A focused multiple reaction monitoring (MRM) quantitative method for bioactive grapevine stilbenes by ultra-high-performance liquid chromatography coupled to triple-quadrupole mass spectrometry (UHPLC-QqQ)

Article abstract of DOI:10.3390/molecules22030418

Grapevine stilbenes are a family of polyphenols which derive from trans-resveratrol having antifungal and antimicrobial properties, thus being considered as phytoalexins. In addition to their diverse bioactive properties in animal models, they highlight a strong potential in human health maintenance and promotion. Due to this relevance, highly-specific qualitative and quantitative methods of analysis are necessary to accurately analyze stilbenes in different matrices derived from grapevine. Here, we developed a rapid, sensitive, and specific analysis method using ultra-high-performance liquid chromatography coupled to triple-quadrupole mass spectrometry (UHPLC-QqQ) in MRM mode to detect and quantify five grapevine stilbenes, trans-resveratrol, trans-piceid, trans-piceatannol, trans-pterostilbene, and trans-?-viniferin, whose interest in relation to human health is continuously growing. The method was optimized to minimize in-source fragmentation of piceid and to avoid co-elution of cis-piceid and trans-resveratrol, as both are detected with resveratrol transitions. The applicability of the developed method of stilbene analysis was tested successfully in different complex matrices including cellular extracts of Vitis vinifera cell cultures, reaction media of biotransformation assays, and red wine.

Full text of DOI:10.3390/molecules22030418

molecules
Article
A Focused Multiple Reaction Monitoring (MRM)
Quantitative Method for Bioactive Grapevine
Stilbenes by Ultra-High-Performance Liquid
Chromatography Coupled to Triple-Quadrupole Mass
Spectrometry (UHPLC-QqQ)
Elías Hurtado-Gaitán 1, Susana Sellés-Marchart 1,2, Ascensión Martínez-Márquez 1,
Antonio Samper-Herrero 1 and Roque Bru-Martínez 1,3,
*
1
Departamento Agroquímica y Bioquímica, Facultad de Ciencias, Universidad de Alicante, 03690 Alicante,
Spain; ehurtadog7@gmail.com (E.H.-G.); susana.selles@ua.es (S.S.-M.); asun.martinez@ua.es (A.M.-M.);
antonio.samper@ua.es (A.S.-H.)
Genomics and Proteomics Unit, SSTTI Universidad de Alicante, 03690 Alicante, Spain
Instituto de Investigación Sanitaria y Biomédica de Alicante ISABIAL-Fundación para el Fomento de la
Investigación Sanitaria y Biomédica de la Comunitat Valenciana FISABIO, 03010 Alicante, Spain
Correspondence: roque.bru@ua.es; Tel.: +34-96-590-3400
2
3
*
Academic Editor: Philippe Jeandet
Received: 23 January 2017; Accepted: 1 March 2017; Published: 7 March 2017
Abstract: Grapevine stilbenes are a family of polyphenols which derive from trans-resveratrol having
antifungal and antimicrobial properties, thus being considered as phytoalexins. In addition to their
diverse bioactive properties in animal models, they highlight a strong potential in human health
maintenance and promotion. Due to this relevance, highly-specific qualitative and quantitative
methods of analysis are necessary to accurately analyze stilbenes in different matrices derived
from grapevine. Here, we developed a rapid, sensitive, and specific analysis method using
ultra-high-performance liquid chromatography coupled to triple-quadrupole mass spectrometry
(UHPLC-QqQ) in MRM mode to detect and quantify five grapevine stilbenes, trans-resveratrol,
trans-piceid, trans-piceatannol, trans-pterostilbene, and trans-ε-viniferin, whose interest in relation
to human health is continuously growing. The method was optimized to minimize in-source
fragmentation of piceid and to avoid co-elution of cis-piceid and trans-resveratrol, as both are detected
with resveratrol transitions. The applicability of the developed method of stilbene analysis was tested
successfully in different complex matrices including cellular extracts of Vitis vinifera cell cultures,
reaction media of biotransformation assays, and red wine.
Keywords: UHPLC; MRM; resveratrol; piceid; piceatannol; epsilon-viniferin; pterostilbene;
cell culture; bioconversion; wine
1. Introduction
Grapevine (Vitis vinifera L.) stilbenes are a family of polyphenols which derive from
trans-Resveratrol (trans-R: 3,5,40-trihydroxystilbene), found in different plant organs, including
berries and, consequently, also in wines. Trans-R is synthesized as an end-product of a branch
of the phenylpropanoid pathway; further trans-R may undergo biochemical modifications, such
as hydroxylation, glycosylation, methylation, or oligomerization, giving rise to several bioactive
derivatives (Figure 1). The synthesis and accumulation of stilbenes is considered to be part of the
general defense mechanism as these are considered as the phytoalexins of Vitis species since they
display strong antifungal and antimicrobial activities [
1–
3]. In fact, trans-R is accumulated in grapevine
Molecules 2017, 22, 418; doi:10.3390/molecules22030418
www.mdpi.com/journal/molecules

