-Based In Silico Fragmentation Library

Chemical structures were drawn for known betalains [15,36,43] using ChemDraw
Professional 16.0 in accordance with the guidelines of the American Chemical Society
(ACS-1996). Next, the structures were placed in the Structure Editor of MassFrontier 7.0
software (Thermo Scientific, Waltham, MA, USA). The charge specification was made
to generate precursor masses [M+H]+ and fragment ions in silico. HighChem ESI Pos
2008 and HighChem Fragmentation Libraries were used. The fragmentation parameters
were established to be steric optimal, allowing resonance reactions for electron sharing,
Molecules 2024, 29, 5485 20 of 24

charge stabilization, and radical isomerization. Fragment peaks and precursor masses were
deposited in Table S3.

4. Conclusions
Collectively, our compiled database of betalains, as provided in the Supplementary
Materials of this manuscript, and the mass spectrometry fingerprints of betanin, beet-
root extract, and red pitaya extract revealed information about betalain fragmentation.
These data can aid in interpreting the mass spectra of betalains, which ultimately not
only improves the identification of betalains but also serves as a guide for mass spectra
annotation. The aglycone betacyanin-derived ions with m/z 389 for betacyanins, m/z 345
for betalain derivatives, and m/z 343 for neobetanin variants can be used for the rapid
identification of decarboxylated and dehydrogenated betalains; further automation on
parallel metabolomics studies will aid in increasing the annotation quality of metabolite
identification. The ion with m/z 211.07 was characteristic of betaxanthins. As betalains are
responsible for biological traits in Caryophyllales plants or for nutritional and biological
activities seen in organisms feeding on raw or processed food containing betalains, further
investigation will benefit from the improved identification of betalains. For red pitaya be-
talains, given the catalog has grown to 86, it is crucial to conduct more research into the
diversity and abundance of betalains in different pitaya varieties with distinct flesh and
skin colors.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/molecules29225485/s1. Table S1: Database of betalains made
in this study consisting of structures and spectral libraries based on the use of in silico fragmenta-
tion with MassFrontier (Thermo Scientific) software; Table S2: Product ions of betanin fragmented
with HCD at 50 eV with a relative abundance of more than 5% highlighted in this study as fin-
gerprints of betalains; Table S3: Chromatographic and mass spectrometry data for betalains from
Beta vulgaris extract. References: [2,15,36,37,40,43,50,57–60,63,65–70,73,74,76–104] are cited in the
Supplementary Materials.
Author Contributions: Conceptualization: J.A.A.-L. and V.A.-H.; methodology, formal analysis and
investigation: J.A.A.-L., R.O.-A., L.G.B.-A., I.S.-d.P. and S.R.P.-S.; software: J.A.A.-L. and V.A.-H.;
validation: R.O.-A., L.G.B.-A., I.S.-d.P. and S.R.P.-S.; supervision: V.A.-H. and I.S.-d.P.; visualization:
J.A.A.-L., I.S.-d.P. and V.A.-H.; resources: I.S.-d.P. and V.A.-H.; writing—original draft: J.A.A.-L.;
writing—review and editing: I.S.-d.P. and V.A.-H. All authors have read and agreed to the published
version of the manuscript.
Funding: CONAHCYT funded this research, grant FORDECYT-PRONACES-15319/2020.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are contained within the article and Supplementary Materials.
Acknowledgments: Sánchez-del Pino I., Peraza-Sánchez S.R., and Aguilar-Hernández V. thank
CONAHCYT for support under the project FORDECYT-PRONACES-15319/2020. Araujo-León J.A.
thanks CONAHCYT for PhD scholarship 800585.
Conflicts of Interest: The authors declare no conflicts of interest.

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