Detection of SO2 derivatives using a new chalco-coumarin derivative in cationic micellar media: Application to real samples

Article abstract of DOI:10.1039/c8ra04526g

A new probe (E)-7-(diethylamino)-3-(3-(thiophen-2-yl)acryloyl)-2H-chromen-2-one (ChC16) was synthesized and studied as a turn-on fluorescent probe, based on a Michael addition mechanism for sensing SO₂ derivatives, which is favored in the presence of cationic micellar media such as cetylpyridinium bromide (CPB). The probe showed high selectivity and sensitivity toward bisulfite over other anions and biothiols, including cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), with a detection limit of 240 nM. Moreover, the probe showed great potential for its practical application in the detection of bisulfite in real samples, such as dry white wine, and in bioimaging.

Full text of DOI:10.1039/c8ra04526g

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PAPER
Detection of SO2 derivatives using a new chalco-
coumarin derivative in cationic micellar media:
application to real samples†
Cite this: RSC Adv., 2018, 8, 31261
ab
a
Veronica Arancibia,a Alexis Moya,a
*
Marisol Gomez,
Margarita E. Aliaga,
´
´
Camilo Segura,c Marco T. Nunez,d Pabla Aguirre,d Edgar Nagles
and Olimpo Garcıa-Beltran
e
˜
*
e
´
´
A
new probe (E)-7-(diethylamino)-3-(3-(thiophen-2-yl)acryloyl)-2H-chromen-2-one (ChC16) was
synthesized and studied as a turn-on fluorescent probe, based on a Michael addition mechanism for
sensing SO2 derivatives, which is favored in the presence of cationic micellar media such as
cetylpyridinium bromide (CPB). The probe showed high selectivity and sensitivity toward bisulfite over
other anions and biothiols, including cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), with
a detection limit of 240 nM. Moreover, the probe showed great potential for its practical application in
the detection of bisulfite in real samples, such as dry white wine, and in bioimaging.
Received 28th May 2018
Accepted 26th August 2018
DOI: 10.1039/c8ra04526g
rsc.li/rsc-advances
At this time, there are several conventional methods for this
purpose, such as iodometric titration,7 chromatography,8,9
1. Introduction
electrochemical analysis10–12 and ow injection analysis
(FIA).13,14 Nevertheless, the principal disadvantage of these
methods is that the majority of them require sample pre-
treatment and the use of multiple reagents. Furthermore, in
some cases, the detection process is very low and depends on
high-cost instruments.
Sulfur dioxide (SO2) derivatives, such as sulte (SO3ꢀ2) and
bisulte (HSO3ꢀ) ions, have been largely used as preservatives
for many foodstuffs, drinks and medications.1 However, it is
well-known that all the SO2 forms can potentially cause health
problems. Specically, it has been reported that elevated
quantities of sulte can cause asthma and allergic reactions in
some people, including difficulty in breathing, urticaria and
gastrointestinal discomfort.2–4
In this context, the Joint FAO/WHO Expert Committee on
Food Additives has determined that an acceptable daily intake
of sultes should be lower than 0.7 mg kgꢀ1 of body weight.5
Besides, since 1986 the FDA in the United States has demanded
that any food or drink that contains a sulte concentration
bigger than 10 mg Lꢀ1 (125 mM)6 should be labelled. Therefore,
the development of analytical methods that allow the detection
and quantication of by-products of SO2 have gained great
interest.
By contrast, the current use of uorescent probes as a detec-
tion technique has acquired great relevance due to their high
sensitivity and accuracy. In this context, novelty uorescent
probes have been developed by the detection of SO2 deriva-
tives,15–19 mainly based on reactions with HSO3ꢀ/SO32ꢀ. Some
examples reported in the literature are summarized in Table S1
(ESI†), and among this information we are interested in those
that undergo a nucleophilic addition to “C]C” double bond, as
a detection mechanism. Interestingly, Zhang et al. (2013)20 re-
ported promising studies in this area, using a cationic cetyl-
trimethylammonium bromide (CTAB) micelle, which created
a hydrophobic microenvironment promoting the addition reac-
tion from the sulte to an activated olen in aqueous solutions.