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vegetative tissues and berries, as well as in cell cultures in response to abiotic and biotic stress [1,4–9].
Stilbenes have been demonstrated to have a large number of bioactive properties in relation to
major human diseases such as cancer, atherosclerosis, or neurodegeneration [10]. Many studies have
reported the potential of trans-R in human health as an antioxidant in model systems of cardiovascular
diseases [11], as a cytotoxic agent for several cancer lines [12
and antioxidant agent [16 17], having a beneficial role on neurological system [18], as well as health
maintenance and survival in adverse scenarios related with obesity and aging [19 20]. Moreover,
–15], as an anti-inflammatory, anti-aging,
,
,
other studies have demonstrated that some trans-R polyhydroxy and polymethoxy derivatives also
exhibit enhanced pharmacological activity and bioavailability [21]. Such potential of trans-R and
other stilbenes have called the attention of numerous companies producing a large variety of dietary
supplements and cosmetics. Stilbenes are presently in natural foods, such as grapes, berries of Vacinum
species, cocoa, and peanuts, and their derived products [22–26]. In this sense, stilbenes, especially
resveratrol, play a key role in wine health-promoting properties (see [27] for a review). Once the
potential health benefits of stilbenes have been demonstrated, it is necessary to have highly specific
qualitative and quantitative methods to accurately analyze stilbenes in different matrices derived
from grapevine.
Figure 1. Grapevine stilbenes: t-Resveratrol and derivatives produced by different biochemical reactions.
Stilbenes have been detected by different analytical techniques coupled to chromatographic
separation. In wine and grape samples, several research groups report the separation and detection
of phenolic compounds using HPLC coupled to detection by either UV-VIS and fluorescence [28],
or MS and MS/MS [29–33]. However, the specificity of detection of these techniques is limited
because different compounds, having similar mass or spectroscopic properties may co-elute in a
chromatographic run. The incorporation of triple quadrupole mass spectrometers (HPLC-QqQ) to
analyze phenolic compounds significantly improved the detection and allowed the simultaneous
quantitation of multiple compounds in complex matrices in a reduced time. Jaitz et al. quantified 11
polyphenolic compounds from red wines in less than 13 min [34]. In other plant matrices, HPLC-QqQ
MS was applied to quantify 28 polyphenols in cocoa extracts [35] and, more recently, 20 polyphenols
from carob (Ceratonia siliqua L.) flour by improving the chromatography separation and speed by using
ultra-high-performance liquid chromatography (UHPLC) [36]. UHPLC allows faster separation and
enhanced chromatographic resolution as compared to classical HPLC, due to reduced particle size and
increased column pressure (up 1000 bar). UHPLC coupled to triple quadrupole mass spectrometry
using multiple reaction monitoring (MRM) mode has been proposed as an alternative for fast analysis

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of phenolic compounds in grapes and wine. UHPLC-QqQ in MRM mode has been used to detect and
quantify 152 polyphenols in rose wine, in 30 min [37], which is quite a noticeable improvement for
the analysis of general wine phenolics. The large increase in the number of compounds analyzed is
achieved at expense of longer chromatographic runs and possibly, some loss of specificity. and/or
sensitivity when different co-eluting compounds share transitions or are present at dramatically
different abundances.
Thus, when focusing on particular compound families (i.e., stilbenes), improved methods that
provide effective compound separation and quantitation in different kinds of matrices in short
chromatographic runs are necessary.
Our study aimed at developing an analytical method for efficient separation and accurate
quantification of stilbenes produced by Vitis vinifera, of application to different matrices, such as
red wine and plant material extracts, among others. Five important bioactive stilbenes were chosen for
analysis by UHPLC-QqQ MS operated in MRM detection mode, in order to improve the detection and
quantitation of stilbenes in a short time of analysis.
2. Results
2.1. Optimization of Ion Source Parameters
To carry out this work we utilized an Agilent 6490 QQQ instrument equipped with a JetStream®
ion source. As this instrument uses a fixed 380 V fragmentor voltage this parameter cannot be
optimized. Glycosylated polyphenols, such as piceid, are prone to in-source fragmentation, thus, it was
necessary to optimize some parameters of the ion source to minimize this effect. To that, a standard
solution of piceid was directly infused into the instrument and several parameters were tested to
maximize the signal intensity. The cell acceleration voltage, tested at 2 and 5 V, was set at 2 V for
subsequent a◦nalysis. Next, the gas curtain temperature was tested at five values in the range of
◦
◦
250 C to 400 C, with 300 C being selected as a compromise between signal intensity and compound
in-source fragmentation. The gas flow had almost no effect on fragmentation, but a clear improvement
in the signal intensity was observed at 12 L/min. Eventually, we tested several capillary voltages
in a range of 2500–4500 V, getting the best result at 3000 V. As the other stilbene compounds do not
undergo in-source fragmentation, the source parameters optimized for piceid were used throughout in
subsequent analysis.
2.2. Selection of MRM Transitions and Optimization of Chromatographic Separation
Five stilbenes found in Vitis species, namely t-Piceid, t-Piceatannol, t-R, t-ε-Viniferin and
t-Pterostilbene were used in this study. MRM conditions were first optimized in positive detection
mode. Each compound was individually infused and, while operating the MS instrument in product
ion scan mode, four discrete collision energies were applied (0, 10, 20, and 30 eV) in order to detect
fragment ions and automatically select the four most intense ones. Then, the collision energy for each
transition was optimized by applying a 0–40 eV ramp. The transitions selected for each compound,
optimal collision energies and intensities are listed in Table 1. Since the transitions selected for piceid
gave rise to non-specific peaks in the chromatogram, the fragment ions were changed for those of
trans-R, thus producing clean chromatographic traces. Only the product ion m/z 229.1 was kept
as being the most abundant transition for piceid. Due to in-source fragmentation, piceid generates
resveratrol parent ions that are detected through the resveratrol transitions; thus, peak overlapping of
resveratrol and piceid must be avoided.

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Table 1. MRM parameters, chromatographic attributes, and quantitative response of stilbene compounds in standard samples.
Precursor
Ion (m/z)
Product Ion
Collision
Energy (eV)
Retention Time
(Min–Max) trans/cis
Linearity b
Compound
Piceid
Formula
Mass (Da)
% Intensity
LOD (mg/L) LOQ (mg/L)
(m/z)
114.8
308.8
20
8
0
86.5
67.5
100
0.93–0.99/1.52–1.63
1.85–1.99/2.55–2.65
1.25–1.35/1.90–2.00
3.00–3.17/2.80–2.90
5.18–5.28/5.26–5.35
0.04
0.12
0.12
0.07
0.06
0.07
0.22
0.15
0.09
0.08
640
C20H22O8
390.13
391.1
229.09
245.08
455.15
257.12
349.9
229.1 a
8
24.4
107.1 a
24
24
8
100
45.7
80.9
35.6
800
91 a
C14H12O3
C14H12O4
C28H22O6
C16H16O3
228.08
244.07
454.14
Resveratrol
Piceatannol
ε-Viniferin
135 a
165 a
28
107.1
135.1
152
20
12
36
24
95.5
100
11.6
38.7
1600
1777
1000
181.1
107.1
215.1
349.1
199.1
32
20
16
24
100
75.0
38.5
32.0
181
133.1
91
40
12
28
40
90.1
100
58.5
72.2
256,11
Pteroestilbene
165.1
a
b
Transitions used for piceid analysis. Ratio between the highest and lowest concentrations in linear range.