Therefore, the selectivity and sensitivity of the probe improved
considerably. More recently, we have reported new probes able to
detect SO2-derivatives via a Michael-type addition reaction, which
was also favored by micellar media.21 In particular, we demon-
strated that typical interferences, such as glutathione (GSH) and
cysteine (Cys) were inhibited by the presence of the cationic
micelles of cetylpyridinium bromide (CPB) and zwitterionic
micelle of sulfobetaine (SB3-14) and the detection limits
improved considerably. Thus, we take advantage of this fact
and we are presenting in this study a new coumarin–chalcone
derivative (E)-7-(diethylamino)-3-(3-(thiophen-2-yl)acryloyl)-2H-
a
´
´
˜
Facultad de Quımica, Ponticia Universidad Catolica de Chile, Vicuna Mackenna
4860, Santiago, 7820436, Chile
b
´
´
Escuela de Obstetricia y Puericultura and Centro Integrativo de Biologıa y Quımica
Aplicada (CIBQA), Universidad Bernardo OHiggins, General Gana 1702, Santiago,
8370993, Chile
cDepartment of Chemistry, Faculty of Sciences, Universidad de Chile, Santiago
7800024, Chile
dBiology Department, Faculty of Sciences, Universidad de Chile, Santiago 7800024,
Chile
e
´
´
Facultad de Ciencias Naturales y Matematicas, Universidad de Ibague, Carrera 22
´
Calle 67, Ibague 730001, Colombia. E-mail: jose.garcia@unibague.edu.co
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c8ra04526g
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Table 1 Photophysical parameters of the tested fluorescent ChC16 in
the absence and presence of CPB and Na2SO3 in aqueous solution
chromen-2-one (ChC16). The latter would undergo a nucleophilic
addition reaction of SO2-derivatives to its “C]C” double bond,
favored by the use of a cationic micelle.
[CBP] (mM) [Na2SO3] (mM) lAbs (nm) lEmi (nm) 3 (Mꢀ1 cmꢀ1
)
F
—
1.5
—
—
—
500
500
450
460
450
460
500
490
500
490
23 888
36 034
28 434
31 546
0.169
0.349
0.381
0.893
2. Results and discussion
2.1 Synthesis and characterization of probe (ChC16)
1.5
We describe here a new probe ChC16 (Scheme 1), which was
designed considering a diethylaminocoumarin unit, as the
uorophore,5 bound via a double bond (C]C) with a thiophene
fragment. The bond (C]C) would be responsible for the
recognition of SO2-derivatives via a nucleophilic addition reac-
tion. The latter based on previous studies, which demonstrated
that other chalco-coumarin derivatives were able to act as
uorescent sensors for biothiols based on Michael addition.22,23
Regarding the thiophene fragment, it is considered as a good
moiety for Michael acceptor. In fact, studies demonstrated that
2-(2-nitrovinyl)thiophene is only a little less reactive than the
well-known Michael acceptor trans b-nitrostyrene.24
we evaluated the changes in the uorescence intensity associ-
ated with it. The latter was carried out in the presence of
Na2SO3, at different pH values. As it can be seen in Fig. 1, the
uorescence response of probe ChC16 alone was almost pH
insensitive, whereas upon the interaction with Na2SO3 impor-
tant changes were observed depending on the pH values,
showing a high uorescence response from pH 6 to 9. In fact,
a pH value of 7 was found to be appropriate for the bisulte/
sulte detection by probe ChC16. The latter suggests that this
2
probe can successfully react with HSO3ꢀ/SO3 and allow for
The new compound ChC16 was synthesized in two steps, as
shown in Scheme 1. Firstly, 4-diethylaminosalicylaldehyde (1)
was condensed with ethyl acetoacetate (Knoevenagel) and
cyclized obtaining the compound 3-acetyl-7-(diethylamino)-2H-
chromen-2-one (2).25 Finally, ChC16 was synthetized by adap-
tations of a literature procedure26 and characterized by 1H-NMR,
13C-NMR and HRMS (S1–S3; ESI†).
them to be detected in physiological conditions.