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To optimize the separation conditions of these five stilbenes in a short chromatographic run, a mix
of standard was injected in a UHPLC-QQQ from Agilent (Santa Clara, CA, USA). Several trials were
conducted with different gradients in the mobile phase, containing water as solvent A and acetonitrile
or acetonitrile:methanol as solvent B. Using several binary gradients starting at 5% acetonitrile resulted
in a poor resolution and late elution of peaks and, in addition, peaks corresponding to cis-Piceid
and trans-R appeared overlapping. To improve the poor separation observed, ternary gradients of
water:acetonitrile:methanol were assayed starting at 35% B for an earlier peak elution. A 10 min
gradient was tested using from 40:60 up to 70:30 acetonitrile:methanol as solvent B, thus achieving a
significant improvement in the chromatographic separation, 70:30 being the proportion that showed
the best separation of all the stilbenes. A key improvement of this chromatographic optimization has
been the cis-Piceid peak neat separation from the trans-R peak, otherwise trans-R quantification would
be overestimated as the sum of the signal of both compounds. As can be seen in Figure 2, the gradient
was finally shortened to 6 min (the total run after equilibration was 8 min) without compromising
resolution. Minor peaks, supposed to be the respective cis-isomers as judged by the co-elution of all
compound-specific transitions, were also detected. As mainly the trans isomers are commercialized
as standards, we exposed the standard mixture to UV light to promote photoisomerization in order
to characterize the peaks corresponding to the cis isomers. Under the optimized chromatographic
conditions, all stilbenes both, as trans and cis isomers were detectable and eluted clearly differentiated
(Figure 2). The only co-eluting compounds were trans-R and cis-piceatannol, but such overlapping is
unambiguously resolved by their specific transitions.
Figure 2. Chromatogram of MRM transitions of stilbenes in the standard mix. 1: trans-Piced;
2: trans-piceatannol; 3: cis-Piceid; 4: trans-Resveratrol; 5: cis-Piceatannol; 6: cis-Resveratrol;
7: cis-Viniferin; 8: trans-Viniferin; 9: trans-Pterostilbene; 10: cis-Pterostilbene. (
exposition; ( ) short exposition to UV light; and ( ) prolonged exposition to UV light. Arrows indicate
the presence of new compounds originated by the prolonged UV exposition.
A): Without UV light
B
C

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2.3. MRM Quantitation of Stilbenes in a Standard Mixture
After MRM has been successfully optimized to detect target stilbenes produced by V. vinifera,
performance of the method to quantify these compounds was assessed. For quantitative MRM,
the quantifier transition chromatogram of each targeted precursor m/z was used, and the rest of
transitions provided the evidence of the presence or absence of the stilbene in the standard mixture.
The quantifier transitions used for each compound were m/z 391.1
m/z 229.09 107.1 for resveratrol, m/z 245.08 135.1 for piceatannol, m/z 455.15
and m/z 257.12 133.1 for pterostilbene. Detailed data of metabolite fragmentation is provided as
→
229.1 for piceid,
107.1 for viniferin,
→
→
→
→
Supplementary Material in file S1. The optimized transition for each specific compound constitute a
robust assay that can be used to quantify the target stilbenes in complex mixtures by MRM analysis.
The estimated concentration of standard stilbene solutions were calculated, results are shown in
Supplementary Material file S1.
In order to explore the linear dynamic range of the instrument’s response and the limits
of detection (LOD) and quantification (LOQ) for each stilbene, serial dilutions of the standard
mixture covering several orders of magnitude were analyzed with the MRM method and results
are summarized in Table 1 and Tables S1–S5 in Supplementary Material, file S2. Linear relationship
between peak area and concentration was calculated for all the stilbenes, considering only the peaks
corresponding to trans isomers. At high concentrations of stilbenoids a loss of linearity was apparent,
holding linearity up to 40 mg/L for piceid and pterostilbene, 50 mg/L for Resveratrol and 160 mg/L for
piceatannol and ε-viniferin. The response of the detector was linear over ca. three orders of magnitude
on average for the set of stilbenes analyzed. To determine LOD and LOQ the signal-to-noise ratio was
used establishing rather conservative cut-off values. The LOD ranged from 0.04 to 0.12 mg/L and the
LOQ from 0.07 to 0.22 mg/L.
2.4. Examples of Applications of the Optimized Method
In order to validate the applicability of the MRM method developed we carried out stilbene
analysis in different experimental scenarios. On the one hand, we applied the method to the analysis
of stilbenes extracted from grapevine cell cultures. V. vinifera cell cultures might produce some of
these five stilbenes incubated either in normal growth medium or under elicitation conditions [38].
To that, several samples of methanolic extracts of elicited cell cultures of V. vinifera were used for
the applications of the method in complex mixtures. Use of external calibration curves with three
levels of standards enable the detection and quantification of all stilbenes assayed with the exception
of pterostilbene, which was absent in this sample. The quantifier transition chromatogram for each
stilbene detected is shown in Figure 3 and the calculated concentrations are in Table 2. As expected,
trans-R was the most abundant stilbene produced by V. vinifera under elicitation, but others including
piceid, piceatannol, and ε-viniferin were also present at significant levels.
Table 2. Quantitation of stilbenes in V. vinifera cell culture extracts upon elicitation.
Concentration (mg/L) in
Compound
Extract #1
Extract #2
Extract #3
Piceid
10.92
46.93
1.44
1.48
n.d.
14.07
62.24
1.46
0.93
n.d.
16.35
50.82
1.38
0.11
n.d.
Resveratrol
Piceatannol
ε-Viniferin
Pteroestilbene

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Figure 3. Quantitation of stilbenes from V. vinifera cell cultures upon elicitation. Transition chromatograms
for each compound are shown. Blank is methanol, 80%.
On the other hand, the method was applied to follow the enzymatic assay of bioconversion of
trans-R through an hydroxylation reaction into a tetrahydroxystilbene [39]. A solution of trans-R
standard and NADPH substrates was incubated in the presence of a protein crude extract from
grapevine cell cultures. Stilbene reaction products after one hour incubation, as well as residual trans-R
substrate, was extracted and analyzed with the developed method. As shown in Figure 4A, trans-R
peak decreased in abundance at the time a new peak at the same retention time as trans-Piceatannol
appeared, thus confirming that grapevine cell extracts have resveratrol hydroxylation activity.
Figure 4. Bioconversion of trans-Resveratrol to trans-Piceatannol. (A) Resveratrol transition chromatogram;
and (B) piceatannol transition chromatogram.

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Eventually, red wine samples were analyzed using the MRM method in order to detect and
quantify the absolute amount of the five targeted stilbenes in this highly complex matrix. To reduce
interferences and increase sensitivity of the assay, stilbenes from both spiked and non-spiked samples
were recovered by solid phase extraction [40]. However, the resulting sample is still highly complex as
evidenced by an HPLC-UV analysis (Figure S2) in conditions described by Martinez-Esteso et al. [
5].
As shown in Figure 5, out of the five stilbenes analyzed, piceid, resveratrol and -viniferin were present,
ε
but only their respective trans isomers were quantified (Table 3). In addition, their cis isomers as well
as other viniferins were also present and, presumably, in higher amounts as judged by the intensity of
their corresponding transition peaks, but the lack of standards precluded their absolute quantification.
Figure 5. Quantitation of stilbenes in red wine extract and in the same red wine extract spiked with a
standard stilbene mixture. Transition chromatograms for each compound are shown.
Table 3. Quantitation of stilbenes in red wine extract.
t-Resveratrol
(mg/L)
t-Piceatannol
(mg/L)
ε-Viniferin
(mg/L)
t-Pterostilbene
(mg/L)
t-Piceid (mg/L)
wine
S/N
0.826
140.24
0.281
60.86
n.d.
0.93
0.014
15.6
n.d.
1.67
n.d. Not detected; S/N Signal-to-Noise.
Although the quantitation limits of the stilbenes in this method can be above the existing amount
in wines, the protocol used for sample preparation combining solid phase extraction, leading to 33-fold
concentration, and several calibration points of standard spiking allows for the confident analysis of
concentrations in wine well below LOQ, as is the case for ε-viniferin.
3. Discussion
In the context of the occurrence of bioactive stilbenes in natural sources, a liquid
chromatography-based effective methodology to analyze both qualitatively and quantitatively the
content of these compounds in different matrices is necessary. The way towards efficient analytical
methods of polyphenols has been focused in making both a more efficient chromatographic separation
and a more specific detection system. Thus, we have currently arrived to UHPLC, that lead to sharp
peak resolution in short runs, and mass detectors, from which the triple quadrupole (QqQ) operated
in SRM/MRM mode offers the highest specificity, excellent sensitivity and an extreme multiplexing
capacity. Overall benefits involve a significant reduction of the analysis time and, thus, of costs.
Using HPLC-MS/MS stilbenes from elicited grapevine cell cultures were qualitatively analyzed,
detecting up to 20 different compounds in ca. 30 min chromatographic runs [33]. There also currently
exist UHPLC-MRM methods focused on stilbenes [41], however these were specifically designed
to analyze resveratrol and piceid metabolites in urine bearing negatively-charged groups such as