Considering these results, we assessed the effect of adding
increasing concentrations of Na2SO3 on the intensity of the emis-
sion band of ChC16 at 490 nm, in the presence of CPB. As shown
in Fig. 2(A), the emission band peak of probe in the presence of
different concentrations of Na2SO3 (0–10 equivalent) gradually
increased. The intensity of emission changed from 110 to 700
arbitrary units (see quantum yields in Table 1) and the reaction
could be completed in 15 min. As a result, a good linear rela-
tionship between them was observed; thus, based on this linearity
(Fig. 2(B)), the detection limit was determined to be 240 nM (S/N ¼
3). This value was lower to those reported in the literature for other
probes for sulte in the absence of the micellar medium.21,27
Regarding the stability of the method, this was mentioned in
the Experimental section. The relative standard deviation (RSD)
of the detection associated with the probe in the presence of the
cationic micelle was 2%. It is important to mention that lesser
values to 5% are accepted.28
2.2 Absorption and emission properties of the probe
(ChC16) in a micellar media of CPB
The absorption and emission properties of the ChC16 derivative
at neutral conditions, 20 mM Britton–Robinson (BR) buffer (pH
z 7) containing 1% (v/v) DMSO were assessed. Probe ChC16
exhibits an absorption band with a maximum at 450 nm
(Fig. S4; ESI†), a molar absorptivity (3450) of 23 888 L molꢀ1 cmꢀ1
(Fig. S5; ESI†) and a characteristic emission ꢁ500 nm. In the
presence of the micelle CPB, the absorption band associated
with ChC16 is well dened and centered at 460 nm. Regarding
the intensity of uorescence emission (centered on 500 nm), it
considerably increased by CPB effect. As shown in Table 1, the
quantum yields were determined to be 0.169 and 0.349 for
ChC16 in absence and presence of CPB, respectively.
2.3 Effect of pH on the uorescence response of the probe
ChC16 toward SO2-derivatives
To ensure the optimal conditions to efficiently detect SO2
derivatives (HSO3ꢀ/SO32ꢀ) with the tested derivative (ChC16),
Fig. 1 Effect of pH on the fluorescence intensity of the ChC16 (5 mM)
Scheme 1 Synthetic route for probe ChC16. Reagents and conditions: alone ( ) and after its reaction with Na2SO3 (10 mM) ( ) at 25 ꢂC after
(a) ethyl acetoacetate, piperidine, AcOH, EtOH, reflux, 6 h; (b) 2-thi- 120 min of reaction. Emission at 485 nm was measured. lex ¼ 450 nm.
ophenecarboxaldehyde, DCM, reflux, 12 h.
Slits 5 nm.
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Fig. 3 Fluorescence intensities (lex ¼ 450 nm) of ChC16 (10 mM) upon
addition of various anions (100 mM) in the presence of 1.5 mM of CPB.
White bar represents: ChC16 alone (1) and grey bars represent: NO +
anions (2) HSO3ꢀ(*); (3) cysteine; (4) glutathione; (5) Fꢀ; (6) Clꢀ; (7) Brꢀꢀ;
(8) Iꢀ; (9) CH3COOꢀ; (10) NO2ꢀ; (11) NO3ꢀ; (12) HCO3ꢀ; (13) H2PO4
;
(14) SO42ꢀ; (15) S2O32ꢀ; (16) S2ꢀ. Slits 5 nm. The symbol (*) represents
the analyte of interest.
anions have no interference with the uorescence response of
2ꢀ
ChC16 toward HSO3ꢀ/SO3
, thereby indicating its high
selectivity.
2.5 Sensing mechanism of the probe ChC16 toward SO2-
derivatives
2ꢀ
Considering that both HSO3ꢀ/SO3
can be acting as
Fig. 2 (A) Fluorescence spectra of ChC16 (10 mM) upon the addition of
increasing concentration of Na2SO3 from 5 to 50 mM in Britton–
Robinson (BR) buffer solution (20 mM, pH 7, 1% DMSO) in the presence
of 1.5 mM of CPB; lex ¼ 450 nm. (B) Changes in fluorescence intensity
of ChC16 at 480 nm upon the gradual addition of Na2SO3 from 5 to 80
mM; lex ¼ 485 nm; slits, 5 nm.
nucleophiles, we proposed that their addition to the double
bond (C]C) of the probe could be responsible for the change in
the uorescence emission.
2.4 Selectivity and competition studies of the probe ChC16
toward SO2-derivatives over other analytes
With the aim to evaluate the selectivity of the tested probe
(ChC16) toward SO2 derivatives, we investigate its uorescence
response to typical interfering anions such as Fꢀ; Clꢀ; Brꢀ; Iꢀ;
NO3ꢀ; NO2ꢀ; SO42ꢀ; SCNꢀ; S2O32ꢀ; S2O42ꢀ; S2ꢀ; CH3COOꢀ;
ꢀ
HCO3ꢀ; H2PO4 and biothiols (i.e. cysteine (Cys) and gluta-
thione (GSH)). As illustrated in Fig. 3, no signicant variations
in the uorescence emission associated to the probe were
observed, for most of these anions, even in the presence of 10
eq. of the anions mentioned above. Except a slight change in the
probe intensity was observed when the biothiol (GSH) was
assessed.