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glucuronides and sulfates derived from phase II reactions. Recent examples of the application of
UHPLC-QqQ to the analysis of polyphenols in specific matrices, as rose wine [37] or several fruits
and beverages [42], have extensively demonstrated these capacities. Trying to exploit these capacities
to extreme has led to develop the simultaneous assay of 152 different polyphenols in non-processed
samples of rose wine [37], but this remarkable achievement was done at the cost of longer time of
analysis, introduction of quantitative uncertainty and loss of specificity for some compounds which
include stilbenes. In particular, the trans and cis configurations of resveratrol, piceid and two viniferin
isomers of unknown configuration included in such analysis eluted between 12 and 18 min. The short
availability of polyphenolic standards and, in particular, most of the cis isomers of stilbenes, precludes
the direct quantification of these configurations. The similarity between cis and trans fragmentation
spectrum allows their easy detection with the major transitions of the trans standards, as seen in
UV-exposed standard mix (Figure 2), but the different relative abundance of fragment ions introduces
uncertainty in their absolute quantitation. By means of an extensive UV exposition we intended
to quantitatively generate the corresponding cis standards but the appearance of new peaks from
almost every compound revealed that such trans/cis photoisomerization is not quantitative as the
reaction may proceed to generate other isomers [43], so we have not performed cis-isomer quantitation.
The same is true for the diversity of viniferins resulting from the coupling of two resveratrol units
producing several dehydrodimers [44] that, in turn, may also have trans and cis configurations.
In-source fragmentation is frequently occurring in glucoside and galactoside derivatives of phenolic
compounds as syringetin [42] or piceid seen here. This phenomenon lead to detect aglycone at the
retention time of the glycosylated form, in addition to the aglycone at its own retention time. Although
we tried to avoid this phenomenon through source parameters optimization, only an improvement
of the piceid signal was achieved, perhaps because a key parameter of the ion source to attain such
goal, i.e., the fragmentor voltage [45], is fixed for this instrument. For MRM detection, co-elution of
analytes is not generally a problem unless these are detected by the same transitions, as is the case for
the glycosylated cis-Piceid and the aglycone trans-Resveratrol due to the in-source fragmentation of
the former, therefore co-elution has to be avoided. Quantitation of trans-Resveratrol, which has been
extensively performed in plant extracts, juices and especially wines during the last two decades for its
relevance as bioactive compound [46], might therefore be deeply affected by this issue. Although these
compounds co-eluted in most of the chromatographic conditions assayed here, we were eventually
able to establish the right conditions for a neat separation, becoming the first UHPLC-MRM method to
solve this problem, thus assigning each transition peak area to a unique stilbene compound. The use
of methanol as solvent in the mobile phase has been reported to increase considerably the response
when the instrument is operating in positive ionization mode (ESI+) [47]. In the most of methods to
determine and quantify polyphenols based on LC-MS or MS/MS [34,37,42,48–50], solvent B consisted
of acetonitrile or methanol, but here we achieved the best chromatographic resolution using a mixture
70:30 acetonitrile:methanol as solvent B.
As mentioned above, resveratrol dimers exist in diverse isomeric forms but only the
ε
-trans-viniferin is commercialized as standard. The cis isomer was produced by UV exposition
and, conversely to the rest of cis isomers for the stilbenes included in this study, it eluted earlier than
the trans (Figure 2). This has some implications on the tentative assignment of structural variants of
these compounds in complex samples i.e., wine, when no standards are available. In previous studies
using HPLC coupled to an ion trap mass spectrometer we found several viniferin dimers eluting
between resveratrol and pterostilbene standards [5,32], thus, the gap between ε-trans-viniferin and
pterostilbene in the current method is not worthy to shorten as complex samples may contain these
isomers, as can be seen in Figure 5.
MRM has proven to be a successful technique for detection and quantitation of target metabolites.
The set of transitions selected for each stilbene show a high specificity, and allow both their
determination and quantitation in an efficient manner in the standard mixture. Although we monitored
all of them only the most intense transition was used as the quantifier, and the peak area of it and the

Molecules 2017, 22, 418
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nominal concentration was used to construct the calibration curves for each compound. The limit of
quantitation (LOQ) and limit of detection (LOD) were based on the S/N of standards and the values,
in the tens of
µg/L range, were well below the levels typically described for these compounds, above
the hundreds of
µg/L range. Only pterostilbene can be a scarcer compound in biological samples as
its accumulation in grapevine tissues occurs locally and in response to pathogen infection [51], and
even in engineered cell lines its cytotoxicity and fast degradation precludes its high accumulation [38].
The response of the detector was linear over ca. three orders of magnitude, thus covering the levels
found in the majority of matrices. In this way, target stilbenes could be quantitatively analyzed in
different types of samples, including grapevine cell extracts, bioconversion reaction media, and wine.
In conclusion, major achievements of the analytical method developed include a higher analysis
throughput by means of a significant shortage of chromatographic runs and a higher specificity and
accuracy provided by a good separation of compounds detected with the same transitions. Focusing on
the particular family of stilbene compounds provides the analytical advantages over broad compound
coverage methods. Many studies were done in the past twenty years on resveratrol and piceid
content in natural sources, such as fruits, juices, and beverages, including wine, as well as cultures of
grapevine cells. There is a renewed interest in other stilbenes such as piceatannol and pterostilbene,
which show enhanced pharmacological and pharmacodynamical properties with respect to its relative
resveratrol and, oligomers of resveratrol begin to being explored as neuroprotecting compounds [52].
The number of compounds quantifiable with this method may increase as new stilbenes become
available as standards.
4. Materials and Methods
4.1. MRM Method Optimization
4.1.1. Chemicals and Reagents
Trans-Resveratrol, trans-Piceid, and trans-Pterostilbene standards were supplied by Chromadex
(Irvine, CA, USA); trans-ε-Viniferin from Actichem (Mountaban, France) and trans-Piceatannol were
from Cayman Chemical Company (Ann Arbor, MI, USA). Thermo Fisher scientific (Waltham, MA,
USA) supplied formic acid, acetonitrile, water, methanol, and trifluoroacetic acid of LC-MS grade.
4.1.2. Preparation of Standard Solutions
Stock solutions of each individual standard stilbene were prepared in 80% methanol, resveratrol
(500 mg/L), piceid (400 mg/mL), piceatannol (160 mg/mL), ε-viniferin (800 mg/mL), and pterostilbene
(400 mg/mL). These stock solutions were used to prepare a mixture containing the following
concentration of standards: resveratrol (50 mg/L), piceid (40 mg/mL), piceatannol (90 mg/mL),
ε
-viniferin (90 mg/mL), and pterostilbene (40 mg/mL). Dilutions of either pure of mixed standards
were carried out in 80% methanol. To generate cis-isomers of the standards, the stilbene mixture was
exposed to UV light using a germicide lamp for 3 h (short exposition) or 24 h (prolonged exposition).
4.1.3. Chromatography and Mass Conditions
LC-MS analysis were performed in an Agilent 1290 Infinity UHPLC coupled to an Agilent
6490 triple quadrupole mass spectrometer through an Agilent Jet Stream® ion source (Agilent,
Santa Clara, CA, USA). Separation of analytes was performed on a Zorbax Extended C18 column
(
2.1 microns × 50 mm, 1.8
µm; 1200 bar maximal pressure; Agilent, Santa Clara, CA, USA).
In optimized conditions the mobile phase consisted in solvent A (0.05% trifluoroacetic acid) and
solvent B (acetonitrile:methanol 70:30 with 0.05% trifluoroacetic acid) using the following gradient:
0 min 35% B. 2 min 35% B, 2.3 min 36.4% B, 3 min 37% B, 4 min 40% B, 5 min 65% B, 6 min 65% B at a
constant flow rate of 0.4 mL/min. Unless otherwise stated, injection volume was 1 µL.