Fig. 4 Fluorescence intensities (lex ¼ 450 nm) of ChC16 (10 mM) upon
addition of various anions (100 mM) in the presence of 1.5 mM of CPB.
White bar represents: ChC16 alone (1) and grey bars represent: ChC16
Furthermore, a competitive analysis of the different tested
2ꢀ
anions with HSO3ꢀ/SO3 was conducted. As shown in Fig. 4,
ꢀ
in the presence of HSO3 (2) and other anions; (3) cysteinꢀe; (4)
aer adding SO2 derivatives into the solution containing other
anions, no signicant variation in uorescence intensity asso-
ciated with ChC16 was found. It can be noted that all the tested
glutathione; (5) Fꢀ; (6) Clꢀ; (7) Brꢀ; (8) Iꢀ; (9) CH3COOꢀ; (10) NO2 ; (11)
NO3ꢀ; (12) HCO3ꢀ; (13) H2PO4ꢀ; (14) SO42ꢀ; (15) S2O32ꢀ; (16) S2ꢀ. Slits
5 nm. The symbol (*) represents the analyte of interest.
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Table 2 Determination of bisulfite concentration in real samples, see
details in Experimental section
In order to conrm the proposed mechanism for the
2ꢀ
response of the probe aer its interaction with HSO3ꢀ/SO3
,
rst we carried out experiments using high resolution mass
spectroscopy (HRMS-ESI).
Fluorometric method
using ChC16 + CPB
AOAC Official
Sample
Method29
Results from the HRMS-ESI (Fig S6; ESI†) show a major ion
peak found at m/z 435.1598 which is nearly identical to the
theoretical molecular mass of the ChC16–bisulte adduct
([ChC16–H]+ calcd 435.0858). These data strongly support the
1 : 1 adduct formation of probe ChC16 mainly with bisulte
(ChC16–SO3H).
Wine 1
Wine 2
104.7 [mg Lꢀ1
115.3 [mg Lꢀ1
]
]
100.3 [mg Lꢀ1
119.6 [mg Lꢀ1
]
]
neuroblastoma cells were incubated with ChC16 (5 mM) for
about 20 min at 37 ꢂC, a low intracellular uorescence was
observed, with cytoplasmic accumulation. Aer treatment of
cells with Na2SO3 (30 mM) for an additional 45 min at 37 ꢂC,
a slight increase in the uorescence signal was detected (not
shown).
However, other experiments demonstrated that, when SH-
SY5Y cells were incubated with ChC16 (5 mM) in the presence
of the cationic micelle CPB (1 mM) and Na2SO3 for an additional
Subsequently, we assessed the interaction mode between
1
ChC16 and bisulte using H-NMR spectroscopy. As shown in
Fig. 5A, the spectrum of ChC16 in the absence of Na2SO3 depicts
two important signals at 7.93 and 7.75 ppm, which could be
attributed to the vinylic protons Hb and Ha, respectively. Upon
the addition of Na2SO3 to a solution containing the probe, the
resulting spectrum presents a new signal that appears at
5.08 ppm (Fig. 5(B)), which is attributed to the proton Hb that
was upeld shied. These data suggest that the reaction
between bisulte and ChC16 is a Michael addition in the b-
carbon of the ketone a,b-unsaturated. This result is consistent
with the formation of the ChC16–SO3H adduct.
ꢂ
15 min at 37 C, a large increase in uorescent intensity could
be observed. Thus, demonstrating that the compound ChC16
acts as a probe “turn on” induced by the mixture CPB-SO2
derivatives. Finally, it is important to mention that the ChC16
compound has a diethylamino group at the C-7 of the coumarin
and this group have shown affinity for the cell membrane.25
However, in this study it was observed that the compound have
no affinity by cell membrane. Moreover, an accumulation in
cytoplasmic structures was observed.
2.6 Validation and application to real samples
In order to demonstrate the applicability of the tested probe,
ꢀ
compound ChC16 was assessed to detect HSO3 in real
samples. In the rst case, the real sample included white wines,
which were analyzed using probe ChC16 in the presence of CPB
as a uorometric method and a reported method to determine
the total sulte concentration.29 As shown in Table 2, the ob-
tained results using these two detection methods for bisulte
perfectly t (error range 4–9%). Thus, these results suggest that
probe ChC16, in the presence of a cationicꢀmicelle as CPB, can
be used for quantitative detection of HSO3 in a real sample.
Furthermore, bearing in mind evidence on the production of
sulfur dioxide in cytosols and mitochondria of cells30 we also
assessed whether the probe can determine SO2 derivatives in
biological systems. As shown in Fig. 6(A), when SH-SY5Y
Fig. 6 Fluorescence images of SH-SY5Y cells. (A) Cells were incubated
Fig. 5 1H-NMR partial of ChC16 in the absence (A) and in the presence with probe ChC16 (5 mM) for 20 min; (B) image of cells after subse-
(B) of Na2SO3 (10 eq.), solvent used DMSO-d6 : D2O (4 : 1).
quent treatment with CPB (1 mM) and Na2SO3 (30 mM) for 15 min.
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4.2.2 Nuclear magnetic resonance (NMR) studies. H and
3. Conclusions
13C NMR spectra were obtained at 25 ꢂC on a Bruker Avance 400
In this work, we synthesized and characterized a new chalco- MHz spectrometer using TMS as an internal standard. The NMR
coumarin derivative and we assessed its photo-physical spectra were processed with MestreNova soware v9.0. All
behavior as a sensor for SO2 derivatives. We found that this solutions were prepared by mixing appropriate volumes of stock
probe, in the presence of a cationic micellar medium, displays solutions of ChC16 in DMSO-d6 and D2O.
a high selectivity for sulte over the other typical interferents
4.2.3 Determining the quantum yield of emission. Fluo-
(biothiols) with a detection limit next to 10ꢀ9 M. Finally, we were rescence quantum yields ChC16 were measured using a solu-
able to carry out experiments in real samples of white wine and tion of quinine sulfate in 0.5 M H2SO4 as standard (Fs ¼ 0.546)31
cell imaging to visualize bisulte by the proposed uorometric for probe. All values were corrected taking into account the
method, using ChC16 in a micellar cationic media.
solvent refraction index. Quantum yields were calculated using
eqn (1),31 where the subscripts x and s denote sample and
standard, respectively, F is the quantum yield, h is the refractive
index, and Grad is the slope from the plot of integrated uo-
rescence intensity vs. absorbance.
4. Experimental
4.1 Synthetic procedures
ꢀ
ꢁꢀ
ꢁ
2
4.1.1 Synthesis of (E)-3-(3-(1H-pyrrol-2-yl)acryloyl)-7-(dieth-
ylamino)-2H-chromen-2-one. 4-Diethylaminosalicylaldehyde (1)
(2.0 g, 10.4 mmol), ethyl acetoacetate (2.6 g, 20 mmol), piperidine
(0.5 mL) and one drop of AcOH, were combined in absolute EtOH
(60 mL) and reuxed for 6 h and cyclized in a single step to afford
obtaining the compound 3-acetyl-7-(diethylamino)-2H-chromen-
2-one (2). Subsequently, compound 2 (0.5 g, 1.9 mmol) and 2-
thiophenecarboxaldehyde (0.2 g, 1.9 mmol) were dissolved in
10 mL of EtOH and 0.3 mL of piperidine were added to this
solution. The mixture was kept at reux temperature for 14 h. The
solution was concentrated under reduced pressure. The solid was
nally puried by CC on silica gel eluting with DCM : AcOEt
15 : 1. The product obtained is a yellow solid (0.478 g, 1.35 mmol)
in 71.0% yield. 1H-NMR (400 MHz, DMSO-d6) d 8.57 (s, 1H), 7.93
(d, 1H, J ¼ 20 Hz, Ar-CH, H30), 7.75 (d, 1H, J ¼ 20 Hz, CO–CH¼,
H20), 7.73 (s, 1H, Ar–H, H4), 7.68 (d, 1H, J ¼ 12 Hz, Ar–H, H400),
7.56 (d, 1H, J ¼ 12 Hz, Ar–H, H6, H8), 7.17 (t, 1H, J ¼ 4.0 Hz, Ar–H,
H6), 6.80 (d, 1H, J ¼ 4.0 Hz, Ar–H, H6), 6.59 (s, 1H), 3.50 (dd, 4H, J
¼ 8.0, –CH2–), 1.15 (s, 6H, J ¼ 8.0, –CH3). 13C-NMR (100 MHz,
DMSO-d6) 12.8, 44.9, 96.3, 108.4, 110.7, 115.6, 124.0, 129.2, 130.2,
133.0, 133.4, 135.5, 140.7, 148.9, 153.3, 158.7, 160.5, 185.3. The m/
z observed value was 354.1148 positive mode, and the calculated
value for C20H19NO3S was 353.1085 (Fig. S3†).