Molecules 2017, 22, 418
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Multiple reaction monitoring (MRM) analysis mode was used to monitor the transitions from
precursor ions to dominant product ions. Several specific transitions were used to determine each
compound, and the most intense transition was used for the quantitation (quantifier transition).
◦
The optimized source parameters were: capilar voltage 3 kV, gas curtain temperature 300 C, gas
flow 12 L/min, cell acceleration voltage 2 V, a nebulizer (40 psi). For each transition, the collision
energy applied was optimized in order to detect the greatest possible intensity. Dwell time 20 ms. Data
acquisition was performed with the software Mass Hunter Workstation Quantitative Analysis version
B06 SP01 (Agilent, Santa Clara, CA, USA).
4.1.4. Calibration Curves and Lower Limit of Quantitation
Method validation was performed by studying the linear dynamic range, precision of the analysis,
and limit of detection (LOD) and quantitation (LOQ) for the standard compounds. The linear dynamic
range was evaluated using serial dilutions of standard stock solutions and plotting the peak area of
the quantifier transition of each stilbene vs. the nominal concentration of each corresponding standard
compound. The upper linear response was determined by fitting the data starting at a moderately
low concentration (from 0.2 to 1.6 mg/L depending on the compound) to a straight line by linear
regression analysis method. High concentration data were removed until R2
LOQs were established for each compound as the concentration of standard providing a signal to noise
ratio 5 and 10, respectively. For the construction of calibration curves of individual stilbenes, serial
≥
0.99. The LODs and
≥
≥
dilutions from the standard mix solution were performed. The volume injected is the same as the one
mentioned above.
4.2. Method Applications
4.2.1. Analysis and Quantitation of Stilbenes Produced by V. vinifera Cell Culture Upon Elicitation
Vitis vinifera cv. Gamay cell cultures were handled and subjected to elicitation as described in [
Samples of stilbenes from the extracellular medium and from cells were prepared as described in [
One µL aliquot was injected for LC-MS MRM analysis.
2
5
].
].
4.2.2. Biotransformation Assays
Crude protein extracts were prepared from elicited V. vinifera cv. Gamay cell cultures as described
]. Resveratrol hydroxylation reaction was assayed as described in [39 53]. The reaction mixture
in [6
,
contained 0.2 mM of trans-R delivered in DMSO (Fluka Chemika-Sigma Aldrich, St. Louis, MO, USA),
1 mM ascorbic acid, 1 mM NADPH, and 100 mM potassium phosphate buffer, pH 7.4, and started by
adding 50 µL◦ of protein extract to complete a final volume of 1 mL. The reaction mixture was incubated
for 1 h at 37 C in the dark and terminated by the addition of 0.5 mL ethyl acetate to extract stilbenoids
twice. The solvent was evaporated in a Speed-vac (Eppendorff, Hamburg, Germany) and the solid
residue resuspended in 0.2 mL 80% methanol. One µL of reaction medium extract was injected for
UHPLC-MS MRM analysis under the optimized conditions.
Absolute concentration of stilbenes in real samples of cell extracts and enzymatic reaction
extract was determined using an external calibration curve performed with three levels of standards
in duplicate.
4.2.3. Analysis and Quantitation of Stilbenes in Red Wine
Red wine (80% Cabernet Sauvignon and 20% Alicante Bouschet), used in this study was elaborated
during the 2012 vintage in a local wine cellar and the analyzed sample was taken right after the alcoholic
fermentation and then clarified by centrifugation at 10,000
×
g for 20 min. Wine samples of 2 mL
were extracted by◦solid phase according to Kallithraka et al. [40]. After that, extracted stilbenes were
evaporated at 30 C in a Speed-Vac centrifuge (Eppendorff, Hamburg, Germany), resuspended in
◦
60
µL methanol 80% and stored at
−
20 C until analysis. One
µL of sample extract was injected

Molecules 2017, 22, 418
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for UHPLC-MS MRM analysis under the optimized conditions. To minimize the effects of sample
preparation on analysis, a sample of wine was spiked with the standard mix, four serial dilutions were
prepared by mixing the spiked sample with fresh wine before proceeding to solid phase extraction.
A non-spiked wine aliquot was extracted in parallel. Then, concentration of the standard added
to the wine sample was plotted against the measured peak area of the quantifier transition of the
extracted sample. In this way the percentage of stilbene recovery from wine was obviated and the real
concentration of each stilbene compound is obtained from the intercept of the fitting straight line with
the standard concentration axis (A = Ao + F.C, where A is the peak area of any sample; Ao is the peak
area of the non-spiked sample, F is the response factor and C standard concentration), as shown in
Figure S1.
Supplementary Materials: The supplementary materials are available online.
Acknowledgments: A.M.-M. acknowledges a grant from Conselleria d’Educacio, Cultura I Sport de la
Generalitat Valenciana (FPA/2013/A/074). This work has been supported by grants from University of Alicante
(VIGROB-105), the Spanish Ministry of Economy and Competitiveness (BIO2014-51861-R), and European funds
for Regional development (FEDER). Technical assistance of Mario Antonazzo is acknowledged.
Author Contributions: R.B.-M. and S.S.-M. conceived and designed the experiments; E.H.-G., S.S.-M., A.M.-M.,
and A.S.-H. performed the experiments and analyzed the data; E.H.-G. and R.B.-M. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Not Available.
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