Gradx
Grads
hx
hx
Fx ¼ Fs
(1)
2
4.2.4 Reproducibility. The stability of the method was
determined into measuring the intensity of uorescence in the
wavelength of the peak of emission in the presence of CPB for
30 successive measures each 5 minutes. The shrunk data were
examined in the soware Origin 8.0.
4.2.5 Detection limit. The detection limit (LOD) was
calculated based on uorescence of the successive additions of
the probe. To determine the S/N ratio, the emission intensity of
each probe with Na2SO3 was measured for three times and the
standard deviation of calibration curve was determined. The
LOD was calculated from LOD(y) ¼ a + 3sx/y and LOD(y) ¼ a +
bLOD, where a is the intercept, sx/y is the random error in x and
y, and b is the slope of the plot of uorescence intensity versus
sample concentration.21 In addition, LOD ¼ LOD(x) ¼ 3sx/y/b,
assuming that errors principally occur in the y-direction.
4.2.6 Preparation of real samples. The studied samples
were commercial white wines. For this, 30 mL of a newly opened
wine bottle was taken and 5 g of activated carbon was added.
Aer 30 min, the sample was ltered with syringe lters 0.45
micron to remove carbon. Subsequently each sample was added
to the uorescence cuvette, it contained Britton–Robinson (BR)
buffer solution (20 mM, pH 7, 1% DMSO) in the presence of
1.5 mM of CPB. Subsequently, successive additions of Na2SO3
were carried out and using the standard addition method. The
concentration of the sample was determined.
4.2.7 Cell culture and uorescence imaging. SH-SY5Y
neuroblastoma cell lines (CRL-2266, American Type Culture
Collection, Rockville, MD) were cultured in MEM/F12 medium
supplemented with 10% fetal bovine serum (FBS), non-essential
amino acids, antibiotic–antimycotic mixture, and 20 mM
HEPES buffer, pH 7.4. The medium was replaced every 2 days.
Cells were exposed and incubated with, ChC16 (5 mM, 20 min)
and the basal uorescence of the probe was measured. In other
cases, adding one mix incubated with ChC16 (5 mM) in the
presence of the cationic micelle CPB (1 mM), Na2SO3 (30 mM) for
about 15 min, and washed with HBSS with calcium and
magnesium salts, aer that the uorescence was determined.
4.2 Materials and methods
Solvents and reagents utilized were Sigma-Aldrich. All solutions
employed in this study were prepared in Britton–Robinson (BR)
buffer solution (20 mM, pH ¼ 7) and the reagents utilized were
Suprapur®. The stock dissolution of the probe was conducted
in DMSO. Absorption and uorescence spectra were obtained
HP-8453 diode array spectrophotometer and Cary Eclipse uo-
rescence spectrophotometer, respectively.
4.2.1 High-resolution mass spectrometry (HRMS-ESI)
studies. High-resolution mass spectra (HRMS-ESI) were extrac-
ted from high resolution mass spectrometer Exative™ Plus
Orbitrap, ThermoFisher Scientic. The analysis for the reaction
products was performed with the following scan parameters:
resolution 140.000; AGC target 1e6; inject time 200. HESI source
was heath gas ow 8; auxiliary gas ow rate 3; sweep gas ow
rate 0; capillary temperature 250 ꢂC, S-lens RF 100; heater
temperature 100 ꢂC, at mode positive with spray voltage 2.8 kV.
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Conflicts of interest
There are no conicts to declare.
Acknowledgements
This work was supported by FONDECYT postdoc grant
#3150196, FONDECYT grants #1170753 and #11170701, and by
COLCIENCIAS Grants #130774559056 Colombia.
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